US20210285952A1

US20210285952A1 - Nanoparticles and distinct exosome subsets for detection and treatment of cancer

Info

Publication number: US20210285952A1
Application number: US16/873,700
Authority: US
Inventors: David C. Lyden; Haiying Zhang
Original assignee: Cornell University
Current assignee: Cornell University
Priority date: 2017-12-01
Filing date: 2018-12-03
Publication date: 2021-09-16
Also published as: US20230266329A1; EP3716986A4; EP3716986A1; WO2019109077A1; CN111971560A

Abstract

The present invention is directed to methods of diagnosing, prognosing, and managing treatment of cancer in a subject. These methods involve selecting a subject having cancer and obtaining, from the selected subject a population of either exomeres having a diameter less than 50 nm, small exosomes having a diameter of 60-80 nm, or large exosomes having a diameter of 90-120 nm, The exomeres, small exosomes, or large exosomes are recovered from the sample, and the exomeres, small exosomes, or large exosomes or portions thereof are contacted with one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more proteins contained in said exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more N-giyeans contained In said exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or mom lipids contained in said exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or mom genetic mutations in nucleic acid molecules associated with cancer and contained in said exomeres, small exosomes, or large exosomes, or (5) combinations thereof. Cancer is then diagnosed, prognosed, or treatment Is modified based on this information.

Description

This application claims the priority benefit of U.S. Provisional Patent Application Ser. Nos. 62/623,992 and 62/593,504, filed Jan. 30, 2018 and Dec. 1, 2017, respectively, which are hereby incorporated by reference in its entirety.
This invention was made with government support under R01 CA218513 and R01 CA169416 awarded by National Institutes of Health; U01 CA 169538 awarded by National Cancer Institute; and W81XWH-13-10249 and W81XWH-13-1-0425 awarded by Department of Defense. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to nanoparticles and distinct exosome subsets for detection and treatment of cancer.

BACKGROUND OF THE INVENTION

Cells secrete a wide variety of soluble factors and extracellular vesicles (EVs) to mediate intercellular communication (locally and systemically) under both physiological and pathological conditions, including cancers (Théry et al., “Exosomes: Composition, Biogenesis and Function,” Nat Rev Immunol 2:569-579 (2002); Andaloussi et al., “Extracellular Vesicles: Biology and Emerging Therapeutic Opportunities,” Nat Rev Drug Discov 12:347-357 (2013); Raposo & Stoorvogel, “Extracellular Vesicles: Exosomes, Microvesicles, and Friends,” J Cell Biol 200:373-383 (2013)). EVs are heterogeneous and comprise various subclasses, including exosomes, which are small (50 nm to 150 nm) extracellular membrane vesicles of endosomal origin, and microvesicles, which are large (150 nm to 500 nm or even larger to >10 μm) vesicles shed directly by budding from the cellular plasma membrane. Cancer cells shed atypically large vesicles, known as large oncosomes (0.5 μm to 10 μm) which result from alterations in specific signaling pathways (e.g. Ras Homolog Family Member A/Rho-associated protein kinase (RhoA:Rock) signaling) (Di Vizio et al., “Oncosome Formation in Prostate Cancer: Association with a Region of Frequent Chromosomal Deletion in Metastatic Disease,” Cancer Res 69:5601-5609 (2009); Morello et al., “Large Oncosomes Mediate Intercellular Transfer of Functional MicroRNA,” Cell Cycle 12:3526-3536 (2013); Minciacchi et al., “MYC Mediates Large Oncosome-Induced Fibroblast Reprogramming in Prostate Cancer,” Cancer Res 77:2306-2317 (2017)). Extensive research has shown that functional molecules, including proteins, genetic material, metabolites and lipids, are selectively recruited and packaged into EVs and horizontally transferred to recipient cells, thereby acting as vehicles of intercellular communication (Balaj et al., “Tumour Microvesicles Contain Retrotransposon Elements and Amplified Oncogene Sequences,” Nat Commun 2:180 (2011); Choi et al., “Proteomics, Transcriptomics and Lipidomics of Exosomes and Ectosomes,” Proteomics 13:1554-1571 (2013); Thakur et al., “Double-Stranded DNA in Exosomes: A Novel Biomarker in Cancer Detection,” Cell Res 24:766-769 (2014); Tetta et al., “Extracellular Vesicles as an Emerging Mechanism of Cell-to-Cell Communication,” Endocrine 44:11-19 (2013)). In addition, through the work described herein, a novel population of non-membranous nanoparticles termed ‘exomeres’ (˜35 nm) have been identified, which are indeed the predominant extracellular nanoparticles (ENPs) secreted by most types of cells.
Exosomes are nanosized extracellular membrane vesicles of endosomal origin secreted by most cell types, including cancer cells (Thery et al., “Exosomes: Composition, Biogenesis and Function,” Nature Reviews. Immunology 2:569-579 (2002); El Andaloussi et al., “Extracellular Vesicles: Biology and Emerging Therapeutic Opportunities,” Nature Reviews. Drug Discovery 12:347-357 (2013); Raposo et al., “Extracellular Vesicles: Exosomes, Microvesicles, and Friends,” The Journal of Cell Biology 200:373-383 (2013)). Proteins, genetic material (e.g., mRNAs, miRNAs, lnRNAs, DNA), metabolites and lipids, are selectively recruited and packaged into exosomes, which horizontally transfer their cargo to recipient cells, thereby acting as vehicles of intercellular communication in both physiological and pathological conditions (Balaj et al., “Tumour Microvesicles Contain Retrotransposon Elements and Amplified Oncogene Sequences,” Nature Communications 2:180 (2011); Choi et al., “Proteomics, Transcriptomics and Lipidomics of Exosomes and Ectosomes,” Proteomics 13:1554-1571 (2013); Thakur et al., “Double-Stranded DNA in Exosomes: a Novel Biomarker in Cancer Detection,” Cell Research 24:766-769 (2014); Tetta et al., “Extracellular Vesicles as an Emerging Mechanism of Cell-to-Cell Communication,” Endocrine 44:11-19 (2013)). Harnessing this knowledge, translational researchers have focused on developing exosome-based diagnostic/prognostic biomarkers and therapeutic strategies.
The present invention is directed to overcoming these and other deficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a method of diagnosing cancer in a subject. This method involves selecting a subject having cancer and obtaining, from the selected subject, a population of either exomeres having a diameter of less than 50 nm, small exosomes having a diameter of 60-80 nm, or large exosomes having a diameter of 90-120 nm. The exomeres, small exosomes, or large exosomes are recovered from the sample, and the exomeres, small exosomes, or large exosomes or portions thereof are contacted with one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more proteins contained in said exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more N-glycans contained in said exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more lipids contained in said exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in said exomeres, small exosomes, or large exosomes, or (5) combinations thereof. Cancer is then diagnosed based on the contacting step.
Another aspect of the present invention is directed to a method of prognosing cancer in a subject. This method involves selecting a subject having cancer and obtaining, from the selected subject, a population of either exomeres having a diameter of less than 50 nm, small exosomes having a diameter of 60-80 nm, or large exosomes having a diameter of 90-120 nm. The exomeres, small exosomes, or large exosomes are recovered from the sample, and the exomeres, small exosomes, or large exosomes or portions thereof are contacted with one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more proteins contained in said exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more N-glycans contained in said exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more lipids contained in said exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in said exomeres, small exosomes, or large exosomes, or (5) combinations thereof. Cancer is then prognosed based on the contacting step.
Another aspect of the present invention is directed to a method of managing treatment in a subject. This method involves selecting a subject undergoing treatment for cancer and obtaining, from the selected subject, a population of either exomeres having a diameter of less than 50 nm, small exosomes having a diameter of 60-80 nm, or large exosomes having a diameter of 90-120 nm. The exomeres, small exosomes, or large exosomes are recovered from the sample, and the exomeres, small exosomes, or large exosomes or portions thereof are contacted with one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more proteins contained in said exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more N-glycans contained in said exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more lipids contained in said exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in said exomeres, small exosomes, or large exosomes, or (5) combinations thereof. Treatment is then modified based on the contacting step.
Another aspect of the present invention relates to a kit suitable for diagnosing cancer. The kit includes one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard or to a sample from a subject, or the presence or absence, of one or more proteins contained in exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard or to a sample from a subject, or the presence or absence, of one or more N-glycans contained in exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard or to a sample rom a subject, or the presence or absence, of one or more lipids contained in exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in exomeres, small exosomes, or large exosomes, or (5) combinations thereof, wherein said exomeres have a diameter of less than 50 nm, said small exosomes have a diameter of 60 to 80 nm and said large exosomes have a diameter of 90 to 120 nm.
Through studies described herein, the influence of several key parameters of AF4 that were shown to be critical for high-resolution separation of distinct exosome subsets were evaluated. These influencing factors included cross-flow, channel height, sample focusing, type of membrane, and the amount of loaded sample. It should be noted that these factors collectively determine fractionation quality, and changing one parameter usually affects the influence of other factors on resolution power. Testing different combinations of these factors, however, can be expensive, time-consuming, and labor-intensive, and thus can be impractical. Understanding the working principles of AF4 and determining the complexity of the analyzed samples will be useful to guide the method development process.
The present invention describes identification of a novel type of nanoparticle secreted by most cell types, including cancer cells, which are termed exomeres. Exomeres are a prominent and heterogeneous population of small (<50 nm hydrodynamic diameter, with a peak about 35 nm), weakly negatively charged (−2.7 mV to −9.7 mV), and highly stiff (˜145-816 MPa) nanoparticles secreted by cells. Structural analysis revealed the lack of external lipid-bilayer membrane structure of exomeres, and molecular characterization showed its composition of a variety of biologically functional molecules, including proteins, lipids, nucleic acids (DNA and RNAs), metabolites, and glycans. Besides exomeres, two distinct subsets of exosomes, namely the small exosomes (Exo-S, 60-80 nm) and large exosomes (Exo-L, 90-120 nm) were separated, by employing the technique of asymmetric flow field-flow fractionation (AF4). In contrast to exomeres, both Exo-S and Exo-L have external lipid-bilayer membrane structures, carry more negative charges, and are softer than exomeres.
The studies described herein reveal that each nanoparticle type, i.e. exomeres, Exo-S and Exo-L, contain unique molecular signatures in comparison to each other. These nanoparticles are secreted into both the surrounding environment of cells and the peripheral circulation system and other types of body fluids. Therefore, these nanoparticles represent a reservoir of biomarkers for cancer diagnosis, prognosis and monitoring disease progression and recurrence post treatment.
Furthermore, both exomeres and exosome subsets can horizontally transfer their cargo to recipient cells, thereby acting as vehicles of intercellular communication in both physiological and pathological conditions, thus representing targets of therapeutics development. Specifically, exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins and specific pathways, such as glycolysis and mTOR signaling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signaling pathways, respectively. Biodistribution examination revealed that exomeres target organs such as liver, spleen, and bone marrow primarily, implicating their potential function in systemic regulation during tumor progression. In distinction to the observation of exomeres, Exo-S demonstrated higher uptake by the lung and Exo-L by lymph nodes, suggesting their potential roles in mediating organ-specific metastasis and immune response during disease progression, respectively.
Taken together, the newly identified exomeres, Exo-S and Exo-L present unique potential of serving as biomarkers and therapeutic targets for cancer patients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G show the identification, via AF4 and EM imaging analysis, of exomeres and two distinct subpopulations of exosomes released by tumor cells. FIG. 1A shows a representative AF4 fractionation profile of B16-F10-derived exosomes. x-axis, time (min); y-axis (scale) and black dots, hydrodynamic radius (nm); red and blue lines illustrate the QELS (DLS) intensity and UV absorbance (shown on a relative scale), respectively. P1-P5 marks the peaks detected based on UV absorbance. Fractions were pooled for exomeres (hydrodynamic diameter <50 nm); Exo-S (60-80 nm); and Exo-L (90-120 nm). FIG. 1B shows a representative correlation function at peak 3 (P3), t=25.1 min. For 1A and 1B the experiment was repeated independently 50 times with similar results. FIG. 1C shows TEM imaging analysis of exosome input mixture (pre-fractionation) and fractionated exomeres, Exo-S and Exo-L subpopulations. Arrows point to exomeres (red), Exo-S (blue) and Exo-L (green). Scale bar, 200 nm. This experiment was repeated 7 times independently with similar results. FIG. 1D shows Western blotting analysis of exosomal marker proteins in fractionated samples. 100 μg of whole cell extract (WCE) and 10 μg of exosome and exomere mixture input and each subset were analyzed. This experiment was done once. FIG. 1E shows measurement of hydrodynamic diameters of exomeres, Exo-S and Exo-L derived from representative cell lines (i.e. B16-F10 (F10), AsPC-1, Pan02, MDA-MB-4175 (4175) and 4T1) in the batch mode using Zetasizer after pooling fractions collected for each subset of nanoparticles from an individual AF4 fractionation, Data are presented as mean±SEM (standard error of the mean), in the order of exomere, Exo-S and Exo-L: B16-F10 (n=10, 9, and 8 independent measurements, respectively); Pan02 (n=11, 6, 11); AsPC-1 (n=5, 5, 5); 4175 (n=3, 5, 3); 4T1 (n=5, 5, 5)). FIG. 1F shows TEM imaging analysis of fractions collected from explant culture of fresh human melanoma tissue. Scale bar, 200 nm. This experiment was performed with two independent specimens with similar results. FIG. 1G shows a batch mode measurement of hydrodynamic diameters of fractions shown in FIG. 1F. Data are presented as mean±SEM (exomeres and Exo-L, n=6; Exo-S, n=7 independent measurements). Unprocessed blots are provided in FIG. 14.

FIGS. 2A-2K shows characterization of AF4 fractions using TEM imaging and NTA analyses and examination of AF4 profiles of nanoparticles derived from cells under different culture and storage conditions. FIG. 2A shows TEM analysis of particles in AF4 peaks P1 and P5 of B16-F10. The experiment was repeated independently 3 times with similar result. Scale bar, 500 nm. FIG. 2B shows a comparison of the hydrodynamic diameter of each fraction determined by AF4-QELS versus NTA. Individual fractions (time slice, 0.5 min/fraction) were taken every 2 minutes from 20 to 44 minutes during the AF4 time course, and subjected to NTA. Results shown are mean±SEM (n=3 independent samples). Mode size from NTA was utilized. X-axis, time course of AF4 (min); Y-axis, hydrodynamic diameter (nm). FIG. 2C shows the size distribution profiles of representative fractions by NTA (input, unfractionated samples; fractions at 20, 32, and 44 minutes). Multiple peaks were detected for fractions at 20 and 44 minutes by NTA. A mode size of 126 nm of input indicates that NTA cannot efficiently resolve polydisperse samples and is biased towards large particles. This experiment was repeated 3 times independently with similar results. FIG. 2D shows the particle concentration of each fraction measured by NTA. The hydrodynamic diameter of the peak fraction (28 minutes) was 77 nm. Results shown are mean±SEM (n=3 independent samples). FIGS. 2E-2I show AF4 profiles of B16-F10 sEVs collected from technical (blue lines, replicate #1; red lines, replicate #2) (FIG. 2E) and biological replicates (red lines, QELS; blue lines, UV; black (replicate #1) and green dots (replicate #2), hydrodynamic radius; Differences in UV and QELS signal intensity is due to the different amount of input samples for two replicates)(FIG. 2F), kept at either 4° C. or −80° C. for one week (red lines, QELS; blue lines, UV; black (fresh) and green dots (frozen), hydrodynamic radius) (FIG. 2G), cells of different passage numbers (blue and red lines, UV of cells at passage 10 and 18, respectively; black dots, hydrodynamic radius) (FIG. 2H), and under hypoxic versus normoxic conditions for 48 h (blue and red lines, UV for samples cultured with 20% and 1% O₂, respectively; black dots, hydrodynamic radius) (FIG. 2I). Experiments were repeated independently 3 times for FIGS. 2E-2G and twice for FIG. 2H with similar results. For FIG. 2I, the experiment was repeated with 3 different cell lines independently with similar results. FIGS. 2J and 2 k show AF4 (FIG. 2J) and TEM (FIG. 2K) analysis of nanoparticles isolated in parallel from the blank media control and CM of 3-day cultures of B16-F10 and MDA-MB-231-4175. This experiment was done once. (Red and Blue lines, UV; black dots, hydrodynamic radius; Scale bar, 200 nm.)

FIGS. 3A-3B show identification of exomeres and exosome subpopulations released by multiple cancer cell lines. Shown are AF4 profiles (FIG. 3A) and representative TEM images (FIG. 3B) of unfractionated input samples and pooled fractions of exomeres, Exo-S and Exo-L that derived from various cancer cell lines, including AsPC-1. Pan02, MDA-MB-231-4175, and 4T1. Multiple independent experiments were conducted with similar results for (FIG. 3A) (repeated times: AsPC-1, 9×; Pan02, 16×; 4175, 17×; 4T1, 10×) and (FIG. 3B) (AsPC-1, 3×; Pan02, 2×; 4175, 1×; 4T1, 4×). Scale bar, 200 nm. x-axis, time (minutes); y-axis (scale) and black dots, hydrodynamic radius (nm); Red and blue lines illustrate the QELS (DLS) intensity and UV absorbance (shown on a relative scale), respectively.

FIGS. 4A-4B show detection of exomeres, Exo-S and Exo-L in samples isolated from the tissue explant cultures. FIG. 4A is an AF4 profile of exosomes isolated from explant culture of fresh human melanoma tissues. Red and blue lines illustrate the QELS (DLS) intensity and UV absorbance, respectively. This experiment was repeated with 4 independent specimens with similar results. FIG. 4B shows TEM images of exosome samples isolated from the explant culture of normal mouse mammary fat pad and lung tissues. This experiment was repeated independently 2 times with similar results. Scale bar, 500 nm.

FIGS. 5A-5D show characterization of physical and mechanical properties of exomeres and exosome subpopulations. Zeta potential (FIG. 5A) and stiffness (FIG. 5B) of exomeres and exosome subpopulations derived from various cancer cells were measured using Zetasizer and AFM indentation, respectively. Young's modulus was used to express particle stiffness. At least 3 and 5 replicates for each group of particles was measured for zeta potential and stiffness, respectively. Data are presented as mean±SEM. For FIG. 5A, in the order of exomere, Exo-S and Exo-L: B16-F10 (n=8, 10, and 12 independent measurements, respectively); Pan02 (n=13, 11, 13); AsPC-1 (n=12, 12, 12); 4175 (n=17, 9, 6); 4T1 (n=13, 3, 9); for FIG. 5B, B16-F10 (n=6, 6, 6 particles measured); Pan02 (n=6, 6, 6); AsPC-1 (n=21, 19, 16); 4175 (n=11, 10, 5); 4T1 (n=9, 8, 9)). FIG. 5C shows a representative AFM image of exomeres derived from B16F10. This experiment was repeated with samples derived from 3 different cell lines with similar results. FIG. 5D shows AFM imaging analysis of the height (z-dimension) of exomeres derived from B16F10 (n=754 particles analyzed), AsPC1 (n=475) and MDA-MB-4175 (n=160). Mean±SEM is depicted.

FIGS. 6A-6G show proteomic profiling of exomeres and exosome subpopulations derived from various cancer cells. FIG. 6A shows a Venn diagram of proteins identified in each subset of particles. FIG. 6B shows principal component analysis and FIG. 6C shows consensus clustering analysis of normalized proteomic mass spectrometry datasets from human (MDA-MB-4175 and AsPC1) and mouse (B16F10, 4T1, and Pan02) cell lines. FIG. 6D shows a heat map illustration of unique proteins specifically associated with exomeres, Exo-S and Exo-L. Scale shown is intensity (area) subtracted by mean and divided by row standard deviation (i.e. A (area-mean)/SD). FIG. 6E shows Western blot analysis of representative signature proteins in fractionated samples. An equal amount (10 μg) of exosome and exomere input mixture and each subset were analyzed. This experiment was done once. FIG. 6F shows a heat map illustration of the relative abundance of conventional exosome markers in exomeres, Exo-S and Exo-L. Scale shown is intensity (area) subtracted by mean and divided by row standard deviation (i.e. Δ (area-mean)/SD). FIG. 6G shows identification of top candidate gene sets enriched in exomere, Exo-S and Exo-L populations by gene set enrichment analysis (GSEA). Proteins in each subset of nanoparticles are ranked by GSEA based on their differential expression level. Whether a pre-specified pathway is significantly overrepresented toward the top or bottom of the ranked gene list in each subset of nanoparticle is evaluated using the normalized enrichment score (the green line). Black vertical lines mark the positions where the members of a particular pathway appear in the ranked list of genes. Proteins that contributed most to the enrichment score are listed below the plot. For all proteomic analysis (FIGS. 6B-6D, FIGS. 6F-6G), a total of 30 samples (3 nanoparticle subtypes derived from 5 different cell lines; and two independent biological replicates for each nanoparticle sample) were subjected to statistical analysis. Unprocessed blots are provided in FIG. 14.

FIGS. 7A-7E show proteomic profiling of exomeres and exosome subpopulations derived from multiple cancer cell lines, FIG. 7A shows principal component analysis of normalized proteomic mass spectrometry data of exomeres, Exo-S and Exo-L derived from multiple cell lines, including MDA-MB-231-4175, AsPC-1, 4T1, B16F-10 and Pan02. Two independent biological replicates were analyzed for each nanoparticle sample. FIG. 7B is a heat map illustration of the relative abundance of the Rab family proteins in exomeres, Exo-S and Exo-L. Scale shown is intensity (area) subtracted by mean and divided by row standard deviation (i.e., Δ (area−mean)/SD). FIG. 7C shows evaluation of the presence of lipoprotein-particle associated proteins (listed in Table 2) among the total proteins detected in the exomere, Exo-S and Exo-L derived from different cell lines. Results shown are mean of 2 biologically independent experiments. FIG. 7D shows TEM imaging analysis of HDL, LDL and VLDL. Scale bar, 200 nm. This experiment was done once with multiple images showing similar results. FIG. 7E shows identification of specific association of signaling pathways including hypoxia (FDR, q value=0.004), microtubule (FDR, q value=0.002) and coagulation (FDR, q value=0.013) with exomeres by GSEA (left panel) and the heat map illustration of the expression level of related proteins in different subsets of nanoparticles (right panel). A total of n=30 samples (3 nanoparticle subtypes derived from 5 different cell lines; and two independent biological replicates for each nanoparticle samples) were subjected for Kolmogorov-Smimov statistical analysis.

FIGS. 8A-8C show characterization of N-glycosylation of proteins associated with exomere, Exo-S and Exo-L. FIG. 8A shows lectin blotting analysis of N-glycan profile of proteins associated with exomeres versus exosome subpopulations Exo-S and Exo-L. Phaseolus vulgaris erythroagglutinin (E-PHNA) and Phaseolus vulgaris leucoagglutinin (L-PHA) recognize bisected and branched N-glycans, respectively. Aleuria aurantia lectin (AAL) recognizes Fucα6GlcNAc and Fucα3GlcNAc. Sambucus nigra lectin (SNA) recognizes α-2,6-linked sialic acid. All experiments were repeated independently twice with similar results except for AAL and E-PHA blotting for B16-F10 and 4175 which were done once. FIG. 8B shows mass spectrometric analysis of N-glycans of glycoproteins present in exomeres, Exo-S and Exo-L subsets of B16F10. One representative experiment of two biologically independent replicates is shown. FIG. 8C shows a comparison of the relative abundance of the top six most abundant N-glycan structures among exomere. Exo-S and Exo-L of B16F10. The assignments (m/z) [charge; neutral exchange] for MALDI-MS and nanoLC-ESI-MS/MS are the following: (2015.8 [−H; 0]; 1007.4^a[−2H; 0]), (2209.8 [−H; 0]; 1104.4^a[−2H; 0]), (2237.7^b[−H; Na−H]; 732.57^a[−3H; 0]), (2365.5^b[−H;4K−4H]; 783.9^a[−3H; 0] and 1182.4^a,b[−2H; 4K−4H]), and (2404.8^b[−H; 2K−2H]; 12010.9^b[−2H; 2K−2H]). Data shown were quantified and normalized to the most abundant structure in the sample. Results are represented as average of three independent analytical measurements of one representative experiment. Unprocessed blots are provided in FIG. 14. Note: ^aThe product ion spectra for this species did not allow a complete structural assignment. ^bAssignments admit neutral exchanges of protons with cations in sialoglycans, including the presence of potassium and sodium.

FIGS. 9A-9J show mass spectrometric analysis of N-glycans enriched in exomeres, Exo-S and Exo-L derived from AsPC-1 and MDA-MB-231-4175. FIG. 9A shows the total protein profile content of the isolated exomere, Exo-S and Exo-L subpopulations derived from AsPC-1, MDA-MB-231-4175 and B16-F10 assessed by silver staining. This experiment was repeated independently twice for B16-F10 and 4175 with similar results and done once for AsPC-1. FIG. 9B shows the N-glycan mass spectra of particles derived from AsPC-1 (left panel) and MDA-MB-231-4175 (right panel), respectively. This experiment was done once with 3 analytic replicates with similar results. FIG. 9C and FIG. 9D shows quantification of the top six most abundant N-glycan structures identified in the study of AsPC-1 and MDA-MB-231-4175 derived particles. Data shown were quantified and normalized to the most abundant structure in the sample. Results are represented as mean of 2 and 3 independent analytical measurements for AsPC-1 for MDA-MB-231-4175, respectively. NanoHPLC-PGC-HRMS extracted ion chromatograms (EIC) and CID-MS/MS spectra for FIGS. 9E-9G the ion at m/z 1007.38(2-), corresponding to a core-fucosylated complex type N-glycan, characteristic of exomere, and FIGS. 9H-9J the ion at m/z 1111.39(2-), corresponding to a disialylated complex-type N-glycan found in all fractions of B16F10. Fragmentation analysis for extracted ion chromatogram m/z 1007.38 (2-) confirming the structure of this N-glycan in exomeres (FIG. 9E and FIG. 9G) and demonstrating the absence of this N-glycan in Exo-S (FIG. 9F). According to the relative retention time on the PGC column, exomeres contain both α2,3-linked and α2,6-linked sialoglycoforms of the ions at m/z 1111.39(2-) (FIG. 9H). The N-glycan m/z 1111.39(2-) from Exo-S showed N-glycans displaying exclusively α2,3-linked sialic acids based on PGC-LC relative retention time (FIG. 9I). This experiment (FIGS. 9E-9J) was done once.

FIGS. 10A-10C show characterization of lipid composition in exomeres and exosome subsets. FIG. 10A shows a comparison of total lipid content of each subset of nanoparticles derived from different cell lines. Total signal intensity of each sample after normalization to sample weight and internal standards was compared to that of exomeres from the same set of samples (expressed as fold change). Data are presented as mean±SEM (n=3 biologically independent samples). FIG. 10B shows the relative abundance of each lipid class present in each subset of nanoparticles from different cell lines. Data are presented as mean±SEM (n=3 biologically independent samples). FIG. 10C shows a heat map illustration of lipid classes specifically associated with exomeres, Exo-S and Exo-L (ANOVA test, q<0.05). Statistical analysis was performed on a total of 9 samples for each cell line (3 different nanoparticle subtypes and 3 independent biological repeats for each nanoparticle sample). Abbreviation: Cer, ceramide; CerG1-3, glucosylceramides; CL, cardiolipin; DG, diglyceride; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPG, lysophosphatidylglycerol; LPI, lysophosphatidylinositol; MG, monoglyceride; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; TG, triglyceride.

FIGS. 11A-11D show characterization of nucleic acid association with exomere and exosome subsets. FIG. 11A shows the relative abundance of DNA associated with each subpopulation of particles from representative fractionations of B16F10, AsPC1 and MDA-MB-4175. FIG. 11B shows Agilent Bioanalyzer analysis of the size distribution of DNA associated with different subsets of particles. Data shown are the electropherograms (left) and electrophoresis images (right) from a representative of two independent experiments on AsPC1-derived particles. Black arrows, internal standards (35 bp and 10380 bp). Red line, exomeres; blue line, Exo-S; green line, Exo-L. FIG. 11C shows the relative abundance of total RNA associated with each subpopulation of particles from representative fractionations of B16F10 and AsPC1. FIG. 11D shows the size distribution of RNA isolated from different fractions of B16F10. Shown are representative profiles from one of two independent experiments. For FIG. 11A and FIG. 11C, data shown are mean (n=2 biologically independent samples).

FIG. 12 shows bioanalyzer analysis of the size distribution of DNA associated with exomere, Exo-S and Exo-L derived from B16-F10 (top) and MDA-MB-231-4175 (bottom). This experiment was repeated twice independently with similar results.

FIGS. 13A-13B show organ biodistribution of B16F10-derived exomeres and exosome subpopulations in syngeneic naïve mice. FIG. 13A shows whole organ imaging of NIR dye-labeled exomeres, Exo-S and Exo-L from a representative experiment using the Odyssey imaging system (LI-COR Biosciences; n=4 independent experiments). The dynamic range of signal intensity was adjusted for each organ so that the differences among these nanoparticle subsets can be easily recognized. Scale bar, 2.5 mm. FIG. 13B shows quantification of the nanoparticle uptake in different organs in one representative experiment. This experiment was repeated independently 4 times with similar results. Signal intensity in each organ was acquired using the Image Studio (LI-COR Biosciences), and normalized to the brain from the same animal due to undetectable uptake of nanoparticles in this organ. Fold changes (y axis) were then calculated for each organ between the experimental group (i.e. input, exomere, Exo-S and Exo-L) versus the mock control. n=3 animals per group, results shown are mean±SEM. Statistical significance determined using one way ANOVA (* p<0.05; ** p<0.01, unmarked, not significant). For lymph nodes, the p value for comparison between input versus Exo-L, exomere versus Exo-L and Exo-S versus Exo-L are 0.022, 0.001 and 0.01 respectively.

FIG. 14 shows unprocessed blots for related figures in FIG. 1, FIG. 6 and FIG. 8.

FIGS. 15A-15C show a schematic illustration of the AF4 working principle. FIGS. 15A-15C show the side views of the AF4 channel, whose height is usually several hundreds of μm. The part size shown in the figure is for illustration only and not drawn to scale. FIG. 15A shows that in the Focus stage, two flows in opposing directions are pumped into the channel from the inlet and outlet ports and balanced near the injection port. Samples are injected during the Focus stage and focused in a thin band. Particles reach level heights related to their diffusion coefficients. FIG. 15B shows that in the Elution stage of the normal mode, particles with small hydrodynamic size and high diffusion coefficient are eluted at an early time point, whereas particles with large hydrodynamic size and low diffusion coefficient elute late. FIG. 15C shows that when the physical size of a particle is too large to be considered as a point mass compared to the channel height, it elutes in the Steric mode. In contrast to the Normal model as shown in FIG. 15B, large particles elute earlier than the smaller ones.

FIGS. 16A-16C show the influence of cross-flow on AF4 fractionation. FIG. 16A shows a representative AF4 fractionation profile of B16-F10 sEVs collected by applying a linear cross-flow gradient with an initial flow rate at 0.5 mL/min within 45 minutes (Peaks are marked as P0-P5; UV (red line), QELS (blue line), R_h(black dots)), or (FIG. 16B) with an initial flow rate at 0.3 mL/min (blue line), 0.5 mL/min (red line) or 1.0 mL/min (black line) and dropping to 0 mL/min over 45 minutes, or (FIG. 16C) with an initial flow rate at 0.5 mL/min and dropping to 0 mL/min over 15 minutes (blue), 30 minutes (black) or 45 minutes (red). Top, QELS at 100°; bottom, UV absorbance at 280 nm. The other AF4 parameters are: channel flow rate, 1.0 mL/min; channel height, 490 μm; sample focus time, 2 minutes; membrane, regenerated cellulose (RC), input amount, 40 μg.

FIG. 17 shows the effect of the channel height upon AF4 fractionation. Shown are AF4 fractionation profiles of B16-F10 sEVs collected using a channel with a spacer of 350 μm (blue) and 490 μm (red). Top, QELS at 100°; bottom, UV absorbance at 280 nm. The other AF4 parameters are: channel flow rate, 1.0 mL/min; a linear gradient of cross-flow decreasing from 0.5 mL/min to 0 mL/min over 45 minutes; sample focus time, 2 minutes; membrane, regenerated cellulose (RC), input amount, 40 μg.

FIG. 18 shows the effect of the focus time upon AF4 fractionation. Shown are AF4 fractionation profiles of B16-F10 sEVs collected using a sample focus time of 2 minutes (red), 5 minutes (blue) or 10 minutes (black). Top, QELS at 100°; bottom, UV absorbance at 280 nm. The other AF4 parameters are: channel flow rate, 1.0 mL/min; a linear gradient of cross-flow decreasing from 0.5 mL/min to 0 mL/min over 45 minutes; channel height, 490 μm; membrane, regenerated cellulose (RC), input amount, 40 μg.

FIG. 19 shows examination of the sample (B16-F10 sEVs) loading capacity for AF4 analysis. Shown are AF4 fractionation profiles of B16-F10 sEVs with an input of 15 μg (black), 40 μg (red); 100 μg (blue), or 150 μg (green). Top, QELS at 100°; bottom, UV absorbance at 280 nm. The other AF4 parameters are: channel flow rate, 1.0 mL/min; a linear gradient of cross-flow decreasing from 0.5 mL/min to 0 mL/min over 45 minutes; channel height, 490 μm; sample focus time, 2 minutes; membrane, regenerated cellulose (RC).

FIG. 20 shows a comparison of the AF4 performance for separating EVs using different membranes: regenerated cellulose (RC, red) versus poly(ether)sulfone (PES, blue). B16-F10 sEVs were analyzed using the following AF4 parameters: channel flow rate, 1.0 mL/min; a linear gradient of cross-flow decreasing from 0.5 mL/min to 0 mL/min over 45 minutes; channel height, 490 μm; sample focus time, 2 minutes; input amount, 40 μg. Top, QELS at 100°; bottom, UV absorbance at 280 nm.

FIGS. 21A-21B show a schematic illustration of the overall procedure and the flow route of AF4. FIG. 21A shows the overview of experimental design for cell culture-derived sEV isolation and AF4 fractionation. FIG. 21B is an illustration of the AF4 flow route and arrangement of online detectors.

FIGS. 22A-22C shows representative AF4 fractionation analysis of B16-F10 sEVs. Shown are representative AF4 fractionation profile of B16-F10 sEVs (FIG. 22A) and autocorrelation functions at specific time points (FIG. 22B). FIG. 22C shows TEM imaging analysis of combined fractions for peaks P2 (exomere), P3 (Exo-S), and P4 (Exo-L). Scale bar, 200 nm. Colored arrows point to representative particles in each subpopulation.

FIGS. 23A-23B show gene expression analysis of the livers from mice 24 hours post injection of B16-F10 derived exomeres, Exo-S and Exo-L, in comparison with PBS control. 10 μg of exomeres, Exo-S. and Exo-L, and equal volume of PBS were intravenously injected into C57Bl/6 mice, respectively. The livers of the mice were collected for RNA extraction and sequencing analysis 24 hours post injection. The total numbers of genes that are differently expressed in each comparison group are listed in FIG. 23A, and Clustering analysis of top 2000 genes that are significantly changed in comparison with the PBS control are shown in FIG. 23B. n=3 per group.

FIGS. 24A-24B show a heatmap illustration of the top 50 upregulated genes (FIG. 24A) and top 50 downregulated genes (FIG. 24B) in the livers of mice treated with exomere, Exo-S or Exo-L, compared with the PBS control group. n=3 per group.

FIGS. 25A-25E show Ingenuity Pathway Analysis (IPA) of differently expressed genes in the livers of mice 24 hours post injection of B16-F10 derived exomeres. Exo-S and Exo-L, in comparison with PBS control. Shown are representative top pathways that are significantly affected between Exomere and PBS (FIG. 25A), Exo-S and PBS (FIG. 25B), Exo-L and PBS (FIG. 25C), Exomere and Exo-S (FIG. 25D), and Exomere and Exo-L (FIG. 25E).

FIGS. 26A-26C show metabolic mass spectrometry analysis of the livers from mice treated with B16-F10-derived exomeres, Exo-S and Exo-L, compared with PBS control. Metabolites whose abundance were significantly changed are identified using unpaired t test (FIG. 26A) and one-way ANOVA analysis (FIGS. 26B-26C) (metabolites differently detected in each group via one-way ANOVA are shown as specific individual data points in (FIG. 26B), or as clusters highlighted in boxes in (FIG. 26C)). n=3 mice per group.

FIGS. 27A-27B shows that metabolites that are upregulated or downregulated in all three groups of exomeres, Exo-S and Exo-L-treated mouse livers in comparison with the PBS control were identified via one-way ANOVA analysis, and the changes in their abundance are displayed in FIG. 27A and FIG. 27B, respectively. n=3 mice per group.

FIGS. 28A-28C show a demonstration of representative metabolites that are specifically upregulated in exomeres (FIG. 28A), Exo-S (FIG. 28B), and Exo-L (FIG. 28C), respectively, compared with the PBS control group. n=3 mice per group.

FIG. 29 shows an immunofluorescence colocalization study that revealed that kupffer cells, the liver resident macrophages, are the primary cell type that uptakes B16-F10 melanoma-derived exomeres. Exomeres that are labeled with green fluorescent lipophilic PKH67 dye or the mock labeling reaction mixture were injected intravenously into naïve, syngeneic C57BL/6 mice and 24 hours post injection, the livers were harvest and fixed for immunofluorescence colocalization analysis. n=3 mice per group. F4:80 was stained (in red) to identify the macrophages in the liver.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is directed to a method of diagnosing cancer in a subject. This method involves selecting a subject having cancer and obtaining, from the selected subject, a population of either exomeres having a diameter of less than 50 nm, small exosomes having a diameter of 60-80 nm, or large exosomes having a diameter of 90-120 nm. The exomeres, small exosomes, or large exosomes are recovered from the sample, and the exomeres, small exosomes, or large exosomes or portions thereof are contacted with one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more proteins contained in said exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more N-glycans contained in said exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more lipids contained in said exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in said exomeres, small exosomes, or large exosomes, or (5) combinations thereof. Cancer is then diagnosed based on the contacting step.
Another aspect of the present invention is directed to a method of prognosing cancer in a subject. This method involves selecting a subject having cancer and obtaining, from the selected subject, a population of either exomeres having a diameter of less than 50 nm, small exosomes having a diameter of 60-80 nm, or large exosomes having a diameter of 90-120 nm. The exomeres, small exosomes, or large exosomes are recovered from the sample, and the exomeres, small exosomes, or large exosomes or portions thereof are contacted with one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more proteins contained in said exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more N-glycans contained in said exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more lipids contained in said exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in said exomeres, small exosomes, or large exosomes, or (5) combinations thereof. Cancer is then prognosed based on the contacting step.
Another aspect of the present invention is directed to a method of managing treatment in a subject. This method involves selecting a subject undergoing treatment for cancer and obtaining, from the selected subject, a population of either exomeres having a diameter of less than 50 nm, small exosomes having a diameter of 60-80 nm, or large exosomes having a diameter of 90-120 nm. The exomeres, small exosomes, or large exosomes are recovered from the sample, and the exomeres, small exosomes, or large exosomes or portions thereof are contacted with one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more proteins contained in said exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more N-glycans contained in said exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more lipids contained in said exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in said exomeres, small exosomes, or large exosomes, or (5) combinations thereof. Treatment is then modified based on the contacting step.
Cancer prognosis as described herein includes determining the probable progression and course of the cancerous condition, and determining the chances of recovery and survival of a subject with the cancer, e.g., a favorable prognosis indicates an increased probability of recovery and/or survival for the cancer patient, while an unfavorable prognosis indicates a decreased probability of recovery and/or survival for the cancer patient. A subject's prognosis can be determined or modified by the availability of a suitable treatment (i.e., a treatment that will increase the probability of recovery and survival of the subject with cancer). Accordingly, another aspect of the present invention includes selecting a suitable cancer therapeutic based on the determined prognosis and administering the selected therapeutic to the subject.
Prognosis also encompasses the metastatic potential of a cancer. For example, a favorable prognosis based on the presence or absence of a protein, N-glycan, lipid, and/or genetic phenotype can indicate that the cancer is a type of cancer having low metastatic potential, and the patient has an increased probability of long term recovery and/or survival. Alternatively, an unfavorable prognosis, based on the presence or absence of a protein, N-glycan, lipid, and/or genetic phenotype can indicate that the cancer is a type of cancer having a high metastatic potential, and the patient has a decreased probability of long term recovery and/or survival.
Prognosis further encompasses prediction of sites of metastasis, determination of the stage of the cancer, or identifying the location of a primary tumor in a subject.
A change in the levels of certain proteins, N-glycans, lipids, and/or the mutational status of genes associated with cancer (e.g., BRAF and/or EGFR) indicates that a cancer is present or a change in the cancer phenotype has occurred with disease progression. For example, detecting the presence or a genetic mutation in an exomere, small exosomal, or large exosomal dsDNA sample from a subject whereas no genetic mutation was detected in an earlier exomere, small exosomal, or large exosomal dsDNA sample obtained from the same subject, can be indicative of a particular site of metastasis or progression to a more advanced stage of the cancer. Therefore, periodic monitoring of exomere, small exosomal, or large exosomal dsDNA mutational status provides a means for detecting primary tumor progression, metastasis, and facilitating optimal targeted or personalized treatment of the cancerous condition.
The detection of certain proteins, N-glycans, lipids, and/or exomere, small exosomal, or large exosomal dsDNA mutations in a metastatic cancer sample can also identify the location of a primary tumor. For example, the detection of one or more BRAF mutations in a metastatic tumor or cancer cell-derived exomere, small exosomal, or large exosomal sample can indicate that the primary tumor or cancer was melanoma or a form of brain cancer, e.g. glioblastoma. The detection of one or mom EGFR mutations in a metastatic tumor or cancer cell derived exomere, small exosomal, or large exosomal dsDNA sample indicates that the primary tumor originated in the lung, or alternatively the primary cancer was head and neck cancer, ovarian cancer, cervical cancer, bladder cancer, or esophageal cancer.
As described above, another aspect of the present invention is directed to a method of managing treatment of a subject having cancer. In accordance with this aspect, cancer treatment is modified based on the contacting step.
In accordance with all aspects of the present invention, a “subject” or “patient” encompasses any animal, but preferably a mammal, e.g., human, non-human primate, a dog, a cat, a horse, a cow, or a rodent. More preferably, the subject or patient is a human. In some embodiments of the present invention, the subject has cancer, for example and without limitation, melanoma, breast cancer, or pancreatic cancer. In some embodiments, the cancer is a primary tumor, while in other embodiments, the cancer is a secondary or metastatic tumor.
“Exosomes” are microvesicles released from a variety of different cells, including cancer cells (i.e., “cancer-derived exosomes”). These small vesicles (50-100 nm in diameter) derive from large multivesicular endosomes and are secreted into the extracellular milieu. The precise mechanisms of exosome release/shedding remain unclear; however, this release is an energy-requiring phenomenon, modulated by extracellular signals. They appear to form by invagination and budding from the limiting membrane of late endosomes, resulting in vesicles that contain cytosol and that expose the extracellular domain of membrane-bound cellular proteins on their surface. Using electron microscopy, studies have shown fusion profiles of multivesicular endosomes with the plasma membrane, leading to the secretion of the internal vesicles into the extracellular environment. The rate of exosome release is significantly increased in most neoplastic cells and occurs continuously. Increased release of exosomes and their accumulation appear to be important in the malignant transformation process.
As described herein, two exosome subpopulations (i.e., Exo-S and Exo-L) have been identified. “Exo-S”, as used herein, refers to a population of small exosomes having a diameter of 60 to 80 nm, an average surface charge of −9.0 mV to −12.3 mV, and a particle stiffness of 70 to 420 mPa. Exo-S are also enriched in genes involved in membrane vesicle biogenesis and transport, protein secretion and receptor signaling “Exo-L”, as used herein, refers to a population of large exosomes having a diameter of 90 to 120 nm, an average surface charge of −12.3 to −16.0 mV, and a particle stiffness of 26 to 73 mPa. Exo-L are also enriched in genes involved in the mitotic spindle, IL-2/Stat5 signaling, multi-organism organelleorganization, and G-protein signaling.
In addition to Exo-S and Exo-L subpopulations, a novel extracellular nanoparticle has also been identified. As used herein, the term “exomere” refers to a non-membranous nanoparticle having a diameter of less than 50 nm, often approximately 35 nm, an average surface charge of −2.7 mV to −9.7 mV, and a particle stiffness of 145 to 816 mPa. Exomeres are enriched in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as proteins involved in glycolysis and mTOR signaling
In accordance with the methods of the present invention, exomeres, small exosomes, and large exosomes can be isolated or obtained from most biological fluids including, without limitation, blood, serum, plasma, ascites, cyst fluid, pleural fluid, peritoneal fluid, cerebrospinal fluid, tears, urine, saliva, sputum, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary trances, breast milk, intra-organ system fluid, conditioned media from tissue explant culture, or combinations thereof.
A population of either exomeres, small exosomes or large exosomes can be obtained from a biological sample using methods described herein. For example, exomeres, small exosomes, or large exosomes may be concentrated or isolated from a biological sample asymmetric flow field-flow fractionation (AF4) (Fraunhofer et al., “The Use of Asymmetrical Flow Field-Flow Fractionation in Pharmaceutics and Biopharmaceutics,” European Journal of Pharmaceutics and Biopharmaceutics 58:369-383 (2004); Yohannes et al., “Asymmetrical Flow Field-Flow Fractionation Technique for Separation and Characterization of Biopolymers and Bioparticles,” Journal of Chromotography. A 1218:4104-4116 (2011)).
Exomeres, small exosomes, or large exosomes isolated from a bodily fluid (i.e., peripheral blood, cerebrospinal fluid, urine) can be enriched for those originating from a specific cell type, for example, lung, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colorectal, breast, prostate, brain, esophagus, liver, placenta, and fetal cells. Because the exomeres, small exosomes, or large exosomes often carry surface molecules such as antigens from their donor cells, surface molecules may be used to identify, isolate or enrich for exomeres, small exosomes, or large exosomes from a specific donor cell type. In this way, exomeres, small exosomes, or large exosomes originating from distinct cell populations can be analyzed for their nucleic acid content, For example, tumor (malignant and non-malignant) exosomes carry tumor-associated surface antigens and these exosomes can be isolated or enriched via these specific tumor-associated surface antigens. In one example, the tumor-associated surface antigen is epithelial-cell-adhesion-molecule (EpCAM), which is specific to exosomes from carcinomas of lung, colorectal, breast, prostate, head and neck, and hepatic origin, but not of hematological cell origin (Balzar et al., “The Biology of the 17-1A Antigen (Ep-CAM),” J Mol Med 77(10): 699-712 (1999); Went et al. “Frequent EpCam Protein Expression in Human Carcinomas,” Hum Pathol 35(1): 122-8 (2004), which are hereby incorporated by reference in their entirety). In another example, the surface antigen is CD24, which is a glycoprotein specific to urine microvesicles (Keller et al. “CD24 is a Marker of Exosomes Secreted into Urine and Amniotic Fluid,” Kidney Int 72(9): 1095-102 (2007), which is hereby incorporated by reference in its entirety). In yet another example, the surface antigen is CD70, carcinoembryonic antigen (CEA), EGFR, EGFRvIII and other variants, Fas ligand, TRAIL, transferrin receptor, p38.5, p97 and HSP72. Alternatively, tumor specific exosomes may be characterized by the lack of surface markers, such as the lack of CD80 and CD86 expression.
The isolation of exomeres, small exosomes, or large exosomes from specific cell types can be accomplished, for example, by using antibodies, aptamers, aptamer analogs, or molecularly imprinted polymers specific for a desired surface antigen. In one embodiment, the surface antigen is specific for a cancer type. In another embodiment, the surface antigen is specific for a cell type which is not necessarily cancerous. One example of a method of exosome separation based on cell surface antigen is provided in U.S. Pat. No. 7,198,923, which is hereby incorporated by reference in its entirety. As described in, e.g., U.S. Pat. No. 5,840,867 to Toole and U.S. Pat. No. 5,582,981 to Toole, which are hereby incorporated by reference in their entirety, aptamers and their analogs specifically bind surface molecules and can be used as a separation tool for retrieving cell type-specific exosomes. Molecularly imprinted polymers also specifically recognize surface molecules as described in, e.g., U.S. Pat. Nos. 6,525,154, 7,332,553 and 7,384,589, which are hereby incorporated by reference in their entirety, and are a tool for retrieving and isolating cell type-specific exosomes. These methods can be adapted for use in isolating exomeres, small exosomes, or large exosomes.
In accordance with this aspect and other aspects of the invention, the recovered exomeres, small exosomes, or large exosomes are then contacted with one or more reagents suitable to detect higher or lower levels, relative to a standard for subjects not having cancer or to a prior sample from a subject having cancer, or the presence or absence of one or more proteins in the exomere, small exosome, or large exosome sample.
For purposes of prognosing or managing treatment of cancer, a subject is selected that has or is undergoing treatment for cancer.
In one embodiment, exomeres are recovered from the sample and the method is carried out by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of PP1D, GANAB, MAT1A, CPYD, FAT4, GMPPB, ERP44, CALR, GPD1, BZW1, PFKL, OLFML3, HGD, LGALS3BP, GCLC, PEPD, MTH4FD1, PGD, ACTR3, XPNPEP1, UGP2, SNX2, ALDOC, SEPT11, HSPA13, AARS, SERPINH1, CNDP2, PDE5A, AGL, EXT1, IDH1, SERPINC1, RRM1, CKB, HMGCS1, HPD, PSMC4, NPEPPS, CAT, EXT2, CORO1C, B4GAT1, RACK1, MAPRE1, PGM1, PD1A3, ADK, SHMT1, ACO1, GSN, ESD, PPP2R1A, ALDH1L1, OLA1, ACLY, EEF1G, FLNB, PSMD11, ANGPTL3, FERMT3, PYGL, MDH1, and EIFA2.
In another embodiment, small exosomes are recovered from the sample and the method is carried out to by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of TTYH3, FLOT1, FLOT2, TSPAN14, LAMC1, CD63. MVB12A, ZDHHC20, VAMP3, VPS37B, ARRDC1, and TGFBR2.
In a further embodiment, large exosomes are recovered from the sample and the method is carried out by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of SQSTM1, STIP1, HINT1, WASF2, RASA3, EPB41L2, GIPC1, S100A10, MPP6, KIF23, RACGAP1, ANXA5, CASK, DLG1, TJP1, BAG5, TXN, ABI1, ANXA1, CAPG, DB1, S100A6, CHMP2B, CHMP3, ANXA2, MYO1C, ANXA4, SNX12, LIN7C, STXBP3, CEP55, ALCAM, VCL, CHMP1A, FARP1, ACSL4, BA1AP2, SH3GL1, DSTN, LGALS1, CYF1P1, CTNNA1, RAB31, ARF6, SLC1A5, EPS8, FMNL2, PGAM1, CNP, CHMP4B, ANXA3, VPS4B, GNG12, PACSIN3, GLG1, VTA1, LYN, VPS37C, CHMP5, F3, DNAJA1, RHOC, GNA13, CHMP2A, ATP2B1, RDX, ATP1B1, CAPZB, EHD1, DNAJA2, and CTNND1.
The methods described herein may be performed to diagnose, prognose, or manage treatment of specific types of cancer. For example, in some embodiments, melanoma, breast cancer, or pancreatic cancer may be diagnosed or prognosed. In other embodiments, treatment of melanoma, breast cancer, or pancreatic cancer may be modified.
In one embodiment, the method is performed to diagnose, prognose, or manage treatment of melanoma. For purposes of prognosing or managing treatment of melanoma, a subject is selected that has or is undergoing treatment for melanoma.
According to this embodiment, exomeres are recovered from the sample and the method is carried out by detecting, higher or lower levels, relative to a standard for subjects not having melanoma or to a prior sample from the subject, or the presence or absence, of one or more of the proteins selected from the group consisting of ITIH2, ITIH3, H2AFX, PMEL, MAT1A, HPD, ALB, B4GAT1, ARF1, GCLC, HGD, PPP2CB, PAH, AGL, RNPEP, PPID, BZW1, ME1, DPYD, CA6, OLFML3, NPEPPS, PREP, ERP44, RELN, GPD1, GFPT1, CNDP2, PFKL, ALDH8A1, ATP6V1A, ENO2, THBS3, CORO1C, EXT1, CAT, XPNPEP1, PYGL, CALR, and LGALS3BP.
In another embodiment, small exosomes are recovered from the sample and the method is carried by detecting, higher or lower levels, relative to a standard for subjects not having melanoma or to a prior sample from the subject, or the presence or absence, of one or more of the proteins selected from the group consisting of TYRP1, SDCBP, SDCBP, CD63, IGSF8, HSPA8, MLANA, HBA1/HBA2, GPNMB, DCT, HSPA2, HSPA1L, HSPA5, Fv4, PDCD61P, RAB7A, ENV1, CD81, GNB1, SYT4, GNB2, HIST1H2AH, GNA12, GAPDH, APOE, BC035947, Hist1h2a1, ACTG1, ACTB, GNB4, GNA13, SLC3A2, ACTC1, GNAS, SLC38A2, HIST2H2BF, ATP1A1, TFRC, TMEM176B, VAMP8, TSPAN10, ADGRG1, Hist1h4a, PMEL, UBL3, PP1A, ACTBL2, CD9, BACE2, and TSPAN4.
In a further embodiment, large exosomes are recovered from the sample and the method is carried out by detecting, higher or lower levels, relative to a standard for subjects not having melanoma, or the presence or absence, of one or more of the proteins selected from the group consisting of HSPA8, TYRP1, SDCBP, HSPA2, RPS27A, HSPA1L, MLANA, HSPA5, CD63, IGSF8, GPNMB, Fv4, ENV1, PDCD61P, HSPA1A/HSPA1B, DCT, ACTG1, ACTB, PP1A, SLC3A2, ACTC1, CD81, ITM2C, RAB7A, GNB1, TSPAN4, DNAJA1, GNB2, TFRC, HBA1/HBA2, GNA12, SYT4, GAPDH, APOE, PMEL, MFGE8, GNB4, GNA13, GNAO1, DNAJA2, ATP1A1, ITGB1, TMEM59, SLC38A2, GNA12, ITM2B, GNAS, HIST1H2AH, LAMP1, and EEF1A1.
In another embodiment, the method is performed to diagnose, prognose, or manage treatment of breast cancer. For purposes of prognosing or managing treatment of breast cancer, a subject is selected that has or is undergoing treatment for breast cancer.
According to this embodiment, exomeres are recovered from the sample and the method is carried out to by detecting, higher or lower levels, relative to a standard for subjects not having breast cancer or to a prior sample from the subject, or the presence or absence, of one or more of the proteins selected from the group consisting of FGB, HIST2H2AB, COMP, HIST1H2BJ, C7, GSTA5, ENO3, ARF3, SULT1C4, E1F4A2, MAT1A, GNB2, UGDH, AKR1B10, MTHFD1, CTSC, DPP3, RPSA, OTUB1, ALDH8A1, F11, CTPS2, MGAT1, HYH1, LGALSL, GSTM3, GSTM5, PSMC1, F8, PRKAR2B, RPL10A, HNRNPK, SEMA6D, SNX5, IARS, LCP2, ARPC4, PPP6C, PSMD6, PTPRS, TIE1, PSMD8, PABPC4, RPS18, CHAD, IPO5, FABP3, GALNT2, QPCT, STAT5A, SEMA3A, NTSC2, IDE, STAT3, DPYSL3, PDXK, ARF5, PSMD5, GNE, NBEAL2, FHL1, TIMP3, POSTN, MAPRE2, IT1H3, C3, ENO2, PPID, O1T3, CAND1, SEPT2, UBE2N, DPYSL2, CKB, PTGES3, DSTN, PKLR, THBS3, RAP1B, HIST2H2AB, ACTBL2, TUBB2A, F10, CNTN1, HPD, ACE, EML2, HSPA13, TNXB, HEXB, CALR, ADH5, GPX1, CFL2, KRT76, TCP1, COTL1, DYNLL1, HGD, ALDOC, EPRS, GLO1, MAN2A1, FLT4, NAP1L4, RARS, HMGCS1, GANAB, SEPT7, FKBP4, COL12A1, ADSL, AKR1C20, VASN, DDX39B, ME1, COMT, ALDH1A1, EIF4A3, CDH11, PRPS113, PNPEP, NPEPPS, SEPT11, CMBL, PSMD1, ACTR1B, PSMD3, GCLC, FAT4, LPL, GPD1L, GCLM, VARS, PHPT1, CACNA2D1, SEPT9, GLRX3, AARS, GMPPB, SNX2, GLOD4, PTPRF, CSAD, PXDN, AGL, DPYD, PRKACB, LARS, PPID, LTA4H, PSMD7, CAPNS1, ETF1, IARS, VPS35, TKFC, HYOU1, PGM2, TKT, HMCN1, CYB5R3, GPS1, UMPS, SND1, RTCB, RPL26, CARM1, PLCG2. P4HA2, CORO6, GMPS, IGSF8, PPP1R7, T1MP3, UXS1, DNM2, MEMO1, RPS3, ARHGD1A, PTGES3, NRP2, RAB1A, HBG2, and YWHAQ.
In another embodiment, small exosomes are recovered from the sample and the method is carried out to by detecting, higher or lower levels, relative to a standard for subjects not having breast cancer or to a prior sample from the subject, or the presence or absence, of one or more of the proteins selected from the group consisting of SDCBP, HIST1H2BN, HIST1H2AH, HBA1/HBA2, ITGB1, Hist1h4a, PDCD61P, HIST3H-2BB, H2AFX, CD9, CD63, ITGA3, IT1H2, MFGE8, H2AFZ, PTGFRN, Hist1h3b, HSPA8, ACTG1, ACTB, ARRDC1, ACTC1, IT1H3, IGSF8, GSN, TUBA4A, HiST1H1D, TUBA1A, HIST1H1C, THBS, HSPA2, ENO1, MVB12A, HTRA1, GAPDH, Hist1h1e, VPS28, TSG101, TUBB, TUBB4A, RAP1B, PFN1, CD81, VPS37B, TUBB6, RAP1A, EPCAM, Hist1h1b, PP1A, ADAM10, HBA1, HIST1H2BK, A2M, ED1L3, SDCBP, MFGE8, GSN, HIST2H2AC, HIST1H2AC, H2AFX, ACTB, THBS1, IT1H4, TUBB, TUBB2A, TUBB4B, F10, H2AFZ, TUBB4A, TUBB6, TUBB1, HSPA8, CD9, CD81, GAPDH, PFN1, HIST1H4A, HSP90AA1, HSP90AB1, HSPA2, HIST2H3A, PGK1, THBS2, EEF1A1, GPX3, ITGB1, PP1A, PDCD61P, EEF1A2, FBLN1, AT1C, CPNE8, TLN1, HSPA5, PKM, HIST1H1C, WDR1, RAN, PYGL, and ITGA3.
In a further embodiment, large exosomes are recovered from the sample and the method is carried out to by detecting, higher or lower levels, relative to a standard for subjects not having breast cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of PDCD61P, SDCBP, EHD1, ITGB1, S100A6, ITGA3, CD9, VPS37C, Hist1h4a, RAP1B, CTNNA1, MSN, HIST1H2AH, ITGA2, PTGFRN, ACTG1, HIST1H2BN, Calm1, EPCAM, ITGA6, YWHAE, HSPA1A/HSPA1B, GNB1, SLC3A2, GNB2, EHD2, H2AFX, PP1A, NT5E, VPS4B, GNB4, Cdc42, SLC1A5, GNA12, CFL1, YWHAH, EEF1A1, YWHAB, Hist1h3b, TSG101, YWHAG, ANXA5, GNA13, F5, H3F3A/H3F3B, CHMP4B, HSPA5, EZR, GAPDH, CD81, ED1L3, HBA1/HBA2, UBC, SDCBP, HSPA8, ITGB1, CD9, HSPA2, ACTC1, ACTB, ACTG1, PDCD61P, AFP, HBG2, ANXA2, ITGA3, HIST1H2BK, GAPDH, CD81, SLC3A2, GNA12, GNA13, GNA11, ATP1A1, HIST2H2AC, CPNE8, IST1, PFN1, TUBA4A, H2AFX, TUBA1C, HSPA5, YWHAZ, ENO1, ANXA5, GNAS, DNAJA1, CHMP5, EEF1A1, RHOA, KRT1, CEP55, GNB1, ACTBL2, ITGA2, EPHA2, GNA13, PP1A, RAP1A, and CD59.
In another embodiment, the method is performed to diagnose, prognose, or manage treatment of pancreatic cancer. For purposes of prognosing or managing treatment of pancreatic cancer, a subject is selected that has or is undergoing treatment for pancreatic cancer.
According to this embodiment, exomeres are recovered from the sample and the method is carried out to by detecting, higher or lower levels, relative to a standard for subjects not having pancreatic cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of SULT1E1, PKLR, ENO2, AKR1B1, FH, MGAT2, GPX1, DPP3, SEMA4B, GPD1, CSAD, NCAM1, PCMT1, NARS, THOP1, UMPS, PDE5A, CACNA2D1, TIE1, CDH11, AOX1, F8, GLB1, RPL10A, ACAP2, UXS1, ADSL, BMP1, PSMD3, LANCL1, GLO1, PPP2CA, ESD, PSMD5, FARSB, PAFAH1B1, SNX5, XPO1, MAPRE1, APRT, NEO1, GBA, THBS1, PYGL, THBS2, FAT4, CNTN1, AKR1C20, E1F4A2, ESD, BPGM, VASN, MAT1A, MAT2A, PFKL, CLIC5, HGD, GLOD4, AGL, PLEKHB2, CLSTN1, ST13, CMBL, AKR1E2, PRKAR2A, GPD1, LGALSL, GLA, IL1RAP, GMPPB, PCSK6, SEPT9, PSMC6, FYN, PAFAH1B1, VPS37C, CTNND1, NRBP1, ERP44, SHMT1, DARS, ADSL, GCLM, ALDOC, EPHA4, PEPD, CKB, PCMT1, UGDH, PRKAR1A, and GNAS.
In another embodiment, small exosomes are recovered from the sample and the method is carried out to by detecting, higher or lower levels, relative to a standard for subjects not having pancreatic cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of SDCBP, PDCD61P, HSPA8, IGSF8, CD9. PTGFRN, ACTC1, LY6E, ACTB, MFGE8, HSPA2, CD81, ITGA3, ITGB1, IT1H2, VPS28, CD63, HTRA1, ENV1, Fv4, GSN, ENO1, ED1L3, MVB12A, IF1TM3, SERPINC1, ACTBL2, TUBA4A, PP1A, HSPA1A/HSPA1B, HSPA5, GAPDH, TSG101, TUBB, PLEKHB2, TUBA1C, TUBB4B, PFN1, GPC1, GJA1, EHD1, GNB2, TSPAN4, GNAI2, SLC3A2, VPS37B, GNA13, RAB7A, EEF1A1, GNAS, ALB, HBA1/HBA2, CD9, UBC, SDCBP, F2, ACTG1, ACTB, CD59, ACTC1, ACTA2, A2M, HIST1H2BK, HIST1H2BJ, ISPA8, TSPAN3, HIST2H2AC, CD55, H3F3A/H3F3B, HIST2H3PS2, PDCD61P, ITGB1, POTEJ, SERINC5, H2AFZ, ARRDC1, CLDN3, NT5E, EPCAM, CDH17, ATP1A1, ALPPL2, HIST2H2AB, ALPP, HSPA2, TSPAN8, MVP, ADAM10, THBS1, VNN1, ITGAV, IGSF8, MYOF, ATP1A2, AHCY, GSN, TSPAN1, PP1A, SDCBP2, and HSPA5.
In a further embodiment, large exosomes are recovered from the sample and the method is carried out to by detecting, higher or lower levels, relative to a standard for subjects not having pancreatic cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of ACTC1, ACTG1, ACTB, MFGE8, ITGB1, IHSPA8, ITGA3, SDCBP, GAPDH, LGALS1, ENV1, Fv4, YWHAZ, PP1A, GNB1, GNA12, GNB2, ACTBL2, GNA13, CFL1, Marcks, GNAS, EEF1A1, ENO, BSG, Calm1, S100A4, MSN, EZR, RDX, PTGFRN, PKM, SLC3A2, HBA1/HBA2, ED1L3, GNA13, RHOA, RHOC, S100A6, YWHAE, ALDOA, PDCD61P, PFN1, HSP90AB1, YWHAQ, ANXA1, ANXA2, ATPIA1, ITGA6, UBC, HBA1/HBA2, CD9, ACTG1, ACTB, CD59, MVP, SDCBP, ACTC1, ACTA2, HIST1H2BK, HIST2H2AC, ALB, HSPA8, HIST1H2BJ, CD55, H3F3A, TSPAN3, HIST2H3PS2, POTEJ, DPP4, NT5E, EPCAM, VNN1, H2AFZ, ITGB1, ALPPL2, HIST2H2AB, ATP1A1, ALPP, IST1, PDCD61P, MUC13, ANXA11, HSPA2, CDH17, GPA33, ANXA2, S100A6, ATP1A2, PP1A, EGFR, TSPAN8, MYOF, GNA11, GNA12, GNA13, S100A4, CLDN3, and A2M.
The one or more protein levels are compared to a “standard” level of the same one or more proteins to identify a subject as one that has cancer or is at risk for metastatic disease. In one embodiment, the standard level of a protein is the average expression level of the protein in exomere, small exosomal, or large exosomal samples taken from a cohort of healthy individuals (i.e., the average level in non-cancerous exomere, small exosomal, or large exosomal samples). In another embodiment, the standard level is the average level of the marker in exomere, small exosomal, or large exosomal samples taken from individuals having a primary tumor, e.g., a gastrointestinal tumor that never metastasized to the liver or other organ of the body. In another embodiment, the standard level of a protein is the level of the protein in an exomere, small exosomal, or large exosomal sample taken from the subject being tested, but at an earlier time point (e.g., a pre-cancerous time point).
In accordance with all aspects of the present invention, a “higher level” refers to an expression level (i.e., protein or gene expression level) that is higher than the standard level. For example, a higher expression level is at least 50% higher than the standard expression level. A “lower level” refers to an expression level (i.e., protein or gene expression level) that is lower than the standard level. For example, a lower expression level is at least 50% lower than the standard expression level.
In accordance with this aspect and other aspects of the invention relating to detecting higher or lower levels or the presence or absence of one or more proteins in the sample, suitable methods for detecting proteins include, but are not limited to, measuring RNA expression level and measuring protein expression levels. These methods are commonly used in the art. For measuring protein expression levels, this method generally involve contacting the sample with one or more detectable reagents that is suitable for measuring protein expression, e.g., a labeled antibody or a primary antibody used in conjunction with a secondary antibody, and measuring protein expression level based on the level of detectable reagent in the sample after normalizing to total protein in the sample. Suitable methods for detecting protein expression level in an exosome sample that are commonly employed in the art include, for example and without limitation, western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescent activated cell sorting (FACS). The measured protein expression level in the sample is compared to the protein expression level measured in a reference exosomal sample and the type of metastatic disease is identified based on this comparison.
Measuring gene expression by quantifying mRNA expression can be achieved using any commonly used method known in the art including northern blotting and in si/u hybridization (Parker et al., “mRNA: Detection by in Situ and Northern Hybridization,” Methods in Molecular Biology 106:247-283 (1999), which is hereby incorporated by reference in its entirety); RNAse protection assay (Hod et al., “A Simplified Ribonuclease Protection Assay,” Biotechniques 13:852-854 (1992), which is hereby incorporated by reference in its entirety); reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., “Detection of Rare mRNAs via Quantitative RT-PCR,” Trends in Genetics 8:263-264 (1992), which is hereby incorporated by reference in its entirety); and serial analysis of gene expression (SAGE) (Velculescu et al., “Serial Analysis of Gene Expression,” Science 270:484-487 (1995); and Velculescu et al., “Characterization of the Yeast Transcriptome,” Cell 88:243-51 (1997), which is hereby incorporated by reference in its entirety).
In other embodiments, the exomeres, small exosomes, or large exosomes are then contacted with one or more reagents suitable to detect higher or lower levels, relative to a standard for subjects not having cancer or to a prior sample from a subject having cancer, or the presence or absence of one or more N-glycans in the exomere, small exosome, or large exosome sample.
For purposes of prognosing or managing treatment of cancer, a subject is selected that has or is undergoing treatment for cancer.
In accordance with this embodiment, exomeres are recovered from the sample and the method is carried out by detecting N-glycans selected from the group consisting of N-glycan (Fucose)+GlcNAcβ1-6(GlcNAcβ1-2)Manα1-6(GlcNA β1-4(GlcNAcβ1-2)Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAeβ1-Asn and N-glycan (Fucose)+Neu5Acα2-8Neu5Acα2-3Galβ1-3/4GlcNAcβ1-2Manα1-3(Manα1-3(Manα1-6))Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn.
In another embodiment, small exosomes are recovered from the sample and the method is carried out by detecting N-glycans.
In a further embodiment, large exosomes are recovered from the sample and the method is carried out by detecting N-glycans.
Methods of analyzing glycoproteins are well known in the art. For example, as a first step, the nanoparticles are lysed and total protein is collected, which contains glycoproteins of interest. The complex carbohydrate portion of the glycoproteins may be readily analyzed if desired, by conventional techniques of carbohydrate analysis. For example, techniques such as lectin blotting, which is well-known in the art, reveal proportions of terminal mannose or other sugars such as galactose. Termination of mono-, bi-, tri-, or tetra-antennary oligosaccharide by sialic acids can be confirmed by release of sugars from the protein using anhydrous hydrazine or enzymatic methods and fractionation of oligosaccharides by ion-exchange or size exclusion chromatography or other methods well-known in the art. The isoelectric point (pi) of the glycoprotein can also be measured, before and after treatment with neuraminidase to remove sialic acids. An increase in pi following neuraminidase treatment indicates the presence of sialic acids on the glycoprotein.
The carbohydrates can be analyzed by any method known in the art including those methods described herein. Several methods are known in the art for glycosylation analysis and are useful in the context of the present invention. Such methods provide information regarding the identity and the composition of the oligosaccharide. Methods for carbohydrate analysis useful in the present invention include but are not limited to lectin chromatography; HPAEC-PAD, which uses high pH anion exchange chromatography to separate oligosaccharides based on charge; NMR; Mass spectrometry; HPLC; GPC; monosaccharide compositional analysis; sequential enzymatic digestion.
In some embodiments, as described in the Examples herein, glycoproteins extracts are reduced, alkylated and digested with sequencing-grade, modified trypsin (Promega) using a standard proteomics protocol (Ferreira et al., “Synthesis and Optimization of Lectin Functionalized Nanoprobes for the Selective Recovery of Glycoproteins from Human Body Fluids,” Analytical Chemistry 83:7035-7043 (2011), which is hereby incorporated by reference in its entirety). The N-glycans can then be analyzed based on a modification of Jensen et al (Kolarich et al., “Isomer-Specific Analysis of Released N-Glycans by LC-ESI MS/MS with Porous Graphitized Carbon,” Methods in Molecular Biology 1321:427-435 (2015), which is hereby incorporated by reference in its entirety). Briefly, N-Linked glycans are released with PNGase F (Elizabethkingia meningoseptica; Sigma), deaminated and partially purified using porous graphitized carbon solid-phase extraction cartridges (PGC-SPE, HyperSep-96-Hypecarb, 25 mg, Thermo Scientific) as described previously (Jensen et al., “Structural Analysis of N- and O-Glycans Released from Glycoproteins,” Nature Protocols 7:1299-1310 (2012), which is hereby incorporated by reference in its entirety). Glycan profiling and characterization may be performed by MALDI TOF/TOF mass spectrometry (4800 Plus, SCIEX) using alpha-cyano-4-hydroxycinnamic acid (CHCA; 10 mg/mL in 50% ACN), operated in reflector negative mode (mass range of m/z 1000 to 5000) with external calibration (TOF/TOF calibration mixture, SCIEX). NanoHPLC-High Resolution Mass Spectrometry (HRMS) may be used to validate the presence of most discriminative ions in MALDI-MS spectra using a nanoHPLC system (Dionex, 3000 Ultimate RSLCnano) coupled on-line to a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific) equipped with a nano-electrospray ion source (Thermo Scientific, EASY-Spray source). N-Glycan chromatographic separation using Porous Graphitized Carbon (PGC) may be adapted from a procedure previously described (Jensen et al., “Structural Analysis of N- and O-Glycans Released from Glycoproteins,” Nature Protocols 7:1299-1310 (2012), which is hereby incorporated by reference in its entirety). A nanoflow PGC column (Hypercarb, 150 mm×75 μm ID, 3 μm particle size, Thermo Scientific) followed by a reversed phase C18 column (EASY-Spray C18 PepMap, 100 Å, 150 mm×75 μm ID and 3 μm particle size, Thermo Scientific) can be combined in series. This allows a better separation of carbohydrates and remaining tryptic peptides, while minimizing salt precipitation events encountered when a nanospray emitter was utilized directly after the PGC column. The mass spectrometer is operated in negative ion mode.
The monosaccharide compositions for the glycan precursors on MALDI-MS spectra may then be predicted using the GlycoMod tool considering mass accuracies below 10 ppm. The possibility of neutral exchanges with Na⁺ and K⁺ was considered for sialoglycans. The glycan structures are assigned based on nanoHPLC-PGC-HRMS analysis considering: i) molecular monoisotopic mass; (ii) CID-MS/MS de novo sequencing; and (iii) PGC-LC relative retention times. In particular, α2,3-linked and α2,6-linked sialylated N-glycans were differentiated based on retention time (α2,6<α2,3) (Kolarich et al., “Isomer-Specific Analysis of Released N-Glycans by LC-ESI MS/MS with Porous Graphitized Carbon,” Methods in Molecular Biology 1321:427-435 (2015), which is hereby incorporated by reference in its entirety). For further validation, MS/MS fragmentation profiles are matched to glycosidic fragments calculated in silico on GlycoWorkBench (Ceroni et al., “GlycoWorkbench: a Tool for the Computer-Assisted Annotation of Mass Spectra of Glycans,” Journal of Proteome Research 7:1650-1659 (2008), which is hereby incorporated by reference in its entirety). General understanding of mammalian N-glycosylation may be used to determine some structural aspects. A semiquantitive approach may be used to compare glycan compositions based on MALDI-MS assignments, taking into account the monoisotopic peak intensity.
In other embodiments, the exomeres, small exosomes, or large exosomes are then contacted with one or more reagents suitable to detect higher or lower levels, relative to a standard for subjects not having cancer or to a prior sample from a subject having cancer, or the presence or absence of one or more lipids in the exomere, small exosome, or large exosome sample.
The term “lipidomics” refers to the use of metabolomics as applied to the evaluation of lipid metabolites in biological samples. Lipid profiling generally involves an evaluation of lipid metabolites in one or more lipid classes (e.g., fatty acids, triglycerides, diglycerides, cholesterol esters, and the phospholipid classes including phosphatidycholine, phosphatidylethanolamine, lysophosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin). As used herein, the term “lipid” is intended broadly and encompasses a diverse range of molecules that are relatively water-insoluble or nonpolar compounds of biological origin, including waxes, triglycerides, free fatty acids, diacylglyercols, fatty-acid derived phospholipids, sphingolipids, glycolipids and terpenoids, such as retinoids, cholesterol, cholesterol esters, and steroids. Some lipids are linear aliphatic molecules, while others have ring structures. Some are aromatic, while others are not.
The lipid profile can be quantitative, semi-quantitative and/or qualitative. For example, the lipid profile can evaluate the presence or absence of a lipid, can evaluate the presence of a lipid(s) above or below a particular threshold, and/or can evaluate the relative or absolute amount of a lipid(s). In some embodiments, a ratio among two, three, four or more lipids is determined. Changes or perturbations in lipid ratios can be advantageous in indicating where there are metabolic blocks (or releases of such blocks) or other alterations in metabolic pathways associated with disease, response to treatment, development of side effects, and the like. Methods of evaluating ratios of lipid precursors and products to evaluate enzyme activities and flow through metabolic pathways are known in the art (see. e.g., Attie et al., (2002) J. Lipid Res. 43:1899-1907 and Pan et al., (1995) J. Clin. Invest. 96:2802-2808, which are hereby incorporated by reference in their entirety).
Ratios of lipid metabolites can be used to reflect or assess changes in lipid metabolism. Generally, if the ratio is calculated from metabolites not present in the same lipid class, quantitative data are used to calculate the ratio. If the lipid metabolites reflected in the numerator and the denominator belong to the same lipid class, then relational data can be used.
In some embodiments, the level of a lipid metabolite is normalized against another lipid metabolite. For example, the ratio between two or more lipid metabolites can be normalized against an index associated with a pathway, enzymatic activity, class of metabolites, and/or status of certain metabolic activities.
Alternatively the level of a lipid metabolite can be normalized against a housekeeping lipid metabolite, e.g., a lipid metabolite that is relatively stable in amount under a variety of conditions in the subject. Quantitative metabolomic data include molar quantitative data, mass quantitative data and relational data by either moles or mass (mole % or weight %, respectively) for individual lipid metabolites or subsets of metabolites. In some embodiments, quantitative aspects of lipidomic analysis can be provided and/or improved by including one or more quantitative internal standards during the analysis, for instance, one standard for each lipid class. Internal standards are described in more detail in U.S. Patent Publication No. 2004/01434612, which is hereby incorporated by reference in its entirety.
Truly quantitative data can be integrated from multiple sources (e.g., the data do not need to be generated with the same assay, in the same location and/or at the same time) into a single seamless database regardless of the number of metabolites measured in each, discrete, individual analysis.
As used herein the term “level” is intended broadly and can mean a quantitative amount (e.g., weight or moles), a semi-quantitative amount, a relative amount (e.g., weight % or mole % within class or a ratio), a concentration, and the like.
For purposes of prognosing or managing treatment of cancer, a subject is selected that has or is undergoing treatment for cancer.
In accordance with this embodiment, exomeres are recovered from the sample and the method is carried out by detecting one or more lipids selected from the group consisting of phospholipids, sphingolipids, and glycerolipids. Exemplary specific lipids include, without limitation, TG, Cer, LPG, LPE, PC, PI, PE, LPI, PS, SM, MG, LPC, PG, DG, CerG3, and CerG1.
In another embodiment, small exosomes are recovered from the sample and the method is carried out by detecting lipids selected from the group consisting of LPE, PC, PI, PE, LPI, PS, PC, SM, and CerG3.
In further embodiment, large exosomes are recovered from the sample and the method is carried out by detecting lipids selected from the group consisting of LPE, PC, PI, PE, LPI, PS, PC, SM, and CerG3.
The lipid profile of the exomere, small exosomal, or large exosomal sample can be determined using any suitable method, The different classes of lipids and methods of detecting and optionally quantifying the same are well known in the art (e.g., thin layer chromatography, gas chromatography, liquid chromatography, mass and NMR spectrometry, and any combination thereof (e.g., GC/MS), and the like). One suitable method of detecting, and optionally quantifying, lipids in a biological sample employs stable isotope tracers to label the lipids. Methods of obtaining lipid profiles from biological samples have been described, see, e.g., U.S. Patent Publication No. 2004/0143461 to Watkins and Watkins et al. (2002) J. Lipid Res. 43(11): 1809-17, which are hereby incorporated by reference in their entirety.
In other aspects of the invention, the exomeres, small exosomes, or large exosomes are then contacted with one or more reagents suitable to detect higher or lower levels, relative to a standard for subjects not having cancer or to a prior sample from a subject having cancer, or the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer in the exomere, small exosome, or large exosome sample.
For purposes of prognosing or managing treatment of cancer, a subject is selected that has or is undergoing treatment for cancer.
In accordance with this aspect, the nucleic acid molecule may be DNA or RNA.
The exomere, small exosome, or large exosome fraction from a bodily fluid of a subject can be pre-treated with DNase to eliminate or substantially eliminate any DNA located on the surface or outside of the exosomes. Without DNAse pre-treatment, short DNA fragments on the outside of the exosomes may remain and co-isolate with nucleic acids extracted from inside the exosomes. Thus, elimination of all or substantially all DNA associated with the outside or surface of the exosomes by pre-treatment of with DNase, has the ability to enrich for internal exomere, small exosome, or large exosome dsDNA. To distinguish DNA strandedness within exomeres, small exosomes, or large exosomes, Shrimp DNase specifically digests double-stranded DNA and SI nuclease specifically digests single-stranded DNA.
In accordance with this and all other aspects of the present invention, DNA may be isolated by extracting the DNA from the exomeres, small exosomes, or large exosomes prior to or for analysis.
The extracted DNA can be analyzed directly without an amplification step. Direct analysis may be performed with different methods including, but not limited to, nanostring technology. NanoString technology enables identification and quantification of individual target molecules in a biological sample by attaching a color coded fluorescent reporter to each target molecule. This approach is similar to the concept of measuring inventory by scanning barcodes. Reporters can be made with hundreds or even thousands of different codes allowing for highly multiplexed analysis. The technology is described in a publication by Geiss et al. “Direct Multiplexed Measurement of Gene Expression with Color-Coded Probe Pairs,” Nat Biotechnol 26(3): 317-25 (2008), which is hereby incorporated by reference in its entirety.
In another embodiment, it may be beneficial or otherwise desirable to amplify the nucleic acid of the exomeres, small exosomes, or large exosomes prior to analyzing it. Methods of nucleic acid amplification are commonly used and generally known in the art. If desired, the amplification can be performed such that it is quantitative. Quantitative amplification will allow quantitative determination of relative amounts of the various exosomal nucleic acids.
Nucleic acid amplification methods include, without limitation, polymerase chain reaction (PCR) (U.S. Pat. No. 5,219,727, which is hereby incorporated by reference in its entirety) and its variants such as in situ polymerase chain reaction (U.S. Pat. No. 5,538,871, which is hereby incorporated by reference in its entirety), quantitative polymerase chain reaction (U.S. Pat. No. 5,219,727, which is hereby incorporated by reference in its entirety), nested polymerase chain reaction (U.S. Pat. No. 5,556,773, which is hereby incorporated by reference in its entirety), self sustained sequence replication and its variants (Guatelli et al, “Isothermal, in vitro Amplification of Nucleic Acids by a Multienzyme Reaction Modeled after Retroviral Replication,” Proc Natl Acad Sci USA 87(5): 1874-8 (1990), which is hereby incorporated by reference in its entirety), transcriptional amplification system and its variants (Kwoh et al. “Transcription-based Amplification System and Detection of Amplified Human Immunodeficiency Virus type I with a Bead-Based Sandwich Hybridization Format,” Proc Natl Acad Sci USA 86(4): 1173-7 (1989), which is hereby incorporated by reference in its entirety), Qb Replicase and its variants (Miele et al. “Autocatalytic Replication of a Recombinant RNA.” J Mol Biol 171(3): 281-95 (1983), which is hereby incorporated by reference in its entirety), cold-PCR (Li et al. “Replacing PCR with COLD-PCR Enriches Variant DNA Sequences and Redefines the Sensitivity of Genetic Testing.” Nat Med 14(5): 579-84 (2008), which is hereby incorporated by reference in its entirety) or any other nucleic acid amplification and detection methods known to those of skill in the art. Especially useful are those detection schemes designed for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In one embodiment, the isolated DNA is contacted with one or more reagents suitable to detect the presence or absence of one or more genetic mutations that are associated with cancer. Exemplary genetic mutations associated with cancer include, but are not limited to, BRAF, EGFR, APC, NOTCH1, HRAS, KRAS, NRAS, MET, p53, PTEN, HER2, FLT3, BRCA1, BRCA2, PIK3CA, KIT, RET, AKT, ABL, CDK4, MYC, RAF, PDGFR, BCR-ABL, NPM1, CEBPalpha, and SRC.
The one or more mutations in the one or more identified genes can be detected using a hybridization assay. In a hybridization assay, the presence or absence of a gene mutation is determined based on the hybridization of one or more allele-specific oligonucleotide probes to one or more nucleic acid molecules in the exomere, small exosomal, or large exosomal DNA sample from the subject. The oligonucleotide probe or probes comprise a nucleotide sequence that is complementary to at least the region of the gene that contains the mutation of interest. The oligonucleotide probes are designed to be complementary to the wildtype, non-mutant nucleotide sequence and/or the mutant nucleotide sequence of the one or more genes to effectuate the detection of the presence or the absence of the mutation in the sample from the subject upon contacting the sample with the oligonucleotide probes. A variety of hybridization assays that are known in the art are suitable for use in the methods of the present invention. These methods include, without limitation, direct hybridization assays, such as northern blot or Southern blot (see e.g., Ausabel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1991), which is hereby incorporated by reference in its entirety). Alternatively, direct hybridization can be carried out using an array based method where a series of oligonucleotide probes designed to be complementary to a particular non-mutant or mutant gene region are affixed to a solid support (glass, silicon, nylon membranes). A labeled exomere, small exosomal, or large exosoma DNA or cDNA sample from the subject is contacted with the array containing the oligonucleotide probes, and hybridization of nucleic acid molecules from the sample to their complementary oligonucleotide probes on the array surface is detected. Examples of direct hybridization array platforms include, without limitation, the Affymetrix GeneChip or SNP arrays and Illumina's Bead Array. Alternatively, the sample is bound to a solid support (often DNA or PCR amplified DNA) and labeled with oligonucleotides in solution (either allele specific or short so as to allow sequencing by hybridization).
Other common genotyping methods include, but are not limited to, restriction fragment length polymorphism assays; amplification based assays such as molecular beacon assays, nucleic acid arrays, high resolution melting curve analysis (Reed and Wittwer, “Sensitivity and Specificity of Single-Nucleotide Polymorphism Scanning by High Resolution Melting Analysis,” Clinical Chem 50(10): 1748-54 (2004), which is hereby incorporated by reference in its entirety); allele-specific PCR (Gaudet et al., “Allele-Specific PCR in SNP Genotyping,” Methods Mol Biol 578: 415-24 (2009), which is hereby incorporated by reference in its entirety); primer extension assays, such as allele-specific primer extension (e.g., Illumina® Infinium® assay), arrayed primer extension (see Krjutskov et al., “Development of a Single Tube 640-plex Genotyping Method for Detection of Nucleic Acid Variations on Microarrays,” Nucleic Acids Res. 36(12) e75 (2008), which is hereby incorporated by reference in its entirety), homogeneous primer extension assays, primer extension with detection by mass spectrometry (e.g., Sequenom® iPLEX SNP genotyping assay) (see Zheng et al., “Cumulative Association of Five Genetic Variants with Prostate Cancer,” N. Eng. J. Med. 358(9):910-919 (2008), which is hereby incorporated by reference in its entirety), multiplex primer extension sorted on genetic arrays; flap endonuclease assays (e.g., the Invader® assay) (see Olivier M., “The Invader Assay for SNP Genotyping,” Mutat. Res. 573 (1-2) 103-10 (2005), which is hereby incorporated by reference in its entirety); 5′ nuclease assays, such as the TaqMan® assay (see U.S. Pat. No. 5,210,015 to Gelfand et al. and U.S. Pat. No. 5,538,848 to Livak et al., which are hereby incorporated by reference in their entirety); and oligonucleotide ligation assays, such as ligation with rolling circle amplification, homogeneous ligation, OLA (see U.S. Pat. No. 4,988,617 to Landgren et al., which is hereby incorporated by reference in its entirety), multiplex ligation reactions followed by PCR, wherein zipcodes are incorporated into ligation reaction probes, and amplified PCR products are determined by electrophoretic or universal zipcode array readout (see U.S. Pat. Nos. 7,429,453 and 7,312,039 to Barany et al., which are hereby incorporated by reference in their entirety). Such methods may be used in combination with detection mechanisms such as, for example, luminescence or chemiluminescence detection, fluorescence detection, time-resolved fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, and electrical detection. In general, the methods for analyzing genetic aberrations are reported in numerous publications, not limited to those cited herein, and are available to those skilled in the art. The appropriate method of analysis will depend upon the specific goals of the analysis, the condition/history of the patient, and the specific cancer(s), diseases or other medical conditions to be detected, monitored or treated.
Alternatively, the presence or absence of one or more mutations identified supra can be detected by direct sequencing of the genes, or preferably particular gene regions comprising the one or more identified mutations, from the patient sample. Direct sequencing assays typically involve isolating DNA sample from the subject using any suitable method known in the art, and cloning the region of interest to be sequenced into a suitable vector for amplification by growth in a host cell (e.g. bacteria) or direct amplification by PCR or other amplification assay. Following amplification, the DNA can be sequenced using any suitable method. A preferable sequencing method involves high-throughput next generation sequencing (NGS) to identify genetic variation. Various NGS sequencing chemistries are available and suitable for use in carrying out the claimed invention, including pyrosequencing (Roche® 454), sequencing by reversible dye terminators (Illumina® HiSeq, Genome Analyzer and MiSeq systems), sequencing by sequential ligation of oligonucleotide probes (Life Technologies® SOLiD), and hydrogen ion semiconductor sequencing (Life Technologies®, Ion Torrent™). Alternatively, classic sequencing methods, such as the Sanger chain termination method or Maxam-Gilbert sequencing, which are well known to those of skill in the art, can be used to carry out the methods of the present invention.
In certain embodiments of the present invention, the selected subject has melanoma, breast cancer, or pancreatic cancer.
The methods described herein may further include selection of a suitable cancer therapeutic and administering the selected cancer therapeutic to a subject. In practicing the methods of the present invention, the administering step is carried out to treat the cancer, achieve inhibition of metastasis or metastatic disease progression. Such administration can be carried out systemically or via direct or local administration to the tumor site. By way of example, suitable modes of systemic administration include, without limitation orally, topically, transdermally, parenterally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, or by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes. Suitable modes of local administration include, without limitation, catheterization, implantation, direct injection, dermal/transdermal application, or portal vein administration to relevant tissues, or by any other local administration technique, method or procedure generally known in the art. The mode of affecting delivery of agent will vary depending on the type of therapeutic agent (e.g., an antibody or an inhibitory nucleic acid molecule) and the disease to be treated.
Effective doses for the treatment of a metastatic disease vary depending upon many different factors, including type and stage of cancer, means of administration, target site, physiological state of the patient, other medications or therapies administered, and physical state of the patient relative to other medical complications. Treatment dosages need to be titrated to optimize safety and efficacy.
Another aspect of the present invention is directed to a kit suitable for diagnosing cancer. The kit includes one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard or to a sample from a subject, or the presence or absence, of one or more proteins contained in exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard or to a sample from a subject, or the presence or absence, of one or more N-glycans contained in exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard or to a sample from a subject, or the presence or absence, of one or more lipids contained in exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in exomeres, small exosomes, or large exosomes, or (5) combinations thereof, wherein said small exosomes have a diameter of 60 to 80 nm and said large exosomes have a diameter of 90 to 120 nm.
In one embodiment, the kit contains one or more reagents suitable for isolating exomeres, small exosomes, or large exosomes. By way of example, suitable markers for isolation of exomeres include, without limitation, HSP90AB1, MTHFD1, ACTR3, PEPD, IDH1, HMGCS1, LGALS3BP, CALR, HSPA13, UGP2, MAT1A, GPD1, PFKL, HGD, GCLC, GSN, CNDP2, FAT4, ERP44, BZW1, AGL, B4GAT1, EXT1, CAT, XPNPEP1, CORO1C, RACK1, HPD, EXT2, ACLY, ADK, PSMC4, ACO1, RRM1, SERPINH1, PYGL, ALDH1L1, PGM1, EEF1G, and PPP2R1A. Suitable markers for isolation of small exosomes include, without limitation, FLOT1, FLOT2, TTYH3, TSPAN14, and VPS37B. Suitable markers for isolation of large exosomes include, without limitation, STIP1, MPP6, DLG1, ABI1, ATP2B1, ANXA4, MYO1C, STXBP3, RDX, ANXA1, ANXA5, GNA13, PACSIN3, VPS4B, CHMP1A, CHMP5, CHMP2A, SH3GL1, CHMP4B, GNG12, and DNAJA1.
In some embodiments, the kit comprises one or more reagents suitable to detect higher or lower levels, relative to a standard or to a sample from a subject, or the presence or absence, of one or more proteins contained in exomeres. Exemplary proteins contained in exomeres are described above.
In some embodiments, the kit comprises one or more reagents suitable to detect higher or lower levels, relative to a standard or to a sample from a subject, or the presence or absence, of one or more proteins contained in small exosomes. Exemplary proteins contained in small exosomes are described above.
In some embodiments, the kit comprises one or more reagents suitable to detect higher or lower levels, relative to a standard or to a sample from a subject, or the presence or absence, of one or more proteins contained in large exosomes. Exemplary proteins contained in large exosomes are described above.
The kit of the present invention may also contain reagents suitable to determine if a subject has a particular type of cancer. In certain embodiments, the kit contains reagents suitable to determine if a subject has melanoma, breast cancer, and/or pancreatic cancer. Exemplary proteins suitable for detection in exomeres, small exosomes, or large exosomes of melanoma, breast cancer, or pancreatic cancer subjects are described above.
A number of kits are contemplated to encompass a variety of methods. These kits optionally include reagents to process a tissue or cell sample for the technique employed by that particular kit. By example, a kit for PCR or PCR enhanced in situ hybridization can include reagents to process the sample and isolate the RNA (for PCR). It will also contain suitable primers to amplify the target sequence and additional probes, if necessary, to detect the desired nucleic acid fragments as well as buffers and reagents for the polymerase chain reaction and the buffers and emulsions required for in situ hybridization methods. Other kits can alternatively include reagents for immunofluorescence or ELISA using antibodies or probes, primers and reagents for modifications of in situ or PCR in situ hybridization methods.
For the purposes of the kits of the present invention, the isolation of nucleic acids from the exomere, small exosomal, or large exosomal sample may be desirable. Accordingly, kits may contain reagents necessary to carry out such methods. Methods of isolating RNA and DNA from biological samples for use in the methods of the present invention are readily known in the art. These methods are described in detail in LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, PART I. THEORY AND NUCLEIC ACID PREPARATION (P. Tijssen ed., Elsevier 1993), which is hereby incorporated by reference in its entirety. Total RNA can be isolated from a given sample using, for example, an acid guanidinium-phenol-chloroform extraction, a guanidinium isothiocyanate-ultracentrifugation method, or lithium chloride-SDS-urea method. PolyA® mRNA can be isolated using oligo(dT) column chromatography or (dT)n magnetic beads (See e.g., SAMBROOK AND RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Laboratory Press, 1989) or CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Fred M. Ausubel et al. eds., 1992) which are hereby incorporated by reference in their entirety). See also WO 2000024939 to Dong et al., which is hereby incorporated by reference in its entirety, for complexity management and other nucleic acid sample preparation techniques.
It may be desirable to amplify the nucleic acid sample prior to detecting protein levels. One of skill in the art will appreciate that a method which maintains or controls for the relative frequencies of the amplified nucleic acids to achieve quantitative amplification should be used.
Typically, methods for amplifying nucleic acids employ a polymerase chain reaction (PCR) (See e.g., PCR TECHNOLOGY: PRINCIPALS AND APPLICATIONS FOR DNA AMPLIFICATION (Henry Erlich ed., Freeman Press 1992); PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS (Michael Innis ed., Academic Press 1990); Mattila et al., “Fidelity of DNA Synthesis by the Thermococcus litoralis DNA Polymerase—An Extremely Heat Stable Enzyme with Proofreading Activity,” Nucleic Acids Res. 19:4967-73 (1991); Eckert et al., “DNA Polymerase Fidelity and the Polymerase Chain Reaction,” PCR Methods and Applications 1:17-24 (1991); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,333,675 all to Mullis et al., which are hereby incorporated by reference in their entireties for all purposes). The sample can also be amplified on an array as described in U.S. Pat. No. 6,300,070 to Boles, which is hereby incorporated by reference in its entirety.
Other suitable amplification methods include the ligase chain reaction (LCR) (e.g. Wu et al., “The Ligation Amplification Reaction (LAR)—Amplification of Specific DNA Sequences Using Sequential Rounds of Template-Dependent Ligation,” Genomics 4:560-9 (1989), Landegren et al., “A Ligase-Mediated Gene Detection Technique,” Science 241:1077-80 (1988), and Barringer et al., “Blunt-End and Single-Strand Ligations by Escherichia coli Ligase: Influence on an In Vitro Amplification Scheme,” Gene 89:117-22 (1990), which are hereby incorporated by reference in their entirety); transcription amplification (Kwoh et al., “Transcription-Based Amplification System and Detection of Amplified Human Immunodeficiency Virus Type I with a Bead-Based Sandwich Hybridization Format,” Proc. Natl. Acad. Sci. USA 86:1173-7 (1989) and WO 88/10315 to Gingeras, which are hereby incorporated by reference in their entirety); self-sustained sequence replication (Guatelli et al., “Isothermal, In Vitro Amplification of Nucleic Acids by a Multienzyme Reaction Modeled After Retroviral Replication,” Proc. Natl. Acad. Sci. USA 87:1874-8 (1990) and WO 90/06995 to Gingeras, which are hereby incorporated by reference in their entirety); selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276 to Burg at al., which is hereby incorporated by reference in its entirety); consensus sequence primed polymerase chain reaction (CP-PCR)(U.S. Pat. No. 5,437,975 to McClelland, which is hereby incorporated by reference in its entirety); arbitrarily primed polymerase chain reaction (AP-PCR)(U.S. Pat. No. 5,413,909 to Bassam, and U.S. Pat. No. 5,861,245 to McClelland which are hereby incorporated by reference in their entirety); and nucleic acid based sequence amplification (NABSA) (See U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603 all to Davey, which are hereby incorporated by reference in their entirety). Other amplification methods that may be used are described in U.S. Pat. No. 5,242,794 to Whiteley; U.S. Pat. No. 5,494,810 to Barany; and U.S. Pat. No. 4,988,617 to Landgren, which are hereby incorporated by reference in their entirety.
The kits may also contain probes or primers which hybridize to complementary nucleic acid molecules in the exomere, small exosomal, or large exosomal sample. The probes comprise nucleotide sequences that are complementary to at least a region of mRNA or corresponding cDNA of the desired proteins. As used herein, the term “hybridization” refers to the complementary base-pairing interaction of one nucleic acid with another nucleic acid that results in formation of a duplex, triplex, or other higher-ordered structure. Typically, the primary interaction is base specific, e.g., A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding. Base-stacking and hydrophobic interactions can also contribute to duplex stability. Conditions for hybridizing detector probes to complementary and substantially complementary target sequences are well known in the art (see e.g., NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH, B. Hames and S. Higgins, eds., IRL Press, Washington, D.C. (1985), which is hereby incorporated by reference in its entirety). In general, hybridization is influenced by, among other things, the length of the polynucleotides and their complements, the pH, the temperature, the presence of mono- and divalent cations, the proportion of G and C nucleotides in the hybridizing region, the viscosity of the medium, and the presence of denaturants. Such variables influence the time required for hybridization. Thus, the preferred hybridization conditions will depend upon the particular application. Such conditions, however, can be routinely determined by the person of ordinary skill in the art without undue experimentation. It will be appreciated that complementarity need not be perfect; there can be a small number of base pair mismatches that will minimally interfere with hybridization between the target sequence and single stranded nucleic acid probe. Thus, what is meant by complementarity herein is that the probes are sufficiently complementary to the target sequence to hybridize under the selected reaction conditions to achieve selective detection and measurement.
Detection of hybridization between probes and corresponding target molecules from an exomere, small exosomal, or large exosomal sample can be performed by several assays known in the art that permit detection of the expression level of the one or more proteins. As described herein, the “expression level” of a protein can be achieved by measuring any suitable value that is representative of the gene expression level. The measurement of gene expression levels can be direct or indirect. A direct measurement involves measuring the level or quantity of RNA or protein. An indirect measurement may involve measuring the level or quantity of cDNA, amplified RNA, DNA, or protein; the activity level of RNA or protein; or the level or activity of other molecules (e.g. a metabolite) that are indicative of the foregoing. The measurement of expression can be a measurement of the absolute quantity of a gene product. The measurement can also be a value representative of the absolute quantity, a normalized value (e.g., a quantity of gene product normalized against the quantity of a reference gene product), an averaged value (e.g., average quantity obtained at different time points or from different sample from a subject, or average quantity obtained using different probes, etc.), or a combination thereof.
In a preferred embodiment, hybridization is detected by measuring RNA expression level. Measuring gene expression by quantifying RNA expression can be achieved using any commonly used method known in the art including northern blotting and In situ hybridization (Parker et al., “mRNA: Detection by in Situ and Northern Hybridization,” Methods in Molecular Biology 106:247-283 (1999), which is hereby incorporated by reference in its entirety); RNAse protection assay (Hod et al., “A Simplified Ribonuclease Protection Assay.” Biotechniques 13:852-854 (1992), which is hereby incorporated by reference in its entirety); reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., “Detection of Rare mRNAs via Quantitative RT-PCR,” Trends in Genetics 8:263-264 (1992), which is hereby incorporated by reference in its entirety); and serial analysis of gene expression (SAGE) (Velculescu et al., “Serial Analysis of Gene Expression,” Science 270:484-487 (1995); and Velculescu et al., “Characterization of the Yeast Transcriptome,” Cell 88:243-51 (1997), which is hereby incorporated by reference in its entirety).
In a nucleic acid hybridization assay, the expression level of nucleic acids corresponding to proteins can be detected using an array-based technique. These arrays, also commonly referred to as “microarrays” or “chips” have been generally described in the an, see e.g., U.S. Pat. No. 5,143,854 to Pirrung et al.; U.S. Pat. No. 5,445,934 to Fodor et al.; U.S. Pat. No. 5,744,305 to Fodor et al.; U.S. Pat. No. 5,677,195 to Winkler et al.; U.S. Pat. No. 6,040,193 to Winkler et al.; U.S. Pat. No. 5,424,186 to Fodor et al., which are all hereby incorporated by reference in their entirety. A microarray comprises an assembly of distinct polynucleotide or oligonucleotide probes immobilized at defined positions on a substrate. Arrays are formed on substrates fabricated with materials such as paper, glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, silicon, optical fiber or any other suitable solid or semi-solid support, and configured in a planar (e.g., glass plates, silicon chips) or three-dimensional (e.g., pins, fibers, beads, particles, microtiter wells, capillaries) configuration. Probes forming the arrays may be attached to the substrate by any number of ways including (i) in situ synthesis (e.g., high-density oligonucleotide arrays) using photolithographic techniques (see Fodor et al., “Light-Directed, Spatially Addressable Parallel Chemical Synthesis,” Science 251:767-773 (1991); Pease et al., “Light-Generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis,” Proc. Natl. Acad. Sci. U.S.A. 91:5022-5026 (1994); Lockhart et al., “Expression Monitoring by Hybridization to High-Density Oligonucleotide Arrays,” Nature Biotechnology 14:1675 (1996); and U.S. Pat. No. 5,578,832 to Trulson; U.S. Pat. No. 5,556,752 to Lockhart; and U.S. Pat. No. 5,510,270 to Fodor, which are hereby incorporated by reference in their entirety); (ii) spotting/printing at medium to low-density (e.g., cDNA probes) on glass, nylon or nitrocellulose (Schena et al., “Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray” Science 270:467-470 (1995), DeRisi et al, “Use of a cDNA Microarray to Analyse Gene Expression Patterns in Human Cancer,” Nature Genetics 14:457-460 (1996); Shalon et al., “A DNA Microarray System for Analyzing Complex DNA Samples Using Two-Color Fluorescent Probe Hybridization,” Genome Res. 6:639-645 (1996); and Schena et al., “Proc. Natl. Acad. Sci. U.S.A. 93:10539-11286) (1995), which are hereby incorporated by reference in their entirety); (iii) masking (Maskos et al., “Oligonucleotide Hybridizations on Glass Supports: A Novel Linker for Oligonucleotide Synthesis and Hybridization Properties of Oligonucleotides Synthesised In Situ,” Nuc. Acids. Res. 20:1679-1684 (1992), which is hereby incorporated by reference in its entirety); and (iv) dot-blotting on a nylon or nitrocellulose hybridization membrane (see e.g., SAMBROOK AND RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Laboratory Press, 1989), which is hereby incorporated by reference in its entirety). Probes may also be noncovalently immobilized on the substrate by hybridization to anchors, by means of magnetic beads, or in a fluid phase such as in microtiter wells or capillaries. The probe molecules are generally nucleic acids such as DNA, RNA, PNA, and cDNA.
Fluorescently labeled cDNA for hybridization to the array may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from exomere, small exosomal, or large exosomal samples. Labeled cDNA applied to the array hybridizes with specificity to each nucleic acid probe spotted on the array. After stringent washing to remove non-specifically bound cDNA, the array is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA samples generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al., “Parallel Human Genome Analysis: Microarray-Based Expression Monitoring of 1000 Genes,” “Proc. Natl. Acad. Sci. USA 93(20):10614-9 (1996), which is hereby incorporated by reference in its entirety).
A nucleic acid amplification assay that is a semi-quantitative or quantitative real-time polymerase chain reaction (RT-PCR) assay can also be performed. Because RNA cannot serve as a template for PCR, the first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avian myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MLV-RT), although others are also known and suitable for this purpose. The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer. Calif., USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.
Although the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonuclease activity. An exemplary PCR amplification system using Taq polymerase is TaqMan® PCR (Applied Biosystems, Foster City, Calif.). Taqman® PCR typically utilizes the 5′-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5′ nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect the nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
TaqMan® RT-PCR can be performed using commercially available equipment, such as, for example, the ABI PRISM 7700® Sequence Detection System® (Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), or the Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany).
In addition to the TaqMan® primer/probe system, other quantitative methods and reagents for real-time PCR detection that are known in the art (e.g. SYBR green, Molecular Beacons, Scorpion Probes, etc.) are suitable for use in the methods of the present invention.
To minimize errors and the effect of sample-to-sample variation, RT-PCR is usually performed using an internal standard or spike in control. The ideal internal standard is expressed at a constant level among different tissues. RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.
Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization and quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR. For further details see, e.g., Heid et al., “Real Time Quantitative PCR,” Genome Research 6:986-994 (1996), which is incorporated by reference in its entirety.
When it is desirable to measure the expression level of proteins by measuring the level of protein expression, the kit may contain reagents suitable for performing any protein hybridization or immunodetection based assay known in the art. In a protein hybridization based assay, an antibody or other agent that selectively binds to a protein is used to detect the amount of that protein expressed in a sample. For example, the level of expression of a protein can be measured using methods that include, but are not limited to, western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent activated cell sorting (FACS), immunohistochemistry, immunocytochemistry, or any combination thereof. Also, antibodies, aptamers, or other ligands that specifically bind to a protein can be affixed to so-called “protein chips” (protein microarrays) and used to measure the level of expression of a protein in a sample. Alternatively, assessing the level of protein expression can involve analyzing one or more proteins by two-dimensional gel electrophoresis, mass spectroscopy (MS), matrix-assisted laser desorption/ionization-time of flight-MS (MALDI-TOF), surface-enhanced laser desorption ionization-time of flight (SELDI-TOF), high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS), protein chip expression analysis, gene chip expression analysis, and laser densitometry, or any combinations of these techniques.
In certain embodiments, kits may contain an antibody that specifically binds a protein of interest. Optionally, the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample. Examples of solid supports include glass or plastic in the form of a microtiter plate, a stick, a bead, or a microbead. Examples of solid supports encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, silicones, and plastics such as polystyrene, polypropylene and polyvinyl alcohol. The sample can be diluted with a suitable diluent or eluant before contacting the sample to the antibody.
After incubating the sample with antibodies, the mixture can be washed and the antibody-antigen complex formed can be detected. This can be accomplished by incubating the washed mixture with a detection reagent. This detection reagent may be a second antibody which is labeled with a detectable label, for example. Exemplary detectable labels include magnetic beads (e.g., DYNABEADS™), fluorescent dyes, radiolabels, enzymes (for example, horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads. Alternatively, the antigens in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound specific, primary antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the antigen (i.e., proteins uniquely associated with exomeres, small exosomes, or large exosomes) is incubated simultaneously with the mixture.
immunoassays can be used to determine presence or absence of proteins in an exomere, small exosomal, and/or large exosomal sample as well as the quantity of the proteins in the sample. If a protein is present in the sample, it will form an antibody-protein complex with an antibody that specifically binds the protein under suitable incubation conditions described above. The amount of an antibody-protein complex can be determined by comparing to a standard. A standard can be a known compound or another protein known to be present in a sample, for example. As noted above, the test amount of antigen (i.e., proteins uniquely associated with exomeres, small exosomes, or large exosomes) need not be measured in absolute units, as long as the unit of measurement can be compared to a control.
In one embodiment, the kit contains one or more reagents suitable to detect higher or lower levels, or the presence or absence of one or more lipids contained in exomeres, small exosomes, or large exosomes.
Exemplary lipids to be detected are described above.
For the purposes of detecting the presence or absence or lipids, the kit comprises reagents and reference compounds suitable for detecting lipids. The reference compounds may be one or more of the following, but are not limited to, lipid standard(s), one or more control marker(s) that is/are regularly measured in a clinical setting, and positive and/or negative controls, internal and/or external standards.
In one embodiment, the lipid concentration(s), lipid ratio(s) or (a) lipid combination(s) thereof in a sample from a subject is (are) determined by using mass spectrometry. The sample may be subjected to purification and/or other sample pre-preparation step(s) before mass spectrometry analysis. The purification step may be, but is not limited to chromatography, for example, high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC) and/or ultra high performance liquid chromatography (UHPLC). The sample pre-preparation step may be, but is not limited to solid-phase extraction (SPE), derivatization, liquid-liquid extraction and/or lipoprotein fractionation. The said mass spectrometry determination may be done by tandem mass spectrometry.
In another embodiment, the kit contains one or more reagents suitable to detect higher or lower levels, or the presence or absence of one or more N-glycans contained in exomeres, small exosomes, or large exosomes.
Exemplary N-glycans to be detected are described above.
In some embodiments, the kit contains one or more lectins for a specific glycan structure, in addition to detection reagents and buffers. In some embodiments, the kit contains reagents for identifying glycosylated protein (e.g., the glycosylation detection reagents) in addition to reagents for identifying glycan structures. In some embodiments, the kit contains all of the components necessary and/or sufficient to perform at least one detection assay, including all controls, directions for performing assays, and any necessary or desired software for analysis and presentation of results. In some embodiments, reagents (e.g., lectins) are fluorescently labeled.

EXAMPLES

The examples below are intended to exemplify the practice of the present invention but are by no means intended to limit the scope thereof.

Materials and Methods for Examples 1-7

Cell lines and cell culture. B16-F10, B16-F1, 4T1, MDA-MB-231 series (parental, −1833, −4175, and −831, gifts from Dr. J. Massaguè), LLC, SW620, HCT116 (Horizon Discovery), PANC-1, AsPC-1, Pan02 (purchased from the National Cancer Institute Tumor Repository), and NIH3T3 cells were cultured in DMEM. Human melanoma cells (SK-Mel103, A375M and A375P were obtained from MSKCC), human prostatic carcinoma cell lines PC3 and DU145, as well as BXPC-3, HPAF-11, PC-9, ET2B (gift from Dr. P. Gao and J. Bromberg), K-562 (DSMZ) and NB-4 (DSMZ) cells were cultured in RPM1, supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) and 10% FBS. Cell lines were obtained from American Type Culture Collection, if not otherwise mentioned, and authenticated using STR profiling by commercial providers. All cells were maintained in a humidified incubator with 5% CO₂at 37° C. and routinely tested and confirmed to be free of Mycoplasma contamination. When collecting conditioned media for exosome isolation, FBS (Gibco, Thermo Fisher Scientific) was first depleted of exosomes by ultracentrifugation at 100,000×g for 90 minutes. Cells were cultured for 3 days before supernatant collection.
Human specimens and processing. Fresh human tumor tissues were obtained from subjects with stage 1-3 melanoma at Memorial Sloan-Kettering Cancer Center (MSKCC) and had histologically confirmed melanoma. All individuals provided informed consent for tissue donation according to a protocol approved by the institutional review board of MSKCC (IRB #11-033A, MSKCC; IRB #0604008488, WCM), and the study is compliant with all relevant ethical regulations regarding research involving human participants. Tissues were cut into small pieces and cultured for 24 h in serum-free RPM1 supplemented with penicillin/streptomycin. Conditioned media was processed for exosome isolation and AF4 fractionation as described below.
Exomere and exosome isolation and nanosight tracking analysis (NTA). SEV were prepared using differential ultracentrifugation methods (Peinado et al., “Melanoma Exosomes Educate Bone Marrow Progenitor Cells Toward a Pro-Metastatic Phenotype through MET,” Nature Medicine 18:883-891 (2012), which is hereby incorporated by reference in its entirety) and resuspended in phosphate buffered saline (PBS, pH7.4) for subsequent analysis and AF4 fractionation. Isolated samples were quantified using BCA assay (Pierce, Thermo Fisher Scientific). NTA analysis of exosome size and particle number was performed using the LM10 or DS500 NanoSight system (Malvern Instruments) equipped with a blue laser (405 nm) following manufacturer's instructions.
AF4 fractionation. The detailed step-by-step AF4 fractionation protocol including sample preparation, AF4 setting parameters and running method, data collection and analysis, and fraction collection and characterization) is provided on ProtocolExchange (Zhang et al., “A Protocol for Asymmetric-Flow Field-Flow Fractionation (AF4) of Small Extracellular Vesicles,” Protocol Exchange (2018), which is hereby incorporated by reference in its entirety).
Transmission electron microscopy (TEM) and atomic force microscopy (AFM). For negative staining TEM analysis, 5 μl of sample solution was placed on a formvar/carbon coated grid and allowed to settle for 1 minute. The sample was blotted and negative stained with 4 successive drops of 1.5% (aqu) uranyl actate, blotting between each drop. Following the last drop of stain, the grid was blotted and air-dried. Grids were imaged with a JEOL JSM 1400 (JEOL, USA, Ltd. Peabody, Mass.) transmission electron microscope operating at 100 Kv. Images were captured on a Veleta 2K×2K CCD camera (Olympus-SIS, Munich, Germany).
For AFM, dilutions were made for each sample and then plated on freshly cleaved mica substrate (SPI) for ˜2 minutes before washing with 10 mL of Molecular Biology Grade H₂O (Fisher BP2819-1) and being blown dry with nitrogen gas. Imaging was performed using an MFP-3D-BIO AFM (Asylum Research), with an Olympus AC240TS-R3 AFM probe (Asylum Research) in tapping mode at room temperature. Images were captured at 1 μm×1 μm. Image analysis was performed using a custom-written ImageJ/F1J1 (NIH) code.
Zeta potential measurement. Fractionated samples were diluted in PBS (Phosphate-buffered saline; 0.01 M phosphate buffer. 0.0027 M KCl, 0.137 M NaCl; pH 7.4 tablets, Sigma) for ζ potential analysis using Zetasizer Nano ZS90 (Malvern Instruments). Samples were freshly prepared prior to loading onto the instrument at a 90° angle (respective to the light source). All experiments were performed at a constant temperature of 25° C.
Stiffness measurement. Freshly cleaved mica coverslips were first coated with Poly-L-lysine (0.1%, w/v in H₂O) for 30 minutes and then incubated with samples on the mica surface for 45 minutes. The samples were then rinsed with 1 ml of MilliPure water, washed three times with PBS buffer, then emerged in a drop of PBS on the mica surface. A stand-alone MFP-3D atomic force microscope (Asylum Research, Santa Barbara, Calif.) was utilized to perform the analysis. The spring constant of cantilever was determined as 559.73 pN/nm by the thermal noise method (Langlois et al., “Spring Constant Calibration of Atomic Force Microscopy Cantilevers with a Piezosensor Transfer Standard,” The Review of Scientific Instruments 78:093705 (2007), which is hereby incorporated by reference in its entirety). The curvature radius of cantilever was ˜15 nm, and the resonant frequency of 325 kHz were used for the stiffness analysis (i.e., indentation of cantilever) and imaging. Force measurements were performed with an approximate force distance of 300 nm and velocity of 500 nm/s.
Western blot analysis. Whole cell extract (WCE) and exosome fractions were lysed directly with SDS sample buffer and lysates were cleared by centrifugation at 14,000×g for 10 minutes. 100 μg of WCE and 10 μg of input and each nanoparticle subset were separated on a Novex 4-12% Bis-Tris Plus Gel (Life Technologies), and transferred onto a PVDF membrane (Millipore). Membranes were blocked for 1 hour at room temperature followed by primary antibody incubation overnight at 4° C. The following antibodies were used for western blot analysis: anti-Tsg101 (Santa Cruz sc-7964); anti-Alix1 (Cell Signaling 2171); anti-Hsp90 (Stressgen ADI-SPA-830-F), anti-MAT1A1 (Abcam ab174687); anti-IDH1 (Proteintech 23309-1-AP); anti-FLOT1 (BD Biosciences 610820); anti-TOLL1P (Abcam ab187198); anti-VPS4B (Santa Cruz sc-32922); anti-DNAJA1 (Abcam ab126774); anti-HSPA8/HSC70 (LifeSpan Biosciences LS-C312344-100). All primary antibodies were used at 1:1,000× dilution. IRDye 800 CW Goat-anti-mouse IgG (LI-COR Biosciences P/N 926-32210, 1:15.000× dilution), HRP-linked Sheep-anti-Mouse IgG (GE Healthcare Life Sciences NA931, 1:2,500× dilution), and HRP-linked Donkey-anti-Rabbit IgG (GE Healthcare Life Sciences NA934, 1:2,500× dilution) were used as secondary antibody. The blot was analyzed either using the Odyssey Imaging system (LI-COR Biosciences) or enhanced chemiluminescence substrates (Thermo Fisher Scientific).
Analysis of Proteomic Profiling Data. Protein mass spectrometry analyses of fractionated exosomes were performed at the Rockefeller University Proteomics Resource Center as described previously (Costa-Silva et al., “Pancreatic Cancer Exosomes Initiate Pre-Metastatic Niche Formation in the Liver,” Nature Cell Biology 17:816-826 (2015); Hoshino et al., “Tumour Exosome Integrins Determine Organotropic Metastasis,” Nature 527:329-335 (2015), which are hereby incorporated by reference in their entirety), and conducted on two independent biological replicates for each sample (exomere, Exo-S and Exo-L) derived from 5 different cell lines (B16-F10, 4T1, Pan02, AsPC-1 and MDA-MB-4175).
For proteomic data processing and Principal Component Analysis (PCA), the proteomic expression data was processed using the ‘Limma’ package of the R program. Proteomic expression data was imported and was normalized using ‘normalizeBetweenArrays’ function (method=quantile) (Bolstad et al., “A Comparison of Normalization Methods For High Density Oligonucleotide Array Data Based on Variance and Bias,” Bioinformatics 19:185-193 (2003), which is hereby incorporated by reference in its entirety). PCA was performed for data reduction, simplifying datasets to three dimensions for plotting purposes using ‘princomp( )’ function with default options, and illustrated using the ‘rgl’ package and ‘plot3d( )’ function.
For clustering and marker selection, Consensus clustering analysis, marker selection for each fraction, and heatmap generation were conducted using GENE-E software. Consensus clustering was conducted to assess whether proteomic expression differs between fraction (Monti et al., “Consensus Clustering: A Resampling-Based Method for Class Discovery and Visualization of Gene Expression Microarray Data,” Mach Learn 52:91-118 (2003), which is hereby incorporated by reference in its entirety). To identify fraction-specific markers, the probe (based on UniProt ID) values were collapsed to protein-level using maximum probe. Only proteins detected in both replicates of a sample were included for further analysis. Proteins were sorted by signal-to-noise statistic, (μ_A−μ_B)/(α_A+α_B) where μ and α represent the mean and standard deviation of proteomic expression, respectively, for each class (Golub et al., “Molecular Classification of Cancer: Class Discovery and Class Prediction by Gene Expression Monitoring” Science 286:531-537 (1999), which is hereby incorporated by reference in its entirety). Next, the signal to noise marker selection tool from GENE-E was used to identify fraction-specific markers with 1,000 permutations. The cutoff to select fraction-specific markers was fold change ≥5, false discovery rate (FDR)<0.05, and mean protein expression ≥10⁸with the positivity in ≥80% (i.e. at least 4 out 5 samples from 5 cell lines for each nanoparticle subset) of the corresponding fraction. Heat maps for visualization of differential protein expression patterns were generated for 65 markers (39 exomere-specific markers; 5 Exo-S markers; 21 Exo-L markers) using GENE-E with relative color scheme (by subtracting each mean protein expression, divide by each standard deviation for each row).
For Gene Set Enrichment Analysis (GSEA) the entire proteomic expression data set (Subramanian et al., “Gene Set Enrichment Analysis: A Knowledge-Based Approach for Interpreting Genome-Wide Expression Profiles,” Proc Natl Acad Sci USA 102:15545-15550 (2005), which is hereby incorporated by reference in its entirety) was used. Gene sets from Molecular signatures database v5.1 were used for GSEA (H: 50 hallmark gene sets; C2:KEGG: 186 canonical pathways from Kyoto Encyclopedia of Genes and Genomes [KEGG] pathway database; C5: 825 gene sets based on Gene Ontology [GO] term) (Liberzon et al., “Molecular Signatures Database (MSigDB) 3.0,” Bioinformatics 27:1739-1740 (2011), which is hereby incorporated by reference in its entirety). The default parameters were used to identify significantly enriched gene-sets (FDR q<0.25).
Glycoprotein extraction and lectin blotting. Nanoparticles were lysed with RapiGest SF (Waters) containing 1 mM sodium orthovanadate and protease inhibitor cocktail (Roche), for 30 minutes on ice and centrifuged at 16,000×g for 20 minutes. For lectin blotting 0.5 μg of total protein extracts were separated using 4-15% gradient gels (Biorad) and transferred onto nitrocellulose membranes. Samples were incubated at room temperature (RT) for 1 hour with the following biotinylated lectins Aleuria aurantia Lectin (AAL; Fucα6GlcNAc and Fucα3GlcNAc), Sambucus nigra Lectin (SNA: Neu5Acα6(Gal or GalNAc)). Phaseolus vulgaris Leucoagglutinin (L-PHA; Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3), and Phaseolus vulgaris Erythroagglutinin (E-PHA; Galβ4GlcNAcβ2Manα6(GlcNAcβ4)(GlcNAcβ4Manα3)Manβ4) (Vector Laboratories, 1:2000 dilution except 1:1000 dilution for L-PHA). Vectastain Elite ABC HRP Kit (Vector Laboratories) was used for signal detection with ECL enhanced chemiluminescence technique (GE Healthcare Life Sciences). The total protein profile of the samples was assessed in parallel on a silver-stained gel (FIG. 9A). (Abbreviations: Fuc, fucose; GlcNAc, N-acetylglucosamine; Man, mannose; Neu5Ac, neuraminic acid; Gal, galactose; GalNAc, N-acetylgalactosamine.)
Glycomics analysis. The glycoproteins extracts from the different fractions were reduced, alkylated and digested with sequencing-grade, modified trypsin (Promega) using a standard proteomics protocol (Ferreira et al., “Synthesis and Optimization of Lectin Functionalized Nanoprobes for the Selective Recovery of Glycoproteins from Human Body Fluids,” Analytical Chemistry 83:7035-7043 (2011), which is hereby incorporated by reference in its entirety). The N-glycans were analyzed based on a modification of Jensen et al (Kolarich et al., “Isomer-Specific Analysis of Released N-Glycans by LC-ESI MS/MS with Porous Graphitized Carbon,” Methods in Molecular Biology 1321:427-435 (2015), which is hereby incorporated by reference in its entirety). Briefly, N-Linked glycans were released with PNGase F (Eliaabethkingia meningoseptica: Sigma), deaminated and partially purified using porous graphitized carbon solid-phase extraction cartridges (PGC-SPE, HyperSep-96-Hypercarb, 25 mg, Thermo Scientific) as described previously (Jensen et al., “Structural Analysis of N- and O-Glycans Released from Glycoproteins,” Nature Protocols 7:1299-1310 (2012), which is hereby incorporated by reference in its entirety). Glycan profiling and characterization was performed by MALDI TOF/TOF mass spectrometry (4800 Plus, SCIEX) using alpha-cyano-4-hydroxycinnamic acid (CHCA; 10 mg/mL in 50% ACN), operated in reflector negative mode (mass range of m/z 1000 to 5000) with external calibration (TOF/TOF calibration mixture, SCIEX). Three independent analytical measurements were performed. NanoHPLC-High Resolution Mass Spectrometry (HRMS) was used to validate the presence of most discriminative ions in MALDI-MS spectra using a nanoHPLC system (Dionex, 3000 Ultimate RSLCnano) coupled on-line to a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific) equipped with a nano-electrospray ion source (Thermo Scientific, EASY-Spray source). N-Glycan chromatographic separation using Porous Graphitized Carbon (PGC) was adapted from a procedure previously described (Jensen et al., “Structural Analysis of N- and O-Glycans Released from Glycoproteins,” Nature Protocols 7:1299-1310 (2012), which is hereby incorporated by reference in its entirety). A nanoflow PGC column (Hypercarb, 150 mm×75 μm ID, 3 μm particle size. Thermo Scientific) followed by a reversed phase C18 column (EASY-Spray C18 PepMap, 100 Å, 150 mm×75 μm ID and 3 μm particle size, Thermo Scientific) were combined in series. This allowed a better separation of carbohydrates and remaining tryptic peptides, while minimizing salt precipitation events encountered when a nanospray emitter was utilized directly after the PGC column. The mass spectrometer was operated in negative ion mode.
The monosaccharide compositions for the glycan precursors on MALDI-MS spectra were predicted using the GlycoMod tool considering mass accuracies bellow 10 ppm. The possibility of neutral exchanges with Na⁺ and K⁺ was considered for sialoglycans. The glycan structures were assigned based on nanoHPLC-PGC-HRMS analysis considering: i) molecular monoisotopic mass; (ii) CID-MS/MS de novo sequencing; and (iii) PGC-LC relative retention times. In particular, α2,3-linked and α2,6-linked sialylated N-glycans were differentiated based on retention time (α2,6<α2,3) (Kolarich et al., “Isomer-Specific Analysis of Released N-Glycans by LC-ESI MS/MS with Porous Graphitized Carbon,” Methods in Molecular Biology 1321:427-435 (2015), which is hereby incorporated by reference in its entirety). For further validation, MS/MS fragmentation profiles were matched to glycosidic fragments calculated in silico on GlycoWorkBench (Ceroni et al., “GlycoWorkbench: a Tool for the Computer-Assisted Annotation of Mass Spectra of Glycans,” Journal of Proteome Research 7:1650-1659 (2008), which is hereby incorporated by reference in its entirety). General understanding of mammalian N-glycosylation was used to determine some structural aspects, yet some structural ambiguity remained in a subset of the reported N-glycans as indicated with brackets. A semiquantitive approach was used to compare glycan compositions based on MALDI-MS assignments, taking into account the monoisotopic peak intensity. Glycan standards and negative controls were analyzed in parallel. These results were validated based on the intensity of each specie on nanoHPLC-HRMS ion chromatograms (EIC) (m/z±0.01).
Lipidomics: sample preparation, mass spectrometry and data analysis. Equal amount of each sample (based on BCA quantification) was subjected to lipidomic analysis. Samples were first sonicated with a Model Q700 QSonica sonicator equipped with an Oasis 180 Chiller (4° C.; Amplitude, 95; process, 5 minutes; pulse-on 30 sec; plus-off 55 sec), centrifuged at 14.800 rpm for 10 minutes at 4° C., and 50 μL of the extract supernatant was spiked with 2 μL 50 μg/mL internal standard mixture (Cer 18:1/12:0; PC 12:0/12:0; PE 14:0/14:0; PG 14:0114:0; PS 14:0/14:0). Subsequently, the samples were analyzed by using the Thermo Q-Exactive MS system (Bremen, Germany) in the Metabolomics Laboratory of Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign. Software Xcalibur 3.0.63 was used for data acquisition and analysis. The Dionex Ultimate 3000 series HPLC system (Thermo, Germering, Germany) was used, and the LC separation was performed on a Thermo Accucore C18 column (2.1×150 mm, 2.6 μm) with mobile phase A (60% acetonitrile: 40% H₂O with 10 mM ammonium formate and 0.1% formic acid) and mobile phase B (90% isopropanol: 10% acetonitrile with 10 mM ammonium formate and 0.1% formic acid) and a flow rate of 0.4 mL/min. The linear gradient was as follows: 0 minutes, 70% A; 4 minutes, 55% A; 12 minutes, 35% A; 18 minutes, 15% A; 20-25 minutes, 0% A; 26-33 minutes, 70% A. The autosampler was set to 15° C. and the column was kept at 45° C. The injection volume was 10 μL. Mass spectra were acquired under both positive (sheath gas flow rate, 50; aux gas flow rate: 13; sweep gas flow rate, 3; spray voltage, 3.5 kV; capillary temperature, 263° C.; Aux gas heater temperature, 425° C.) and negative electrospray ionization (sheath gas flow rate, 50; aux gas flow rate: 13; sweep gas flow rate, 3; spray voltage, −2.5 kV; capillary temperature, 263° C.; Aux gas heater temperature, 425° C.). The full scan mass spectrum resolution was set to 70,000 with the scan range of m/z 230˜m/z 1,600, and the AGC target was 1E6 with a maximum injection time of 200 msec. For MS/MS scan, the mass spectrum resolution was set to 17,500, and the AGC target was 5E4 with a maximum injection time of 50 msec. Loop count was 10. Isolation window was 1.0 m/z with NCE of 25 and 30 eV. For data analysis, LipidSearch (v.4.1.30, Thermo) was used for lipid identification. The lipid signal responses were normalized to the corresponding internal standard signal response. For those lipid classes without corresponding internal standard, positive lipid ion signals were normalized with the signal of internal standard Cer 18:1/12:0 and negative ion signals were normalized with the signal of internal standard PG 14:0/14:0. The percentage of lipid classes within a sample was calculated by adding that of each of the individual molecular species quantified within a specific lipid class, and the relative abundance was represented by the mean percentage of 3 replicates for each group of samples. Differences among different subpopulations of particles derived from the same cell line were analyzed using ANOVA test (q<0.05).
Nucleic acid analysis. DNA was extracted from nanoparticles using the AMPure XP beads (Agencourt) following the manufacturers protocol. An equal volume of nanoparticles in PBS and lysis buffer AL (QIAGEN) were mixed and incubated with Proteinase K (20 μg/ml, QIAGEN) at 56° C. for 10 minutes. The mixture was mixed with one volume of each, AMPure beads, isopropanol and PEG solution (Beckman), and incubated for 5 minutes at RT. DNA bound to the beads was then separated from the solution/supernatant on magnet for 5 minutes at RT. The supernatant was removed by pipetting and bead-bound DNA was washed twice with freshly prepared 80% ethanol, then air dried for 5 minutes. Lastly, DNA was eluted from beads with nuclease free water and quantified using QuBit assay (Life Technology). DNA extraction was performed for two independent biological replicates of each sample.
RNA was extracted using the Ambion mirVarna kit (Life technology), following the manufacturer's protocol with one modification: one volume of nanoparticles in PBS was first lysed with 7 volumes of lysis buffer. The samples were analyzed using Agilent Total RNA Pico kits. RNA extraction was performed for two independent biological replicates of each sample.
Biodistribution assessment. Fractionated nanoparticles were first labeled with the near infrared dye CellVue N1R815 (eBioscience) following manufacturer's protocol, followed by washing with 20 ml of PBS and pelleting by ultracentrifugation at 100,000×g for 70 minutes at 10° C. 10 μg of labeled nanovesicles resuspended in 100 μl of PBS, or an equivalent volume of mock reaction mixture was retro-orbitally injected into naïve mice (6-week-old female C57BL6 mice purchased from Jackson Labs). 24 hours post injection, tissues were collected and analyzed using the Odyssey imaging system (LI-COR Biosciences). Two independent experiments with 3 animals per group were performed. No statistical method was used to predetermine sample size. The experiments were neither randomized, nor blinded. All animal experiments were performed in compliance with ethical regulations and in accordance with Weill Cornell Medicine institutional, IACUC and AAALAS guidelines, approved for animal protocol 0709-666A.
Statistics and Reproducibility. Error bars in graphical data represent means±SEM. Statistical significance is determined using one way ANOVA. P<0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism software. For lipid class analysis, ANOVA test (q<0.05) was performed using Qlucore Omics Explorer (Sweden). For proteomic analysis, proteins were sorted by signal-to-noise statistic, (μA−μB)/(αA+αB) where μ and α represent the mean and standard deviation of proteomic expression, respectively. The cutoff to select fraction-specific markers was fold change ≥5, false discovery rate (FDR)<0.05, and mean protein expression 0.10 with the positivity in ≥80% (i.e. at least 4 out 5 samples from 5 different cell lines for each subset of nanoparticles) of the corresponding fraction. For GSEA, Kolmogorov-Smirnov statistic was calculated to evaluate whether proteins from a pre-determined pathway are significantly overrepresented towards the top or bottom of the ranked gene list (FDR q<0.25).
Multiple AF4 analyses were performed for each cell line studied in this work: B16-F10.>50× (repeated times); AsPC-1, 9×; Pan02, 16×; MDA-MB-4175 (4175), 17×; and 4T1, 10×. TEM imaging analysis of fractionated particles were conducted for B16-F10. 7×; AsPC-1, 3×; Pan02, 2×; 4175, 1×; and 4T1, 4×. Four independent human melanoma specimens were analyzed using AF4 and two of them were analyzed by TEM. Proteomic profiling of exomeres. Exo-S and Exo-L was performed on two biologically independent samples of each particle derived from five different cell lines (B16-F10: AsPC-1: Pan02; 4175; and 4T1). Western blotting validation of specific signature proteins of each particle subtype was done once (noted in the legend for FIG. 1D). For A-glycan study, lectin blotting was repeated independently twice except for AA L and E-PH A blotting for B16-F10 and 4175 which were done once (FIG. 8A). Glycomic MS was performed on two biologically independent B16-F10 samples and one sample of AsPC-1 and 4175 (FIGS. 9B-9D). Quantification of top 6 most abundant glycans was based on 3 independent analytical measurements of one experiment (FIG. 8C, FIGS. 9C and 9D). Silver stained-PAGE analysis was repeated independently twice for B16-F10 and 4175 and once for AsPC-1 (FIG. 9A). NanoHPLC-PGC-HRMS was done once (FIGS. 9E-9I). Lipidomic analysis was conducted on 3 biologically independent samples. DNA and RNA analyses of each particle subtype were repeated twice. Organ biodistribution analysis of each particle subtype was repeated 4× independently. NTA analysis was conducted using 3 biologically independent samples. TEM analysis was repeated 3 times for AF4 peaks P1 and P5 and once for HDL, LDL and VLDL (FIG. 7D). AF4 analysis of B16-F10 sEVs collected from technical and biological replicates, and samples kept at either 4° C. or −80° C. were repeated independently 3 times, cells of different passage numbers twice, and under hypoxic versus normoxic conditions was repeated with 3 different cell lines independently. AF4 and TEM analysis of particles isolated from the blank media control and CM of B16-F10 and 4175 was done once (FIGS. 2J and 2K).
Independent measurements of hydrodynamic diameters of exomeres, Exo-S and Exo-L derived from different cell lines in batch mode were repeated (in the order of exomere, Exo-S and Exo-L): B16-F10 (n=10, 9, and 8 independent measurements, respectively); Pan02 (n=11, 6, 11); AsPC-1 (n=5, 5, 5); 4175 (n=3, 5, 3); 4T1 (n=5, 5, 5)). For zeta potential, independent measurements were repeated: B16-F10 (n=8, 10, 12); Pan02 (n=13, 11, 13); AsPC-1 (n=12, 12, 12); 4175 (n=17, 9, 6); 4T1 (n=13, 3, 9). For stiffness, B16-F10 (n=6, 6, 6); Pan02 (n=6, 6, 6); AsPC-1 (n=21, 19, 16); 4175 (n=11, 10, 5); 4T1 (n=9, 8, 9). For AFM imaging analysis of the height of exomeres: B16F10 (n=754 particles analyzed), AsPC1 (n=475) and 4175 (n=160). AFM imaging of exomeres was repeated with samples derived from 3 different cell lines.
For all experiments described above, all attempts at replication were successful with similar results.
Data Availability. The datasets for proteomic analysis of exomeres, Exo-S and Exo-L subpopulations derived from various cancer cell lines have been deposited.
Proteins that are uniquely associated with or among the top 50 most abundant proteins in exomere, Exo-S and Exo-L derived from different cancer cell lines are shown in Table 1 below.

TABLE 1

Top 50 most abundant proteins identified in each subset of particles.

B16F10

Pan02

4T1

exomere	Exo-S	Exo-L	exomere	Exo-S	Exo-L	exomere	Exo-S

HBA1/HBA2	TYRP1	HSPA8	HBA1/HBA2	SDCBP	ACTC1	Hbb-b1	SDCBP
ITIH2	SDCBP	TYRP1	ITIH2	PDCD6IP	ACTG1	HBA1/HBA2	HISTIH2BN
GSN	SDCBP	SDCBP	ACTB	HSPA8	ACTB	HIST1H2BN	HIST1H2AH
ITIH3	CD63	HSPA2	ACTC1	IGSF8	MFGE8	ITIH2	HBA1/HBA2
GAPDH	IGSF8	RPS27A	GSN	CD9	ITGB1	HIST1H2AH	ITGB1
ACTG1	HSPA8	HSPA1L	SERPINC1	PTGFRN	HSPA8	HBG2	Hist1h4a
HIST1H2AH	MLANA	MLANA	HTRA1	ACTC1	ITGA3	H2AFX	PDCD6IP
H2AFX	HBA1/HBA2	HSPA5	ENO1	LY6E	SDCBP	Hist1h4a	HIST3H2BB
ACTC1	GPNMB	CD63	SDCBP	ACTB	GAPDH	ACTG1	H2AFX
TYRP1	DCT	IGSF8	THBS1	MFGE8	LGALS1	ACTB	CD9
SDCBP	HSPA2	GPNMB	TUBB	HSPA2	ENV1	ACTC1	CD63
ENO1	HSPA1L	Fv4	TUBB4B	CD81	Fv4	GSN	ITGA3
Pzp	HSPA5	ENV1	GAPDH	ITGA3	YWHAZ	HIST2H2AB	ITIH2
HSP90AB1	Fv4	PDCD6IP	HSPA8	ITGB1	PPIA	H2AFZ	MFGE8
HSPA2	PDCD6IP	HSPA1A/	TUBB6	ITIH2	GNB1	Hist1h3b	H2AFZ
		HSPA1B
TUBA4A	RAB7A	DCT	TUBA4A	VPS28	GNAI2	Hist1h3a	PTGFRN
HSPA1L	ENV1	ACTG1	HSP90AB1	CD63	GNB2	ACTBL2	Hist1h3b
SERPINC1	CD81	ACTB	PGK1	HTRA1	ACTBL2	ENO1	HSPA8
APOE	GNB1	PPIA	CD9	ENV1	GNAI3	GAPDH	ACTG1
TUBB	SYT4	SLC3A2	EEF1A1	Fv4	CLF1	PFN1	ACTB
TUBB4B	GNB2	ACTC1	TUBB4A	GSN	Marcks	HTRA1	ARRDC1
TUBB4A	HIST1H2AH	CD81	LGALS38P	ENO1	GNAS	TUBB	ACTC1
HSPA5	GNAI2	ITM2C	ITGB1	EDIL3	EEF1A1	TUBB2A	ITIH3
GALK1	GAPDH	RAB7A	PPIA	MVB12A	ENO1	TUBA4A	IGSF8
PKM	APOE	GNB1	PKM	IFITM3	BSG	EZR	GSN
IGSF8	BC035947	TSPAN4	TLN1	SERPINC1	Calm1	TUBB4B	TUBA4A
ALDH1L1	Hist1h2al	DNAJA1	HSPA5	ACTBL2	S100A4	TUBB4A	HIST1H1D
RAN	ACTG1	GNB2	FLNA	TUBA4A	MSN	HSP90AB1	TUBA1A
PPIA	ACTB	TFRC	PDCD6IP	PPIA	EZR	HIST1H1D	HIST1H1C
ALB	GNB4	HBA1/HBA2	CD81	HSPA1A/	RDX	HIST1H1C	THBS1
				HSPA1B
EEF1A1	GNAI3	GNAI2	WDR1	HSPA5	PTGFRN	Hist1h1e	HSPA2
TLN1	5LC3A2	SYT4	CPNI	GAPDH	PKM	TUBB6	ENO1
PDCD6IP	ACTC1	GAPDH	IFITM3	TSG101	PKM	PGK1	MVB12A
PGK1	GNAS	APDE	ENO3	TUBB	SLC3A2	TLN1	HTRA1
THBS3	SLC38A2	PMEL	PGK2	PLEKHB2	HBA1/HBA2	PKM	GAPDH
ALDH9A1	HIST2H2BF	MFGE8	HSP90B1	TUBA1C	EDIL3	SDCBP	Hist1h1e
RAB7A	ATP1A1	GNB4	ITGA3	TUBB4B	GNAI3	PPIA	VPS28
ACD1	TFRC	GNAI3	FBLN1	PFN1	RHOA	EEF1A1	TSG101
Hist1h3b	TMEM176B	GNAD1	IGSF8	GPC1	RHOC	HSPA2	TUBB
CPN1	VAMP8	DNAJA2	RAP1B	GJA1	S100A6	FLNA	TUBB4A
GPNMB	TSPAN10	ATP1A1	GmS424	EHD1	YWHAE	RAN	RAP1B
GDIZ	ADGRG1	ITGB1	MFGE8	GNB2	ALDOA	RAP1B	PFN1
DCT	Hist1h4a	TMEM59	ILK	TSPAN4	PDCD6IP	HSPA5	CD81
Gm5424	PMEL	SLC36A2	ADK	GNAI2	PFN1	FBLN1	VPS37B
LGALS3BP	UBL3	GNA12	PYGL	SLC3A2	HSP90AB1	PGK2	TUBB6
PGD	PPIA	ITM2B	HPD	VP537B	YWHAQ	CPN1	RAP1A
EEF1A2	ACTBL2	GNAS	GNB1	GNAI3	ANXA1	WDR1	EPCAM
PYGL	CD9	HIST1H2AH	ATIC	RAB7A	ANXA2	ENO3	Hist1h1b
PHGDH	BACE2	LAMP1	AKR1B1	EEF1A1	ATP1A1	EEF2	PPIA
Hist1h4a	TSPAN4	EEF1A1	THB53	GNAS	ITGA6	PYGL	ADAM10

4T1

MDA-MB-4175

AsPC1

Exo-L	exomere	Exo-S	Exo-L	exomere	Exo-S	Exo-L

PDCD6IP	HBA1/HBA2	HBA1	EDIL3	ALB	ALB	UBC
SDCBP	HBA1	HIST1H2BK	HBA1/HBA2	HBA1/HBA2	HBA1/HBA2	HBA1/HBA2
EHD1	C3	A2M	UBC	A2M	CD9	CD9
ITGB1	AFP	EDIL3	SDCBP	F2	UBC	ACTG1
S100A6	ITIH4	SDCBP	HSPA8	ACTG1	SDCBP	ACTB
ITGA3	HBG2	MFGE8	ITGB1	ACTB	F2	CD59
CD9	GSN	GSN	CD9	ACTC1	ACTG1	MVP
VPS37C	ITIH3	HIST2H2AC	HSPA2	ACTA2	ACTB	SDCBP
Hist1h4a	A2M	HIST2H2AC	ACTC1	POTEJ	CD59	ACTC1
RAP1B	ACTG1	H2AFX	ACTB	GSN	ACTC1	ACTA2
CTNNA1	ACTC1	ACTB	ACTG1	AHCY	ACTA2	HIST1H2BK
MSN	ACTBL2	THBS1	PDCD6IP	ENO1	A2M	HIST2H2AC
HIST1H2AH	FGB	ITIH4	AFP	THBS1	HIST1H2BK	ALB
ITGA2	THBS1	TUBB	HBG2	LGALS3BP	HIST1H2BJ	HSPA8
PTGFRN	ENO1	TUBB2A	ANXA2	HSP90AA1	HSPA8	HIST1H2BJ
ACTG1	HIST2H2AC	TUBB4B	ITGA3	HSP90AB1	TSPAN3	CD55
HIST1H2BN	HIST1H2AB	F10	HIST1H2BK	F5	HIST2H2AC	H3F3A
Calm1	HIST1H2BK	H2AFZ	GAPDH	PKM	CD55	TSPAN3
EPCAM	COMP	TUBB4A	CD81	EEF1A1	H3F3A/H3F3B	HIST2H3PS2
ITGA6	LGALS3BP	TUBB6	SLC3A2	TLN1	HIST2H3PS2	POTEJ
YWHAE	HIST1H2BJ	TUBB1	GNAI2	AHCYL2	PDCD6IP	DPP4
HSPA1A/	GAPDH	HSPA8	GNAI3	AHCYL1	ITGB1	NT5E
HSPA1B
GNB1	C7	CD9	GNAI1	EEF1A2	POTEJ	EPCAM
SLC3A2	TUBA4A	CD81	ATP1A1	TUBA4A	SERINC5	VNN1
GNB2	PGK1	GAPDH	HIST2H2AC	PFN1	H2AFZ	H2AFZ
EHD2	HSP90AB1	PFN1	CPNE8	TUBA1A	ARRDC1	ITGB1
H2AFX	TUBB	HIST1H4A	IST1	HIST1H2BK	CLDN3	ALPPL2
PPIA	TUBB2A	HSP90AA1	PFN1	PRSS23	NT5E	HIST2H2AB
NT5E	TUBB4B	HSP90AB1	TUBA4A	HSP90AB4P	EPCAM	ATP1A1
VPS4B	HIST2H2AB	HSPA2	H2AFX	HIST1H2BJ	CDH17	ALPP
GNB4	H2AFZ	HIST2H3A	TUBA1C	TUBB4B	ATP1A1	IST1
Cdc42	FBLN1	PGK1	HSPA5	TUBB	ALPPL2	PDCD6IP
SLC1A5	EEF1A1	THBS2	YWHAZ	ASS1	HIST2H2AB	MUC13
GNAI2	TLN1	EEF1A1	ENO1	PGK1	ALPP	ANXA11
CFL1	HIST1H4A	GPX3	ANXA5	HSPAB	HSPA2	HSPA2
YWHAH	HSPA8	ITGB1	GNAS	ACO1	TSPAN8	CDH17
EEF1A1	TUBB4A	PPIA	DNAJA1	GAPDH	MVP	GPA33
YWHAB	GPX3	PDCD6IP	CHMP5	TUBB2A	ADAM10	ANXA2
Hist1h3b	PKM	EEF1A2	EEF1A1	TUBB4A	THBS1	S100A6
TSG101	F10	FBLN1	RHOA	THBS2	VNN1	ATP1A2
YWHAG	THBS2	ATIC	KRT1	H3F3A/H3F3B	ITGAV	PPIA
ANXA5	RAN	CPNE8	CEP55	APOM	IGSF8	EGFR
GNAI3	TUBB6	TLN1	GNB1	HIST2H3PS2	MYOF	TSPANB
F5	GSTP1	HSPA5	ACTBL2	AKR1A1	ATP1A2	MYOF
H3F3A/H3F3B	PYGL	PKM	ITGA2	TUBB6	AHCY	GNAI1
CHMP4B	EEF1A2	HIST1H1C	EPHA2	TUBB3	GSN	GNAI2
HSPA5	ASS1	WDR1	GNA13	RAN	TSPAN1	GNAI3
EZR	WDR1	RAN	PPIA	CAP1	PPIA	S100A4
GAPDH	FLNA	PYGL	RAP1A	F10	SDCBP2	CLDN3
CD81	HIST2H3A	ITGA3	CD59	PPIA	HSPA5	A2M

Proteomics analysis of lipoprotein particles are shown in Table 2.

TABLE 2

Accession	Gene Symbol	Gene Name	HDL	LDL	VLDL

P02768	ALB	Serum albumin	1.114E8	1.714E7	1.365E6
P02760	AMBP	Protein AMBP	5.229E6		4.802E7
P02647	APOA1	Apolipoprotein A-II	1.251E11	1.046E8	4.111E8
P02652	APOA2	Apolipoprotein A-II	6.485E10	8.811E7	3.404E8
P06727	APOA4	Apolipoprotein A-IV	7.529E8	1.501E7	3.977E7
A0A087WTM7	APOB	Apolipoprotein B-100	7.138E6	2.401E9	1.118E10
P04114	APOB	Apolipoprotein B-100	7.138E6	2.401E9	1.118E10
K7ERI9	APOC1	Truncated apolipoprotein C-1 (Fragment)	1.144E10	2.412E8	9.354E9
P02656	APOC3	Apolipoprotein C-III	1.545E10	1.204E9	4.632E10
K7ER74	APOC4	Apolipoprotein C-IV	8.593E9	2.013E8	2.683E10
P55056	APOC4	Apolipoprotein C-IV	1.389E6	3.439E6	1.013E9
P05090	APDD	Apolipoprotein D	8.067E9	2.328E8	1.093E9
P02649	APOE	Apolipoprotein E	6.179E8	6.243E7	1.026E10
Q13790	APOF	Apolipoprotein F	5.347E8	1.056E7
P02749	APOH	Beta-2-glycoprotein 1			3.014E7
O14791	APOL1	Apolipoprotein L1	7.094E8	8.268E6	2.637E7
O95445	APOM	Apolipoprotein M	3.924E9	3.639E7	2.070E8
P61769	B2M	Bets-2-microglobulin	6.130E6		2.207E7
P01024	C3	Complement C3	2.058E8	2.987E7	7.496E7
B0UZ83	C4A	Complement C4 beta chain	1.006E8		5.074E7
F5GXS0	C4B	C4b-B	8.376E7		5.074E7
P49913	CAMP	Cathelicidin antimicrobial peptide	2.654E7		1.211E8
Q92496	CFHR4	Complement factor H-related protein 4			1.383E8
P10909	CLU	Clusterin	4.354E7		1.295E8
P02671	FGA	Fibrinogen alpha chain		5.793E6	2.376E7
A0A087WU08	HP	Haptoglobin	2.265E6		6.358E6
A0A075B6H6	IGKC	Ig kappa chain C region (Fragment)	1.104E7		2.19457
P60985	KRTDAP	Keratinocyte differentiation-associated protein	8.309E6		5.140E6
P04180	LCAT	Phosphatidylcholine-sterol acyltransferase	2.616E7
P08519	LPA	Apolipoprotein(a)		9.171E6	6.704E7
Q9UHG3	PCYOX1	Prenylcysteine oxidase 1	3.599E7	6.826E6	1.179E8
Q13093	PLA2G7	Platelet-activating factor acetylhydrolase	3.283E6		3.818E7
P55058	PLTP	Phospholipid transfer protein	2.825E7
P27169	PON1	Serum paraoxonase/arylesterase 1	8.966E8	1.531E6	9.747E7
Q15166	PON3	Serum paraoxonase/lactonase 3	3.961E8		1.029E8
P0DJI8	SAA1	Serum amyloid A-1 protein	2.726E9	1.030E7	1.102E7
P0DJI9	SAA2	Serum amyloid A-2 protein	9.455E8	2.518E6	6.650E6
A0A096LPE2	SAA2-SAA4	Protein SAA2-SAA4	1.620E10	4.256E7	3.075E9
P01009	SERPINA1	Alpha-1-antitrypsin	1.175E9	2.892E6	1.536E7
P02766	TTR	Transthyretin	4.141E6		9.975E6
P04304	VTN	Vitronectin	1.668E7		1.276E7

Gene set enrichment analysis (GSEA) of proteins associated with exomeres, Exo-S and Exo-L derived from various cancer cell lines are shown in Tables 3-5.

TABLE 3

GSEA for Exomeres.

					Fdr Q
					Value
				P	(Cutoff <
Name	Size	Es	Nes	Value	0.05)

Hallmark_Mtorc1_Signaling	105	0.45	2.60	<0.001	<0.001
Hallmark_Glycolysis	90	0.44	2.46	<0.001	<0.001
Hallmark_Fatty_Acid_Metabolism	53	0.43	2.11	<0.001	0.002
Hallmark_Myc_Targets_VI	114	0.36	2.18	<0.001	0.002
Hallmark_Xenobiotic_Metabolism	75	0.39	2.12	<0.001	0.002
Hallmark_Hypoxia	75	0.37	1.99	<0.001	0.004
Hallmark_Unfolded_Protein_Response	32	0.42	1.86	0.011	0.010
Hallmark_Adipogenesis	48	0.36	1.81	0.003	0.012
Hallmark_Coagulation	62	0.34	1.78	0.003	0.013
Hallmark_Bile_Acid_Metabolism	23	6.42	1.67	0.023	0.025
Hallmark_Reactive_Oxigen_Species_Pathway	24	0.39	1.60	0.032	0.037
Kegg_Glycolysis_Gluconeogenesis	32	0.72	3.22	<0.001	<0.001
Kegg_Amino_Sugar_And_Nucleotide_Sugar_Metabolism	23	0.77	3.12	<0.001	<0.001
Kegg_Aminoacyl_Trna_Biosynthesis	20	0.73	2.80	<0.001	<0.001
Kegg_Fructose_And_Mannose_Metabolism	16	0.78	2.78	<0.001	<0.001
Kegg_Pentose_Phosphate_Pathway	18	0.74	2.67	<0.001	<0.001
Kegg_Starch_And_Sucrose_Metabolism	18	0.69	2.58	<0.001	<0.001
Kegg_Proteasome	28	0.59	2.48	<0.001	<0.001
Kegg_Drug_Metabolism_Cytochrome_P450	17	0.66	2.36	0.003	0.000
Kegg_Glutathione_Metabolism	29	0.56	2.36	<0.001	0.000
Kegg_Metabolism_Of_Xenobiotics_By_Cytochrome_P450	20	0.57	2.13	0.003	0.002
Kegg_Cysteine_And_Methionine_Metabolism	15	0.58	2.07	<0.001	0.002
Kegg_Purine_Metabolism	40	0.40	1.93	0.003	0.007
Kegg_Tyrosine_Metabolism	15	0.52	1.83	0.003	0.013
Kegg_Complement_And_Coagulation_Cascades	31	0.39	1.69	0.018	0.030
Go_Organic_Acid_Metabolic_Process	252	0.52	3.52	<0.001	<0.001
Go_Cellular_Amino_Acid_Metabolic_Process	100	0.58	3.36	<0.001	<0.001
Go_Adp_Metabolic_Process	22	0.83	3.34	<0.001	<0.001
Go_Oxidation_Reduction_Process	171	0.51	3.33	<0.001	<0.001
Go_Coenzyme_Metabolic_Process	82	0.60	3.33	<0.001	<0.001
Go_Nucleotide_Phosphorylation	26	0.77	3.31	<0.001	<0.001
Go_Oxidoreduction_Coenzyme_Metabolic_Process	44	0.69	3.30	<0.001	<0.001
Go_Cofactor_Metabolic_Process	98	0.58	3.28	<0.001	<0.001
Go_Cofactor_Binding	54	0.63	3.27	<0.001	<0.001
Go_Carbohydrate_Catabolic_Process	44	0.67	3.23	<0.001	<0.001
Go_Atp_Generation_From_Adp	21	0.83	3.19	<0.001	<0.001
Go_Generation_Of_Precursor_Metabolites_And_Energy	60	0.61	3.18	<0.001	<0.001
Go_Monosaccharide_Biosynthetic_Process	25	0.76	3.11	<0.001	<0.001
Go_Hexose_Catabolic_Process	24	0.74	3.08	<0.001	<0.001
Go_Carbohydrate_Biosynthetic_Process	50	0.63	3.08	<0.001	<0.001
Go_Alpha_Amino_Acid_Metabolic_Process	67	0.58	3.08	<0.001	<0.001
Go_Cellular_Modified_Ammo_Acid_Metabolic_Process	76	0.56	3.07	<0.001	<0.001
Go_Monosaccharide_Metabolic_Process	59	0.58	3.03	<0.001	<0.001
Go_Proteasome_Accessory_Complex	21	0.75	3.01	<0.001	<0.001
Go_Pyruvate_Metabolic_Process	27	0.71	2.98	<0.001	<0.001
Go_Hexose_Metabolic_Process	48	0.62	2.97	<0.001	<0.001
Go_Nadh_Metabolic_Process	22	0.74	2.95	<0.001	<0.001
Go_Oxidoreductase_Activity	124	0.49	2.95	<0.001	<0.001
Go_Small_Molecule_Metabolic_Process	418	0.41	2.95	<0.001	<0.001
Go_Carbohydrate_Metabolic_Process	167	0.45	2.95	<0.001	<0.001
Go_Small_Molecule_Biosynthetic_Process	118	0.49	2.92	<0.001	<0.001
Go_Monosaccharide_Catabolic_Process	28	0.70	2.88	<0.001	<0.001
Go_Sulfur_Compound_Metabolic_Process	105	0.49	2.85	<0.001	<0.001
Go_Nucleobase_Containing_Small_Molecule_Metabolic_Process	152	0.45	2.85	<0.001	<0.001
Go_Ribonucleoside_Diphosphate_Metabolic_Process	31	0.63	2.85	<0.001	<0.001
Go_Dicarboxylic_Acid_Metabolic_Process	27	0.68	2.84	<0.001	<0.001
Go_Monocarboxylic_Acid_Metabolic_Process	114	0.48	2.82	<0.001	<0.001
Go_Amino_Acid_Activation	23	0.71	2.82	<0.001	<0.001
Go_Glucose_Metabolic_Process	37	0.60	2.79	<0.001	<0.001
Go_Ligase_Activity_Forming_Carbon_Oxygen_Bonds	20	0.73	2.79	<0.001	<0.001
Go_Glutathione_Metabolic_Process	29	0.65	2.79	<0.001	<0.001
Go_Coenzyme_Binding	35	0.61	2.78	<0.001	<0.001
Go_Small_Molecule_Catabolic_Process	71	0.53	2.77	<0.001	<0.001
Go_Serine_Family_Amino_Acid_Metabolic_Process	17	0.75	2.75	<0.001	<0.001
Go_Tansferase_Activity_Trans-	25	0.66	2.75	<0.001	<0.001
ferring_Alkyl_Or_Aryl_Other_Than_Methyl_Groups
Go_Nucleoside_Monophosphate_Metabolic_Process	69	0.52	2.74	<0.001	<0.001
Go_Nad_Binding	20	0.71	2.73	<0.001	<0.001
Go_Nad_Metabolic_Process	28	0.65	2.73	<0.001	<0.001
Go_Cellular_Carbohydrate_Metabolic_Process	49	0.55	2.73	<0.001	<0.001
Go_Purine_Containing_Compound_Metabolic_Process	102	0.47	2.68	<0.001	<0.001
Go_Glucose_Catabolic_Process	17	0.76	2.68	<0.001	<0.001
Go_Organic_Acid_Biosynthetic_Process	68	0.52	2.68	<0.001	<0.001
Go_Oxidoreductase_Activity_Act-	38	0.58	2.67	<0.001	<0.001
ing_On_The_Ch_Oh_Group_Of_Do-
nors_Nad_Or_Nadp_As_Acceptor
Go_Oxidoreductase_Activity_Act-	40	0.58	2.67	<0.001	<0.001
ing_On_Ch_Oh_Group_Of_Donors
Go_Glutathione_Transferase_Activity	15	0.77	2.66	<0.001	<0.001
Go_Carbohydrate_Derivative_Metabolic_Process	267	0.39	2.65	<0.001	<0.001
Go_Trna_Metabolic_Process	25	0.65	2.65	<0.001	<0.001
Go_Sulfur_Compound_Biosynthetic_Process	61	0.51	2.62	<0.001	<0.001
Go_Carbohydrate_Binding	68	0.50	2.61	<0.001	<0.001
Go_Lyase_Activity	39	0.56	2.61	<0.001	<0.001
Go_Organic_Acid_Catabolic_Process	27	0.61	2.60	<0.001	<0.001
Go_Alpha_Amino_Acid_Biosynthetic_Process	25	0.63	2.60	<0.001	<0.001
Go_Nucleoside_Diphosphate_Metabolic_Process	37	0.57	2.59	<0.001	<0.001
Go_Monosaccharide_Binding	29	0.58	2.55	<0.001	0.000
Go_Single_Organism_Catabolic_Process	225	0.38	2.57	<0.001	0.000
Go_Nucleotide_Sugar_Metabolic_Process	16	0.73	2.58	<0.001	0.000
Go_Glycosyl_Compound_Metabolic_Process	104	0.43	2.51	<0.001	0.000
Go_Polysaccharide_Metabolic_Process	31	0.58	2.52	<0.001	0.000
Go_Energy_Derivation_By_Oxidation_Of_Organic_Compounds	38	0.54	2.52	<0.001	0.000
Go_Cellular_Amino_Acid_Biosynthetic_Process	27	0.61	2.53	<0.001	0.000
Go_Cellular_Amide_Metabolic_Process	248	0.38	2.54	<0.001	0.000
Go_Nucleobase_Metabolic_Process	21	0.64	2.54	<0.001	0.000
Go_Cytosolic_Part	105	0.43	2.49	<0.001	0.000
Go_Endoplasmic_Reticulum_Lumen	47	0.51	2.49	<0.001	0.000
Go_Protein_Homotetramerization	17	0.68	2.49	<0.001	0.000
Go_Cellular_Aldehyde_Metabolic_Process	33	0.55	2.48	<0.001	0.000
Go_Alpha_Amino_Acid_Catabolic_Process	77	0.62	2.46	<0.001	0.000
Go_Iron_Ion_Binding	18	0.65	2.47	<0.001	0.000
Go_Cellular_Amino_Acid_Catabolic_Process	22	0.62	2.47	<0.001	0.000
Go_Single_Organism_Biosynthetic_Process	288	0.36	2.44	<0.001	0.000
Go_Oxidoreductase_Activity_Act-	18	0.65	2.45	<0.001	0.000
ing_On_The_Aldehyde_Or_Oxo_Group_Of_Donors
Go_Glutamine_Family_Amino_Acid_Metabolic_Process	20	0.63	2.46	<0.001	0.000
Go_Peptide_Metabolic_Process	203	0.36	2.38	<0.001	0.000
Go_Positive_Regulation_Of_Dna_Biosynthetic_Process	20	0.62	2.38	<0.001	0.000
Go_Transferase_Activity_Transferring_Hexosyl_Groups	39	0.52	2.36	<0.001	0.000
Go_Regulation_Of_Cellular_Amino_Acid_Metabolic_Process	35	0.52	2.35	<0.001	0.000
Go_Hydrolase_Activity_Hydrolyzing_O_Glycosyl_Compounds	26	0.56	2.34	<0.001	0.000
Go_Hydrolase_Activity_Acting_On_Glycosyl_Bonds	31	0.54	2.34	<0.001	0.001
Go_Protein_Activation_Cascade	27	0.55	2.34	<0.001	0.001
Go_Protein_Tetramerization	34	0.52	2.32	<0.001	0.001
Go_Organonitrogen_Compound_Biosynthetic_Process	309	0.34	2.32	<0.001	0.001
Go_Polysaccharide_Biosynthetic_Process	18	0.63	2.30	<0.001	0.001
Go_Organonitrogen_Compound_Catabolic_Process	90	0.40	2.29	<0.001	0.001
Go_Cellular_Carbohydrate_Biosynthetic_Process	24	0.55	2.28	<0.001	0.001
Go_Proteasome_Complex	43	0.48	2.28	<0.001	0.001
Go_Udp_Glycosyltransferase_Activity	29	0.53	2.28	<0.001	0.001
Go_Secretory_Granule_Lumen	25	0.57	2.28	<0.001	0.001
Go_Vesicle_Lumen	37	0.49	2.27	<0.001	0.001
Go_Sulfur_Amino_Acid_Metabolic_Process	17	0.63	2.26	<0.001	0.001
Go_Nucleoside_Triphosphate_Metabolic_Process	52	0.46	2.26	<0.001	0.001
Go_Maintenance_Of_Location	38	0.48	2.23	<0.001	0.001
Go_Purine_Containing_Compound_Biosynthetic_Process	40	0.47	2.23	<0.001	0.001
Go_Oxidoreductase_Activity_Acting_On_The_Alde-	15	0.64	2.22	<0.001	0.001
hyde_Or_Oxo_Group_Of_Donors_Nad_Or_Nadp_As_Acceptor
Go_Cellular_Modified_Amino_Acid_Biosynthetic_Process	22	0.57	2.22	<0.001	0.001
Go_Electron_Carrier_Activity	21	0.57	2.21	<0.001	0.001
Go_Regulation_Of_Telomere_Mainte-	17	0.61	2.20	<0.001	0.001
nance_Via_Telomere_Lengthening
Go_Erad_Pathway	15	0.63	2.19	<0.001	0.002
Go_Regulation_Of_Cellular_Amine_Metabolic_Process	42	0.46	2.17	<0.001	0.002
Go_Carbohydrate_Derivative_Biosynthetic_Process	154	0.34	2.17	<0.001	0.002
Go_Microtubule	88	0.38	2.16	<0.001	0.002
Go_Organophosphate_Metabolic_Process	221	0.32	2.14	<0.001	0.002
Go_Carboxylic_Ester_Hydrolase_Activity	25	0.52	2.12	<0.001	0.003
Go_Hydro_Lyase_Activity	15	0.60	2.11	<0.001	0.003
Go_Regulation_Of_Telomere_Maintenance	19	0.57	2.11	<0.001	0.003
Go_Carbon_Oxygen_Lyase_Activity	19	0.55	2.11	<0.001	0.003
Go_Glucan_Metabolic_Process	24	0.51	2.11	<0.001	0.003
Go_Posttranscriptional_Regulation_Of_Gene_Expression	143	0.34	2.09	<0.001	0.004
Go_Tansferase_Activity_Transferring_One_Carbon_Groups	19	0.55	2.09	<0.001	0.004
Go_Structural_Constituent_Of_Cytoskeleton	44	0.44	2.07	0.003	0.005
Go_Response_To_Endoplasmic_Reticulum_Stress	54	0.41	2.06	<0.001	0.005
Go_Nucleoside_Monophosphate_Biosynthetic_Process	30	0.48	2.06	0.003	0.005
Go_Regulation_Of_Cellular_Ketone_Metabolic_Process	46	0.42	2.05	0.003	0.006
Go_Antigen_Processing_And_Presentation_Of_Pep-	52	0.41	2.04	<0.001	0.006
tide_Antigen_Via_Mhc_Class_I
Go_Unfolded_Protein_Binding	47	0.41	2.03	<0.001	0.006
Go_Hydrolase_Activity_Acting_On_Carbon_Nitro-	23	0.51	2.02	0.006	0.007
gen_But_Not_Peptide_Bonds
Go_Nucleoside_Phosphate_Biosynthetic_Process	55	0.40	2.02	<0.001	0.007
Go_Serine_Hydrolase_Activity	50	0.40	2.01	0.003	0.007
Go_Cellular_Catabolic_Process	392	0.28	2.00	<0.001	0.008
Go_Purine_Nucleoside_Monophosphate_Biosynthetic_Process	21	0.50	1.99	0.003	0.008
Go_Monocarboxylic_Acid_Biosynthetic_Process	36	0.43	1.99	0.003	0.008
Go_Protein_Localization_To_Nucleus	35	0.44	1.99	0.003	0.008
Go_Aspartate_Family_Amino_Acid_Metabolic_Process	17	0.55	1.99	0.003	0.008
Go_T_Cell_Receptor_Signaling_Pathway	57	0.39	1.99	<0.001	0.008
Go_Amide_Biosynthetic_Process	174	0.31	1.98	<0.001	0.009
Go_Metalloexopeptidase_Activity	16	0.55	1.98	0.003	0.009
Go_Nucleotidyltransferase_Activity	15	0.55	1.98	0.005	0.009
Go_Exopeptidase_Activity	37	0.43	1.97	0.003	0.009
Go_Ligase_Activity_Forming_Carbon_Nitrogen_Bonds	17	0.52	1.94	0.014	0.011
Go_Nuclear_Export	27	0.47	1.94	<0.001	0.011
Go_Anaphase_Promoting_Complex_Dependent_Catabolic_Process	42	0.40	1.94	<0.001	0.011
Go_Positive_Regulation_Of_Canonical_Wnt_Signaling_Pathway	53	0.38	1.93	<0.001	0.012
Go_Regulation_Of_Dna_Biosynthetic_Process	26	0.45	1.92	0.006	0.013
Go_Coenzyme_Biosynthetic_Process	29	0.45	1.92	0.006	0.013
Go_Meiotic_Cell_Cycle	17	0.52	1.91	0.003	0.014
Go_Organic_Cyclic_Compound_Catabolic_Process	143	0.31	1.90	<0.001	0.015
Go_Blood_Microparticle	50	0.37	1.89	<0.001	0.016
Go_Aminopeptidase_Activity	18	0.51	1.89	0.003	0.016
Go_Antigen_Processing_And_Presentation_Of_Exoge-	39	0.42	1.89	<0.001	0.016
nous_Peptide_Antigen_Via_Mhc_Class_I
Go_Platelet_Alpha_Granule_Lumen	19	0.49	1.88	0.003	0.017
Go_Innate_Immune_Response_Activating_Cell_Sur-	53	0.37	1.88	<0.001	0.017
face_Receptor_Signaling_Pathway
Go_Energy_Reserve_Metabolic_Process	28	0.43	1.87	0.008	0.018
Go_Antigen_Receptor_Mediated_Signaling_Pathway	63	0.36	1.87	<0.001	0.018
Go_Cellular_Ketone_Metabolic_Process	18	0.49	1.87	0.011	0.018
Go_Transferase_Activity_Transferring_Glycosyl_Groups	55	0.38	1.87	<0.001	0.018
Go_Myelin_Sheath	99	0.32	1.86	<0.001	0.019
Go_Sarcoplasm	19	0.49	1.86	0.008	0.019
Go_Heparin_Binding	45	0.38	1.85	0.009	0.020
Go_Pyrimidme_Containing_Compound_Metabolic_Process	22	0.47	1.85	0.003	0.020
Go_Cofactor_Biosynthetic_Process	35	0.40	1.84	0.006	0.021
Go_Organic_Acid_Binding	56	0.36	1.84	<0.001	0.021
Go_Regulation_Of_Reproductive_Process	24	0.45	1.83	0.012	0.022
Go_Sulfur_Compound_Binding	63	0.35	1.83	<0.001	0.022
Go_Ribonucleoprotein_Complex_Localization	18	0.50	1.82	0.016	0.023
Go_Positive_Regulation_Of_Chromosome_Organization	30	0.42	1.82	0.003	0.023
Go_Extracellular_Matrix	126	0.30	1.83	<0.001	0.023
Go_Inclusion_Body	20	0.47	1.82	0.010	0.023
Go_Regulation_Of_Cellular_Amide_Metabolic_Process	97	0.32	1.82	<0.001	0.023
Go_Positive_Regulation_Of_Wnt_Signaling_Pathway	60	0.35	1.81	<0.001	0.024
Go_Positive_Regulation_Of_Dna_Replication	27	0.42	1.81	0.008	0.025
Go_Fc_Epsilon_Receptor_Signaling_Pathway	57	0.36	1.80	<0.001	0.027
Go_Hormone_Metabolic_Process	35	0.40	1.80	<0.001	0.027
Go_Actin_Filament_Binding	57	0.35	1.79	<0.001	0.027
Go_Cell_Redox_Homeostasis	18	0.47	1.79	0.020	0.028
Go_Nik_Nf_Kappab_Signaling	45	0.37	1.78	0.015	0.029
Go_Cellular_Lipid_Catabolic_Process	25	0.43	1.78	0.008	0.030
Go_Ncrna_Metabolic_Process	102	0.31	1.78	0.003	0.030
Go_Regulation_Of_Dna_Replication	36	0.39	1.77	0.012	0.031
Go_Isomerase_Activity	43	0.38	1.76	<0.001	0.032
Go_Sister_Chromatid_Cohesion	18	0.49	1.75	0.017	0.035
Go_Purine_Containing_Compound_Catabolic_Process	15	0.51	1.75	0.010	0.036
Go_Rna_Localization	33	0.39	1.74	0.014	0.037
Go_Extracellular_Matrix_Structural_Constituent	19	0.45	1.74	0.026	0.037
Go_Response_To_Topologically_Incorrect_Protein	43	0.37	1.74	0.015	0.037
Go_Negative_Regulation_Of_Cellular_Amide_Metabolic_Process	29	0.40	1.74	0.006	0.038
Go_Organic_Hydroxy_Compound_Metabolic_Process	102	0.29	1.73	<0.001	0.038
Go_Adenyl_Nucleotide_Binding	425	0.24	1.73	<0.001	0.040
Go_Protein_Serine_Threonine_Phosphatase_Activity	16	0.48	1.72	0.015	0.041
Go_Antigen_Processing_And_Presentation_Of_Peptide_Antigen	80	0.31	1.72	0.003	0.041
Go_Glycosyl_Compound_Biosynthetic_Process	35	0.38	1.72	0.020	0.042
Go_Mitochondrion	256	0.25	1.71	<0.001	0.044
Go_Proteoglycan_Metabolic_Process	18	0.46	1.71	0.011	0.044
Go_Supramolecular_Fiber	153	0.27	1.69	<0.001	0.048
Go_Translation_Factor_Activity_Rna_Binding	40	0.37	1.69	0.012	0.049

Es, Enrichment Score;
Nes, Normalized Enrichment Score;
Fdr, False Discovery Rate

TABLE 4

GSEA for Exo-S

					Fdr Q
				P	Value
Name	Size	Es	Nes	Value	(Cutoff < 0.05)

Hallmark_Apical_Surface	16	0.58	2.16	<0.001	0.005
Hallmark_Protein_Secretion	69	0.33	1.90	<0.001	0.023
Kegg_Ecm_Receptor_Interaction	47	0.52	2.73	<0.001	<0.001
Keg_Snare_Interactions_In_Vesicular_Transport	23	0.53	2.26	<0.001	0.004
Kegg_Small_Cell_Lung_Cancer	25	0.45	1.96	<0.001	0.024
Go_Intrinsic_Component_Of_Plasma_Membrane	383	0.36	2.75	<0.001	0.001
Go_Late_Endosome_Membrane	59	0.51	2.77	<0.001	0.001
Go_Endosomal_Part	214	0.36	2.58	<0.001	0.001
Go_Vacuolar_Membrane	293	0.33	2.49	<0.001	0.001
Go_Phagocytic_Vesicle_Membrane	32	0.54	2.46	<0.001	0.001
Go_Late_Endosome	106	0.40	2.50	<0.001	0.001
Go_Phagocytic_Vesicle	46	0.50	2.50	<0.001	0.001
Go_Organelle_Membrane_Fusion	39	0.50	2.46	<0.001	0.001
Go_Lytic_Vacuole_Membrane	139	0.38	2.51	<0.001	0.002
Go_Endosome	341	0.33	2.53	<0.001	0.002
Go_Extracellular_Structure_Organization	119	0.38	2.45	<0.001	0.002
Go_Secretory_Granule_Membrane	34	0.52	2.41	<0.001	0.002
Go_Single_Organism_Membrane_Fusion	44	0.46	2.39	<0.001	0.002
Go_Vacuolar_Part	332	0.31	2.39	<0.001	0.002
Go_Organelle_Fusion	46	0.45	2.37	<0.001	0.002
Go_Monovalent_Inorganic_Cation_Transport	64	0.41	2.36	<0.001	0.003
Go_Receptor_Activity	216	0.32	2.33	<0.001	0.004
Go_Phagosome_Maturation	23	0.57	2.31	<0.001	0.004
Go_Lipid_Transporter_Activity	25	0.54	2.31	<0.001	0.004
Go_Vacuolar_Transport	119	0.34	2.24	<0.001	0.007
Go_Atpase_Activity_Coupled_To_Movement_Of_Substances	49	0.43	2.25	<0.001	0.007
Go_Sterol_Homeostasis	24	0.52	2.23	<0.001	0.007
Go_Snap_Receptor_Activty	23	0.53	2.23	<0.001	0.008
Go_Regulation_Of_Membrane_Lipid_Distribution	16	0.62	2.21	<0.001	0.008
Go_Growth_Factor_Receptor_Binding	32	0.47	2.20	<0.001	0.009
Go_Phagosome_Acidification	15	0.59	2.17	<0.001	0.010
Go_Monovalent_Inorganc_Cation_Transmembrane_Transporter_Activity	53	0.40	2.17	<0.001	0.010
Go_Signaling_Receptor_Activity	160	0.32	2.18	<0.001	0.010
Go_Vacuole_Organization	71	0.39	2.18	<0.001	0.010
Go_active_Transmembrane_Transporter_Activity	93	0.35	2.14	<0.001	0.012
Go_Establishment_Of_Protein_Localization_To_Plasma_Membrane	51	0.40	2.12	0.003	0.014
Go_Sodium_Ion_Transport	30	0.45	2.10	<0.001	0.016
Go_Vacuole	491	0.25	2.08	<0.001	0.017
Go_Extracellular_Matrix_Disassembly	33	0.45	2.09	<0.001	0.017
Go_Establishment_Of_Protein_Localization_To_Vacuole	17	0.55	2.08	0.003	0.017
Go_Regulation_Of_Receptor_Mediated_Endocytosis	25	0.48	2.07	<0.001	0.018
Go_Movement_In_Environment_Of_Other_Organism_Involved_In_Symbiotic_Interaction	45	0.41	2.07	<0.001	0.018
Go_Cellular_Monovalent_Inorganic_Cation_Homeostasis	35	0.43	2.06	<0.001	0.018
Go_Protein_Localization_To_Vacuole	29	0.45	2.06	0.006	0.019
Go_Multivesicular_Body	19	0.52	2.04	0.003	0.022
Go_Monovalent_Inorganic_Cation_Homeostasis	36	0.42	2.04	<0.001	0.022
Go_Positive_Regulation_Of_Exocytosis	37	0.41	2.03	<0.001	0.023
Go_Endodermal_Cell_Differentiation	15	0.55	2.01	0.003	0.027
Go_Endocytic_Vesicle	110	0.31	2.00	<0.001	0.027
Go_Membrane_Fusion	52	0.36	1.98	0.003	0.029
Go_Gdp_Binding	37	0.40	1.99	<0.001	0.029
Go_Multivescular_Body_Organization	28	0.44	1.98	<0.001	0.030
Go_Positive_Regulation_Of_Cell_Cell_Adhesion	58	0.36	1.98	<0.001	0.030
Go_Regulation_Of_Muscle_Cell_Differentiation	33	0.43	1.97	0.006	0.031
Go_Protein_Localization_To_Cell_Periphery	66	0.35	1.97	<0.001	0.031
Go_Transmembrane_Receptor_Protein_Kinase_Activity	46	0.37	1.94	0.003	0.032
Go_Cation_Transmembrane_Transporter_Activity	104	0.31	1.95	<0.001	0.032
Go_Gastrulation	47	0.37	1.96	0.003	0.032
Go_Ph_Reduction	19	0.48	1.94	0.009	0.032
Go_Positive_Regulation_Of_Endocytosis	41	0.39	1.95	0.003	0.032
Go_Regulation_Of_Cellular_Response_To_Growth_Factor_Stimulus	72	0.33	1.94	<0.001	0.032
Go_Regulation_Of_Cellular_Ph	27	0.44	1.94	<0.001	0.032
Go_Lytic_Vacuole	231	0.27	1.95	<0.001	0.032
Go_Positive_Regulation_Of_Synaptic_Transmission	18	0.50	1.96	0.009	0.032
Go_Plasma_Membrane_Raft	43	0.38	1.96	0.007	0.032
Go_Snare_Complex	28	0.43	1.95	0.006	0.032
Go_Extracellular_Matrix_Component	49	0.37	1.93	<0.001	0.033
Go_Lytic_Vacuole_Organization	19	0.48	1.93	0.003	0.033
Go_Early_Endosome	129	0.29	1.92	<0.001	0.034
Go_Regulation_Of_Ph	28	0.43	1.91	<0.001	0.035
Go_Recycling_Endosome	57	0.35	1.91	<0.001	0.036
Go_Membrane_Protein_Complex	279	0.25	1.91	<0.001	0.036
Go_Cytokine_Receptor_Binding	45	0.36	1.89	<0.001	0.040
Go_Regulation_Of_Cellular_Response_To_Transforming_Growth_Factor_Beta_Stimulus	37	0.39	1.88	0.003	0.040
Go_Sh3_Domain_Binding	38	0.38	1.88	0.003	0.041
Go_Trivalent_Inorganic_Cation_Transport	24	0.44	1.87	0.015	0.043
Go_Regulation_Of_Toll_Like_Receptor_Signaling_Pathway	15	0.51	1.87	0.006	0.044
Go_Endosome_Organization	37	0.38	1.86	0.003	0.044
Go_Appendage_Development	30	0.41	1.86	0.009	0.044
Go_Escrt_Complex	22	0.46	1.86	0.003	0.044
Go_Virus_Receptor_Activity	34	0.40	1.85	0.006	0.046
Go_Lysosomal_Transport	25	0.42	1.85	0.006	0.048
Go_Lipid_Homeostasis	28	0.42	1.84	<0.001	0.048

Es, Enrichment Score;
Nes, Normalized Enrichment Score;
Fdr, False Discovery Rate

TABLE 5

GSEA for Exo-L.

					Fdr Q
				P	Value
Name	Size	Es	Nes	Value	(Cutoff < 0.25)

Hallmark_Mitotic_Spindle	72	0.40	1.87	<0.001	0.011
Hallmark_Uv_Response_Dn	55	0.41	1.83	0.002	0.014
Hallmark_112_Stat5_Signaling	64	0.41	1.89	<0.001	0.019
Hallmark_P53_Pathway	53	0.40	1.75	<0.001	0.026
Kegg_Long_Term_Depression	29	0.49	1.87	0.008	0.020
Kegg_Cell_Cycle	22	0.55	1.88	<0.001	0.022
Kegg_Regulation_Of_Actin_Cytoskeleton	110	0.37	1.94	0.001	0.023
Kegg_Endocytosis	105	0.39	2.05	<0.001	0.024
Kegg_Tight_Junction	61	0.44	1.98	<0.001	0.025
Kegg_Phosphatidylinositol_Signaling_System	18	0.57	1.88	0.002	0.025
Kegg_Melanogenesis	38	0.46	1.91	0.002	0.026
Kegg_Chemokine_Signaling_Pathway	65	0.39	1.78	0.003	0.037
Kegg_Snare_Interactions_In_Vesicular_Transport	23	0.50	1.74	0.006	0.048
Go_Multivesicular_Body_Organization	28	0.69	2.63	<0.001	<0.001
Go_Endosome_Organization	37	0.63	2.55	<0.001	<0.001
Go_Cell_Separation_After_Cytokinesis	16	0.77	2.51	<0.001	0.000
Go_Small_Gtpase_Mediated_Signal_Transduction	145	0.44	2.41	<0.001	0.000
Go_Multi_Organism_Organelle_Organization	21	0.70	2.41	<0.001	0.000
Go_Endomembrane_System_Organization	178	0.42	2.31	<0.001	0.000
Go_Ras_Protein_Signal_Transduction	64	0.50	2.29	<0.001	0.000
Go_Multi_Organism_Membrane_Organization	23	0.66	2.31	<0.001	0.001
Go_Metaphase_Plate_Congression	19	0.70	2.30	<0.001	0.001
Go_Escrt_Complex	22	0.69	2.45	<0.001	0.001
Go_Cytokinesis	36	0.58	2.33	<0.001	0.001
Go_Cytoplasmic_Side_Of_Membrane	70	0.48	2.26	<0.001	0.001
Go_Virion_Assembly	31	0.59	2.24	<0.001	0.001
Go_Intercalated_Disc	26	0.60	2.24	<0.001	0.001
Go_Mitotic_Sister_Chromatid_Segregation	28	0.59	2.22	<0.001	0.001
Go_Mitotic_Cytokinesis	20	0.65	2.21	<0.001	0.002
Go_Regulation_Of_Exosomal_Secretion	15	0.70	2.20	<0.001	0.002
Go_Signal_Release	39	0.54	2.20	<0.001	0.002
Go_Chromosome_Localization	21	0.62	2.17	<0.001	0.003
Go_Nucleus_Organization	37	0.53	2.17	<0.001	0.003
Go_Midbody	64	0.47	2.17	<0.001	0.003
Go_Neurotransmitter_Transport	37	0.53	2.15	<0.001	0.003
Go_Autophagy	115	0.41	2.15	<0.001	0.003
Go_Presynaptic_Process_Involved_In_Synaptic_Transmission	31	0.56	2.14	<0.001	0.003
Go_Macromolecular_Complex_Disassembly	36	0.54	2.14	<0.001	0.003
Go_Synaptic_Signaling	78	0.44	2.13	<0.001	0.003
Go_Protein_Domain_Specific_Binding	189	0.37	2.13	<0.001	0.003
Go_Cell_Cell_Contact_Zone	35	0.52	2.12	<0.001	0.004
Go_Extrinsic_Component_Of_Cytoplasmic_Side_Of_Plasma_Membrane	40	0.51	2.12	0.002	0.004
Go_Regulation_Of_I_Kappab_Kinase_Nf_Kappab_Signaling	62	0.46	2.11	<0.001	0.004
Go_Regulation_Of_Exocytosis	69	0.45	2.11	<0.001	0.004
Go_Cell_Division	123	0.40	2.10	<0.001	0.004
Go_Regulation_Of_Cytokinesis	19	0.62	2.09	<0.001	0.005
Go_Regulation_Of_Centrosome_Cycle	17	0.64	2.08	<0.001	0.005
Go_Basolateral_Plasma_Membrane	84	0.42	2.07	<0.001	0.006
Go_Apical_Junction_Complex	42	0.50	2.07	<0.001	0.006
Go_Cytoskeleton_Dependent_Cytokinesis	23	0.58	2.06	<0.001	0.006
Go_Regulation_Of_Cell_Division	62	0.44	2.04	<0.001	0.007
Go_Heterotrimeric_G_Protein_Complex	17	0.63	2.04	0.002	0.007
Go_Cell_Cell_Signaling	126	0.39	2.03	<0.001	0.008
Go_Calcium_Dependent_Phospholipid_Binding	19	0.60	2.02	0.002	0.009
Go_Regulation_Of_Centrosome_Duplication	15	0.65	2.00	<0.001	0.010
Go_Plasma_Membrane_Organization	95	0.40	2.00	<0.001	0.010
Go_Extrinsic_Component_Of_Plasma_Membrane	61	0.44	2.00	<0.001	0.011
Go_Positive_Regulation_Of_Viral_Process	29	0.52	1.98	0.002	0.013
Go_G_Protein_Coupled_Receptor_Signaling_Pathway_Cou-	21	0.58	1.98	<0.001	0.013
pled_To_Cyclic_Nucleotide_Second_Messenger
Go_Positive_Regulation_Of_Cell_Division	29	0.52	1.98	<0.001	0.013
Go_Regulation_Of_Ras_Protein_Signal_Transduction	52	0.45	1.97	<0.001	0.014
Go_Nucleoside_Triphosphatase_Regulator_Activity	82	0.40	1.97	0.002	0.014
Go_Positive_Regulation_Of_I_Kappab_Kinase_Nf_Kappab_Signaling	50	0.45	1.96	<0.001	0.015
Go_Amino_Acid_Transport	31	0.52	1.94	0.002	0.017
Go_Cell_Division_Site	24	0.55	1.95	<0.001	0.017
Go_Pdz_Domain_Binding	32	0.50	1.95	<0.001	0.018
Go_Cytoskeletal_Protein_Binding	272	0.33	1.94	<0.001	0.018
Go_Membrane_Budding	65	0.42	1.94	<0.001	0.018
Go_Filopodium	52	0.44	1.92	0.002	0.019
Go_Extrinsic_Component_Of_Membrane	95	0.39	1.92	<0.001	0.020
Go_Regulation_Of_Small_Gtpase_Mediated_Signal_Transduction	80	0.40	1.93	<0.001	0.020
Go_Organelle_Assembly	123	0.36	1.92	<0.001	0.020
Go_Late_Endosome_Membrane	59	0.42	1.93	0.002	0.020
Go_Positive_Regulation_Of_Cell_Cycle_Process	56	0.43	1.92	<0.001	0.021
Go_Sister_Chromatid_Segregation	40	0.46	1.91	0.002	0.021
Go_Rho_Guanyl_Nueleotide_Exchange_Factor_Activity	55	0.61	1.91	<0.001	0.021
Go_Intracellular_Signal_Transduction	426	0.31	1.90	<0.001	0.023
Go_G_Protein_Coupled_Receptor_Signaling_Pathway	100	0.37	1.90	<0.001	0.023
Go_Cell_Projection_Assembly	67	0.41	1.90	<0.001	0.023
Go_Side_Of_Membrane	148	0.35	1.89	<0.001	0.025
Go_Organelle_Localization	139	0.35	1.88	<0.001	0.025
Go_Leukocyte_Migration	79	0.40	1.89	<0.001	0.025
Go_Positive_Regulation_Of_Hydrolase_Activity	207	0.33	1.88	<0.001	0.025
Go_Postsynapse	89	0.38	1.88	0.002	0.025
Go_Synapse_Part	149	0.35	1.88	<0.001	0.025
Go_Plasma_Membrane_Protein_Complex	140	0.35	1.89	<0.001	0.025
Go_Adenylate_Cyclase_Modulating_G_Protein_Coupled_Receptor_Signaling_Pathway	20	0.57	1.88	<0.001	0.025
Go_Synapse	194	0.33	1.87	<0.001	0.027
Go_Regulation_Of_Nuclear_Division	37	0.46	1.87	<0.001	0.027
Go_Positive_Regulation_Of_Peptidyl_Serine_Phosphorylation	18	0.56	1.87	0.003	0.027
Go_Atpase_Activity_Coupled_To_Transmembrane_Movement_Of_Ions_Phosphory-	18	0.57	1.86	0.002	0.028
lative_Mechanism
Go_Regulation_Of_Gtpase_Activity	153	0.34	1.86	<0.001	0.028
Go_Metal_Ion_Transmembrane_Transporter_Activity	67	0.40	1.86	0.002	0.029
Go_Establishment_Of_Cell_Polarity	31	0.48	1.85	0.002	0.030
Go_Heat_Shock_Protein_Binding	30	0.49	1.85	0.002	0.031
Go_Protein_Localization_To_Cell_Periphery	66	0.40	1.84	0.002	0.031
Go_Ruffle	85	0.37	1.84	<0.001	0.031
Go_Snare_Complex	28	0.49	1.84	0.002	0.031
Go_Negative_Regulation_Of_Cellular_Protein_Localization	39	0.45	1.84	0.006	0.033
Go_Neuron_Spine	37	0.45	1.83	0.002	0.033
Go_Regulation_Of_Protein_Complex_Disassembly	70	0.38	1.83	<0.001	0.033
Go_Cytoskeleton_Organization	262	0.31	1.83	<0.001	0.033
Go_Snare_Binding	47	0.42	1.83	<0.001	0.033
Go_Anchoring_Junction	328	0.30	1.83	<0.001	0.033
Go_Sodium_Ion_Transmembrane_Transporter_Activity	29	0.48	1.82	0.003	0.035
Go_Synaptic_Vesicle_Cycle	24	0.51	1.82	<0.001	0.035
Go_Cell_Junction	470	0.29	1.82	<0.001	0.035
Go_Vesicle_Organization	130	0.34	1.82	<0.001	0.035
Go_Vacuole_Organization	71	0.39	1.82	0.002	0.036
Go_Enzyme_Activator_Activity	112	0.35	1.81	0.001	0.037
Go_Regulation_Of_Organelle_Assembly	44	0.43	1.81	<0.001	0.038
Go_Membrane_Region	384	0.29	1.80	<0.001	0.040
Go_Negative_Regulation_Of_Dephosphorylation	15	0.57	1.80	0.005	0.040
Go_Trans_Golgi_Network_Transport_Vesicle	17	0.56	1.80	0.005	0.041
Go_Calcium_Dependent_Protein_Binding	30	0.46	1.79	0.002	0.042
Go_Regulation_Of_Rho_Protein_Signal_Transduction	31	0.46	1.79	0.002	0.042
Go_Cell_Leading_Edge	180	0.32	1.79	<0.001	0.042
Go_Myeloid_Cell_Homeostasis	23	0.50	1.78	0.005	0.048
Go_Regulation_Of_Tumor_Necrosis_Factor_Mediated_Signaling_Pathway	16	0.55	1.78	0.008	0.048

Es, Enrichment Score;
Nes, Normalized Enrichment Score;
Fdr, False Discovery Rate

Lipid classes identified in exomeres and exosome subsets derived from different cell lines (raw data and normalized data) are shown in Tables 6-8.
The values in the tale are relative signal response (signal's peak area count is normalized to sample weight and peak area count of the internal standard signal)

TABLE 6

B16-F10 Exomere	B16-F10 Exo-S	B16-F10 Exo-L

Exomere

Exo-S

Exo-L

Lipid

Fatty Acid

replicate

Lipid Ion

Lipid Group

Class

Chain

1

2

3

1

2

3

1

2

3

AcCa(14:0)+H	AcCa(14:0)+H	AcCa	(14:0)	126403	74344	58450	450142	480444	253736	817233	627321	575713
AcCa(16:0)+H	AcCa(16:0)+H	AcCa	(16:0)	371349	132864	127435	848312	949956	635288	1613560	1298747	1254263
AcCa(18:0)+H	AcCa(18:0)+H	AcCa	(18:0)	271015	122806	111390	815152	1342063	663069	1274337	1373484	1164727
AcCa(18:1)+H	AcCa(18:1)+H	AcCa	(18:1)	177867	148820	162353	488628	684148	419700	727711	1055935	632886
Cer(d18:1/10:0)+H	Cer(d28:1)+H	Cer	(d18:1/10:0)	3285931	2456489	3071965	2826972	2522661	2574269	3282319	2529012	2859390
Cer(d17:1/12:0)+H	Cer(d29:1)+H	Cer	(d17:1/12:0)	2136705	1658319	9710	2183085	2150140	16991	2086917	1905596	8847
Cer(d18:0/12:0)+H	Cer(d30:0)+H	Cer	(d18:0/12:0)	4662128	4717794	258831	5173465	4949391	308432	5318447	4537723	487676
Cer(d18:1/13:0)+H	Cer(d31:1)+H	Cer	(d18:1/13:0)	2061473	2314284	1781159	2389813	2394060	18082	2511543	1995862	12407
Cer(d18:1/14:0)+H	Cer(d32:1)+H	Cer	(d18:1/14:0)	16487509	17733378	11996128	27269296	20032631	24321417	24060587	19624829	17533611
Cer(d17:1/16:0)+H	Cer(d33:1)+H	Cer	(d17:1/16:0)	244972	145280	0	738922	511889	9284	404845	219652	11209
Cer(d18:1/16:0)+H	Cer(d34:1)+H	Cer	(d18:1/16:0)	1931662	2910272	592159	9687638	4994022	3596046	4689271	3395960	54370
Cer(d18:2/16:0)+H	Cer(d34:2)+H	Cer	(d18:2/16:0)	977946	515813	17723	5339763	3254669	219675	4266995	2334043	100282
Cer(d18:1/18:0)+H	Cer(d36:1)+H	Cer	(d18:1/18:0)	10496	8067	75944	181246	13750	344641	0	0	88007
Cer(d18:2/18:0)+H	Cer(d36:2)+H	Cer	(d18:2/18:0)	482498	373915	0	1612597	973300	43568	969446	468632	0
Cer(d18:2/22:0)+H	Cer(d40:2)+H	Cer	(d18:2/22:0)	103724	240980	0	236699	101293	11125	133378	55772	0
Cer(d18:1/24:1)+H	Cer(d42:2)+H	Cer	(d18:1/24:1)	89835	285185	75499	512209	187476	443205	262724	210714	112551
Cer(d18:2/24:0)+H	Cer(d42:2)+H	Cer	(d18:2/24:0)	21377	93184	7980	74370	140785	223620	50536	140395	215214
Cer(d18:2/24:1)+H	Cer(d42:3)+H	Cer	(d18:2/24:1)	586547	1726557	69366	1299561	635994	928491	700013	847586	439139
CerG1(d18:1/16:0)+H	CerG1(d34:1)+H	CerG1	(d18:1/16:0)	2132797	3004216	265833	6754715	5248635	651347	6085606	7925052	471225
CerG1(d18:2/16:0)+H	CerG1(d34:2)+H	CerG1	(d18:2/16:0)	68290	60670	0	322732	399905	7897	486115	632481	13728
CerG1(d42:2)+H	CerG1(d42:2)+H	CerG1	(d42:2)	10524	102212	32440	53143	11325	16572	14912	48875	0
CerG1(d42:3)+H	CerG1(d42:3)+H	CerG1	(d42:3)	131300	207192	0	207152	84636	8467	183967	151961	0
CerG2(d42:2)+H	CerG2(d42:2)+H	CerG2	(d42:2)	57300	162235	116293	35705	61052	46664	63418	54882	10286
CerG3(d34:1)+H	CerG3(d34:1)+H	CerG3	(d34:1)	0	0	0	0	0	0	0	0	0
CL(14:0/14:0/14:0/14:0)−H	CL(56:0)−H	CL	(14:0/14:0/14:0/14:0)	130991	219438	306512	167571	239006	404403	118811	273244	327125
CL(18:2/14:0/14:0/14:0)−H	CL(60:2)−H	CL	(18:2/14:0/14:0/14:0)	2672710	2676445	2708643	1805136	2788450	2750814	2682683	1866518	3229918
CL(63:3)−H	CL(63:3)−H	CL	(63:3)	17718077	24443433	32685664	19052791	38979323	32187596	19983783	21116809	25096959
cPA(18:0)−H	cPA(18:0)−H	cPA	(18:0)	0	0	0	55012	68477	69059	61342	117027	47556
DG(9:0/9:0)+NH4	DG(18:0)+NH4	DG	(9:0/9:0)	947852	155466	0	387446	240845	9824	322159	429779	13833
DG(10:0/10:0)+NH4	DG(20:0)+NH4	DG	(10:0/10:0)	306478	40445	0	145346	58658	0	186129	106451	0
DG(16:0/14:0)+NH4	DG(30:0)+NH4	DG	(16:0/14:0)	23130	492892	0	204445	204748	3103	130175	449852	0
DG(16:0/16:0)+NH4	DG(32:0)+NH4	DG	(16:0/16:0)	312394	3034249	65213	1134564	992674	2401	639777	1625717	273211
DG(18:0/16:0)+NH4	DG(34:0)+NH4	DG	(18:0/16:0)	414414	1717524	81035	898847	870506	16324	625271	1282433	33465
DG(16:0/18:1)+NH4	DG(34:1)+NH4	DG	(16:0/18:1)	249570	5066477	56636	952503	1496482	194460	703700	3431420	216575
DG(16:0/18:2)+NH4	DG(34:2)+NH4	DG	(16:0/18:2)	155579	3053493	52764	455631	705511	205165	573647	1999847	99975
DG(16:1/18:1)+NH4	DG(34:2)+NH4	DG	(16:1/18:1)	52935	821634	607008	606602	1224260	966471	510087	2108477	747255
DG(18:0/18:0)+NH4	DG(36:0)+NH4	DG	(18:0/18:0)	420808	483464	44835	493869	434736	26845	456878	602752	38767
DG(18:0/18:1)+NH4	DG(36:1)+NH4	DG	(18:0/18:1)	128946	994984	36401	358221	522720	169726	311026	1296730	97520
DG(18:1/18:1)+NH4	DG(36:2)+NH4	DG	(18:1/18:1)	371258	7033571	211640	2544291	4636620	954483	3523810	10142038	1024211
DG(18:0/20:4)+NH4	DG(38:4)+NH4	DG	(18:0/20:4)	51341	592569	0	3687436	8075822	379742	2484564	19634348	336286
LPA(16:0)−H	LPA(16:0)−H	LPA	(16:0)	22497	11628	5227	339398	380104	125253	473472	590477	218149
LPA(18:0)−H	LPA(18:0)−H	LPA	(18:0)	6067	15389	0	565585	664690	308216	751856	867404	354941
LPC(12:0)+H	LPC(12:0)+H	LPC	(12:0)	24826009	20995058	22215516	21792204	25883459	17184909	22080168	22679379	19563762
LPC(14:0)+H	LPC(14:0)+H	LPC	(14:0)	569880	382042	516804	2624194	2882195	2196724	4225292	3498731	2526682
LPC(15:0)+H	LPC(15:0)+H	LPC	(15:0)	197761	98604	105982	1131940	1442497	728764	1721614	1366126	979909
LPC(16:0)+H	LPC(16:0)+H	LPC	(16:0)	15828556	960967	8112646	87241945	118375088	64524003	131528490	14660735	86067477
LPC(16:0e)+H	LPC(16:0e)+H	LPC	(16:0e)	744785	349930	426467	5245638	7599868	4354204	7220750	7627865	5423455
LPC(16:0p)+H	LPC(16:0p)+H	LPC	(16:0p)	353470	95568	171801	2491768	2689720	1637153	2900592	2414546	2045586
LPC(16:1)+H	LPC(16:1)+H	LPC	(16:1)	280901	236419	341572	3367394	4859169	2381778	4343171	1967391	213444
LPC(17:0)+H	LPC(17:0)+H	LPC	(17:0)	647030	283200	306780	3784759	4389526	3249965	4225274	3911969	3897263
LPC(17:1)+H	LPC(17:1)+H	LPC	(17:1)	183377	168289	235204	641128	575440	249858	480601	379388	279067
LPC(18:0)+H	LPC(18:0)+H	LPC	(18:0)	1467413	856904	7242653	14028247	15991647	76847887	15435268	14024927	91960591
LPC(18:0e)+H	LPC(18:0e)+H	LPC	(18:0e)	357153	198069	193513	3017626	4339017	2074027	3502395	3365314	2386033
LPC(18:0p)+H	LPC(18:0p)+H	LPC	(18:0p)	240653	111201	168571	2529943	3891950	2081436	3033057	3016781	2337886
LPC(18:1)+H	LPC(18:1)+H	LPC	(18:1)	860980	408803	2199425	3995280	4396377	15853525	5050416	4586403	19619579
LPC(18:1p)+H	LPC(18:1p)+H	LPC	(18:1p)	30139	11829	0	379412	428169	38755	442946	310262	56709
LPC(18:3)+H	LPC(18:3)+H	LPC	(18:3)	612289	319665	593584	4110256	5583184	4216789	6334338	6122347	6072820
LPC(19:0)+H	LPC(19:0)+H	LPC	(19:0)	200029	108013	9599	2275904	2890343	1519032	2665408	2793374	1626281
LPC(19:1)+H	LPC(19:1)+H	LPC	(19:1)	88384	39189	13736	416823	715551	431350	1208577	584080	344782
LPC(20:0)+H	LPC(20:0)+H	LPC	(20:0)	607197	396865	403428	5726753	7985971	4099321	6209335	7234817	5152981
LPC(20:0e)+H	LPC(20:0e)+H	LPC	(20:0e)	634835	261588	9643	4931406	4772196	4124047	5174992	4586907	4079437
LPC(20:1)+H	LPC(20:1)+H	LPC	(20:1)	387845	189452	238530	3051230	3817347	2514088	3507684	3502342	3087582
LPC(20:2)+H	LPC(20:2)+H	LPC	(20:2)	36252	19249	15234	898457	912537	484067	1091403	789470	505463
LPC(20:3)+H	LPC(20:3)+H	LPC	(20:3)	491004	374138	408366	4823463	7201017	5170072	5206771	7586043	6895817
LPC(20:4)+H	LPC(20:4)+H	LPC	(20:4)	336922	135435	115338	1472907	1239627	607968	1194764	774417	499731
LPC(22:0)+H	LPC(22:0)+H	LPC	(22:0)	297854	293971	119072	2195548	3766079	97011	1560850	2891930	964677
LPC(22:3)+H	LPC(22:3)+H	LPC	(22:3)	0	0	0	108584	158539	0	259681	302983	225370
LPC(22:4)+H	LPC(22:4)+H	LPC	(22:4)	130051	71536	70972	757447	902950	355253	670269	564642	346774
LPC(22:5)+H	LPC(22:5)+H	LPC	(22:5)	289181	147999	101529	1393529	1193595	542897	1095475	852414	590041
LPC(22:6)+H	LPC(22:6)+H	LPC	(22:6)	282143	118195	109571	1509678	1099101	490193	1051577	654953	475235
LPC(24:0)+H	LPC(24:0)+H	LPC	(24:0)	269284	469608	98479	2031193	3748373	810926	1846605	2876090	683853
LPC(24:1)+H	LPC(24:1)+H	LPC	(24:1)	172249	195506	32267	1375894	3447011	49999	1521861	3181981	506378
LPC(26:1)+H	LPC(26:1)+H	LPC	(26:1)	180284	203029	267546	934476	1965024	869741	1107557	1536510	879935
LPC(28:0)+H	LPC(28:0)+H	LPC	(28:0)	281142	271278	152632	616403	519593	165653	1217321	725813	196125
LPE(16:0p)−H	LPE(16:0p)−H	LPE	(16:0p)	112766	50167	73845	1005794	858598	946677	1406618	1160918	1004722
LPE(18:0)−H	LPE(18:0)−H	LPE	(18:0)	3921	1952	1640	118233	107913	99044	161225	165661	97721
LPE(20:1)−H	LPE(20:1)−H	LPE	(20:1)	14764	2049	3055	167698	122170	114049	231657	194669	150107
LPE(20:4)−H	LPE(20:4)−H	LPE	(20:4)	65288	12725	20948	468527	289518	235996	373155	284721	250355
LPG(14:0)−H	LPG(14:0)−H	LPG	(14:0)	548800	382595	407893	795783	463367	358026	604048	492052	342052
LPG(16:0)−H	LPG(16:0)−H	LPG	(16:0)	22384	1817	3695	95584	97453	88169	150234	144409	94669
LPG(18:0)−H	LPG(18:0)−H	LPG	(18:0)	7108	5883	2154	145050	199730	118565	180290	279239	125173
LPI(16:0)−H	LPI(16:0)−H	LPI	(16:0)	13139	8286	12324	160039	189966	142107	243454	316147	195268
LPI(18:0)−H	LPI(18:0)−H	LPI	(18:0)	129537	106151	124225	867843	1230904	952013	1366778	1930014	1195908
LPI(18:1)−H	LPI(18:1)−H	LPI	(18:1)	72701	47431	61357	460751	514012	440566	566097	674850	626513
MG(14:0)+H	MG(14:0)+H	MG	(14:0)	375837	88177	35665	448878	402458	5718	372448	550789	6515
MG(16:0)+H	MG(16:0)+H	MG	(16:0)	50604436	13533642	239524	33457708	25341368	552953	44808270	44882937	1700412
MG(18:0)+H	MG(18:0)+H	MG	(18:0)	73255584	24991877	36894	49725813	29224771	121072	61152875	53425253	1679694
MG(18:1)+H	MG(18:1)+H	MG	(18:1)	544195	230230	28730	394439	311141	83987	583537	594474	89301
MG(18:2)+H	MG(18:2)+H	MG	(18:2)	4536076	1085419	10175	3024785	1303318	27108	3187788	2514511	117708
MG(20:0)+H	MG(20:0)+H	MG	(20:0)	603839	158129	0	278723	212540	0	352665	369659	0
PA(16:0/18:1)−H	PA(34:1)−H	PA	(16:0/18:1)	353279	1205140	767645	1083038	3038689	3051987	180867	3819385	3323318
PA(18:0/18:1)−H	PA(36:1)−H	PA	(18:0/18:1)	240583	545416	440200	917938	1190688	1413768	826834	1819319	1686643
PA(18:1/18:1)−H	PA(36:2)−H	PA	(18:1/18:1)	1146268	1346006	847163	870462	3412350	878946	3287797	1861009	1386024
PA(18:0/20:3)−H	PA(38:3)−H	PA	(18:0/20:3)	45040	305269	144750	32540	730895	353903	438562	1004844	522715
PA(18:0/20:4)−H	PA(38:4)−H	PA	(18:0/20:4)	1991604	1909483	752042	5211107	6353043	5173947	5216243	8408667	5567100
PC(16:1)+H	PC(16:1)+H	PC	(16:1)	711019	434839	562537	1292297	861033	891424	1090124	926343	1162390
PC(19:1)+H	PC(19:1)+H	PC	(19:1)	1103262	630501	684980	2531329	1712233	1404432	1883603	1278262	1261451
PC(19:3)+H	PC(19:3)+H	PC	(19:3)	140482	77031	94151	852857	1017233	660854	1057654	995593	611132
PC(22:0)+H	PC(22:0)+H	PC	(22:0)	1548077	1231441	1142048	1521914	1408181	1495404	1505859	1549520	1333985
PC(23:0)+H	PC(23:0)+H	PC	(23:0)	4548177	5231098	5636416	5055545	5205865	6244353	4873030	4752521	4671780
PC(14:0e/10:1)+H	PC(24:1e)+H	PC	(14:0e/10:1)	72125	212115	34016	1441291	2716842	105506	1380547	2972553	92509
PC(25:0)+H	PC(25:0)+H	PC	(25:0)	4682423	4220728	5093812	5029168	4921421	5523937	5163837	4899195	5012193
PC(26:0)+H	PC(26:0)+H	PC	(26:0)	9653915	10088128	9496506	12563166	12315442	11517050	12538366	12117458	10426953
PC(28:0)+H	PC(28:0)+H	PC	(28:0)	16142640	11250710	16890226	94512141	106893440	22203878	132708155	121460509	125699546
PC(28:1)+H	PC(28:1)+H	PC	(28:1)	949819	273215	600528	3358965	7404361	4706427	5146089	3037283	3884449
PC(29:0)+H	PC(29:0)+H	PC	(29:0)	3037216	2192472	3374639	16274102	12464795	21180937	23520211	17179205	23219766
PC(29:0e)+H	PC(29:0e)+H	PC	(29:0e)	500495	620882	20456	6004551	2453423	8616426	6489613	5032334	5676874
PC(11:0/18:1)+H	PC(29:1)+H	PC	(11:0/18:1)	709162	825698	1232231	2810267	3051219	8703188	3405470	2395242	8338689
PC(29:1)+H	PC(29:1)+H	PC	(29:1)	324087	372942	141198	1381426	1260238	952507	2420045	2388402	709915
PC(29:2)+H	PC(29:2)+H	PC	(29:2)	213308	195757	145685	1034052	1185309	619448	2054678	1895639	885229
PC(30:0)+H	PC(30:0)+H	PC	(16:0/14:0)	56691753	74998071	26091350	683027646	682017930	1255178435	881535926	616691128	193155930
PC(30:0e)+H	PC(30:0e)+H	PC	(30:0e)	4056224	7328194	6465647	28560737	27282829	63835289	25395469	27671702	32831475
PC(14:0p/16:0)+H	PC(30:0p)+H	PC	(14:0p/16:0)	629949	1061563	313243	5377894	5372113	2369887	4458921	4577410	1452818
PC(30:1)+H	PC(30:1)+H	PC	(16:1/14:0)	17130717	14079326	2801921	102492171	127551929	24616112	137777254	135604383	20610998
PC(30:1e)+H	PC(30:1e)+H	PC	(30:1e)	127004	106123	28357	532623	2368063	2282784	2994764	997185	2495350
PC(30:2)+H	PC(30:2)+H	PC	(30:2)	117858	51494	196119	1201953	2059520	1026838	1179636	1741418	884183
PC(30:3)+H	PC(30:3)+H	PC	(30:3)	478052	475953	130266	4145335	5388556	3875521	4350801	5055605	1524862
PC(31:0)+H	PC(31:0)+H	PC	(31:0)	5049251	6718203	7639633	42945618	31875039	65082122	47136067	37808345	49514068
PC(31:0e)+H	PC(31:0e)+H	PC	(31:0e)	304982	294402	1022945	446518	1492814	8392308	1738976	1983714	2628902
PC(31:0p)+H	PC(31:0p)+H	PC	(31:0p)	513233	308136	284850	5057611	4515500	6587243	5809117	4369020	5014597
PC(31:1)+H	PC(31:1)+H	PC	(31:1)	5485783	2847101	5842640	42743922	23288029	67963014	70199810	60046556	64138703
PC(31:2)+H	PC(31:2)+H	PC	(31:2)	5945625	3622633	1209331	24297871	19202789	9524323	40465789	29104789	10177023
PC(31:3)+H	PC(31:3)+H	PC	(31:3)	437194	451232	199097	1920300	2302406	1975122	1628067	1532974	1632507
PC(32:0)+H	PC(32:0)+H	PC	(16:0/16:0)	20914846	51875855	56025652	272222966	231440946	606142584	236810344	283458124	365355799
PC(32:0e)+H	PC(32:0e)+H	PC	(32:0e)	1678417	5491350	6736720	18836697	16249536	50019063	11061880	16511316	17616549
PC(32:1)+H	PC(32:1)+H	PC	(16:0/16:1)	381297293	292555198	257928169	2854649412	2788422049	3838279002	3906107098	3661791470	3307385068
PC(32:1e)+H	PC(32:1e)+H	PC	(16:0e/16:1)	29860775	32204686	25604326	179356767	190641317	283723830	173372115	179521106	182837168
PC(14:0p/18:1)+H	PC(32:1p)+H	PC	(14:0p/18:1)	4384602	4502370	3698127	32728425	32189364	43232252	22998160	15556946	23904388
PC(16:1/16:1)+H	PC(32:2)+H	PC	(16:1/16:1)	4364874	2633675	5055540	24904167	31306125	45184481	33133633	39017602	41647218
PC(18:1/14:1)+H	PC(32:2)+H	PC	(18:1/14:1)	4433024	1566012	1162020	14242256	12004576	5683688	19537697	10150760	4489765
PC(21:1/11:1)+H	PC(32:2)+H	PC	(21:1/11:1)	3317305	2457734	1751989	21068787	24306239	13490753	25989753	28806059	11913899
PC(32:2)+H	PC(32:2)+H	PC	(32:2)	853461	541674	133672	3198542	2183848	842944	4588838	2710601	1006746
PC(32:3)+H	PC(32:3)+H	PC	(32:3)	3880729	4838259	2174541	45142873	51367960	24333865	54761013	61607918	77516103
PC(33:0)+H	PC(33:0)+H	PC	(33:0)	2491221	4292191	2094083	9890003	8931707	21094550	17597727	11350499	11113297
PC(33:0e)+H	PC(33:0e)+H	PC	(33:0e)	179591	323333	127786	939808	767556	1925575	692899	578714	910127
PC(33:0p)+H	PC(33:0p)+H	PC	(33:0p)	5514046	5886622	3348197	29905909	26959389	46907116	25793887	20775585	24327880
PC(17:1/16:0)+H	PC(33:1)+H	PC	(17:1/16:0)	42531094	31192660	27351928	183770305	134000588	266593855	205479288	170692737	57118640
PC(33:1)+H	PC(33:1)+H	PC	(33:1)	2555311	1544070	330171	7479162	4246649	2664600	10271628	6775857	2096224
PC(33:2)+H	PC(33:2)+H	PC	(33:2)	24133640	13315080	8893252	58426262	32978893	3957620	94144054	51180448	63916967
PC(33:3)+H	PC(33:3)+H	PC	(33:3)	2890157	1836002	2189731	14117303	10932598	21356198	18716142	14843963	19206746
PC(33:5)+H	PC(33:5)+H	PC	(33:5)	166778	103006	0	885005	888997	130504	1375772	1181957	88887
PC(34:0)+H	PC(34:0)+H	PC	(18:0/16:0)	236340	1799346	2419061	6617209	5443136	19151215	2961868	6195521	7251254
PC(34:0e)+H	PC(34:0e)+H	PC	(34:0e)	32434	340654	688738	1323097	1158873	3721258	735292	1313962	1078602
PC(34:1)+H	PC(34:1)+H	PC	(16:0/18:1)	702164261	683856165	459756300	4090124888	3266409301	5915612417	5156552228	4423740802	4814401416
PC(16:1/18:1)+H	PC(34:2)+H	PC	(16:1/18:1)	95398188	53096879	102332012	1259473250	1426517920	1530374649	1586240129	1288377681	1251331204
PC(34:2e)+H	PC(34:2e)+H	PC	(34:2e)	13437906	12654633	10113848	86846844	92800877	131845330	78230081	78466379	78768625
PC(16:1p/18:1)+H	PC(34:2p)+H	PC	(16:1p/18:1)	2886633	2301135	2578631	18247260	15908218	21615372	12458478	10544133	12531595
PC(12:0/22:3)+H	PC(34:3)+H	PC	(12:0/22:3)	2468111	1650994	1285424	17210347	16231266	18500053	20808370	18756574	16911357
PC(16:1/18:2)+H	PC(34:3)+H	PC	(16:1/18:2)	3827115	2184772	2443733	24237687	21051078	23434834	26957441	23196963	19148407
PC(34:3)+H	PC(34:3)+H	PC	(34:3)	1229610	2883678	4153532	13447228	14653650	44646699	12392809	16525324	3647163
PC(34:3p)+H	PC(34:3p)+H	PC	(34:3p)	309194	1462040	999891	8502590	9797227	18800853	7779018	10339160	11746995
PC(34:4)+H	PC(34:4)+H	PC	(34:4)	20475903	16110930	17293334	178336632	207525148	346713684	66644722	129599970	355127450
PC(34:4p)+H	PC(34:4p)+H	PC	(34:4p)	113809	73055	14857	1300014	976116	165784	1122693	710962	25528
PC(35:0)+H	PC(35:0)+H	PC	(35:0)	16436	82270	0	504377	182394	594817	575443	395473	307532
PC(35:0p)+H	PC(35:0p)+H	PC	(35:0p)	1632829	1320223	587682	3109538	4330640	5998432	4098800	3798103	3825167
PC(19:1/16:0)+H	PC(35:1)+H	PC	(19:1/16:0)	10484048	9928273	10530183	41897018	27918903	104647960	46798441	33327744	72186646
PC(17:0/18:1)+H	PC(35:1)+H	PC	(17:0/18:1)	5988186	3990995	6267876	28487945	16672598	65506113	34058556	19802587	25971953
PC(35:1p)+H	PC(35:1p)+H	PC	(35:1p)	1406045	1075172	780889	6214976	5934505	8837847	5501205	5518786	4576235
PC(19:1/16:1)+H	PC(35:2)+H	PC	(19:1/16:1)	6284334	3958153	1938327	38909168	31351637	14302206	40489367	32418462	23110869
PC(24:1/11:1)+H	PC(35:2)+H	PC	(24:1/11:1)	11336192	5493494	3466525	60657254	43008092	45664196	56058697	39110045	37049797
PC(35:2)+H	PC(35:2)+H	PC	(35:2)	270421	275264	399753	434132	342684	1731724	513537	324179	1291443
PC(35:2p)+H	PC(35:2p)+H	PC	(35:2p)	576576	405357	85747	2841268	4165956	6393422	2198415	3444787	394399
PC(35:3)+H	PC(35:3)+H	PC	(35:3)	890824	501653	134820	7341169	5110940	3697459	6873364	3882500	2474602
PC(35:4)+H	PC(35:4)+H	PC	(35:4)	4495931	3539087	1677683	14508832	9745045	13337298	19081414	13281655	11465781
PC(35:5)+H	PC(35:5)+H	PC	(35:5)	6177999	3293915	714103	32574085	24526286	2758482	44623939	32556959	3676579
PC(35:6)+H	PC(35:6)+H	PC	(35:6)	1730356	1078550	986403	12240209	8969559	8085536	25142649	13256355	6341940
PC(36:1)+H	PC(36:1)+H	PC	(18:0/18:1)	57594289	89875958	35364313	223467745	155762078	372979351	214701069	199982778	234112087
PC(36:1e)+H	PC(36:1e)+H	PC	(36:1e)	1847007	6001714	1695860	8139694	7383334	18083551	6069833	7533265	6988582
PC(20:1p/16:0)+H	PC(36:1p)+H	PC	(20:1p/16:0)	13855149	15166017	9015476	71011723	59946900	96568675	58680699	49404053	53153592
PC(36:2)+H	PC(36:2)+H	PC	(18:1/18:1)	219418964	149657065	113577755	1071681085	698669045	1306180416	1589090062	1186492716	1321526178
PC(36:2e)+H	PC(36:2e)+H	PC	(36:2e)	825058	2288822	294483	8515582	3268628	7341682	3029268	4351222	3631295
PC(18:2p/18:0)+H	PC(36:2p)+H	PC	(18:2p/18:0)	2449211	2105691	1581612	16482738	20454763	22202405	12080750	11324342	11224758
PC(36:2p)+H	PC(36:2p)+H	PC	(36:2p)	6876459	6830048	3760873	33276198	32117954	48103421	24897739	23696285	23387299
PC(36:3)+H	PC(36:3)+H	PC	(16:0/20:3)	19311332	10605539	18324108	149395954	99927597	203371700	148867851	105689176	144632970
PC(18:2p/18:1)+H	PC(36:3p)+H	PC	(18:2p/18:1)	9411864	7043705	8995220	65432592	64555739	34393429	46639296	42312142	59873318
PC(36:4)+H	PC(36:4)+H	PC	(16:0/20:4)	113125173	61796800	65443777	970392843	693468583	1084594991	833128331	554307442	579110379
PC(36:4e)+H	PC(36:4e)+H	PC	(36:4e)	2874252	5227401	2853530	13162899	13730432	35503097	8762435	10529267	15277003
PC(36:4p)+H	PC(36:4p)+H	PC	(36:4p)	429555	226608	186510	3247187	2450934	1938168	2500553	1822372	778538
PC(18:4/18:1)+H	PC(36:5)+H	PC	(18:4/18:1)	3674145	2141956	5301653	32576186	30079311	142867872	40885853	36775111	110522230
PC(36:5)+H	PC(36:5)+H	PC	(36:5)	3528133	4889012	6301483	43142563	43544554	154900750	54354927	51833930	11233573
PC(16:0e/20:5)+H	PC(36:5e)+H	PC	(16:0e/20:5)	9308764	6840060	6189331	75709670	68192259	88245724	48894220	37579300	41683517
PC(36:5p)+H	PC(36:5p)+H	PC	(36:5p)	300012	145095	200932	2676755	2129820	2012616	2121500	1438064	1139635
PC(36:6)+H	PC(36:6)+H	PC	(36:6)	278220	120514	175270	1752752	1469996	26512	2222469	1627227	207469
PC(36:6p)+H	PC(36:6p)+H	PC	(36:6p)	713070	281961	16664	4485149	4233808	256022	3465606	2856681	4331
PC(37:1)+H	PC(37:1)+H	PC	(37:1)	635507	1068698	248544	2118291	1839614	4592553	3160798	2464688	2145896
PC(37:2)+H	PC(37:2)+H	PC	(37:2)	5342789	4118992	2523957	18907786	12841693	24974380	22033493	15068689	17763612
PC(37:3)+H	PC(37:3)+H	PC	(37:3)	2127614	1681616	213678	11875476	7635188	4086475	10217137	6773494	2316787
PC(37:4)+H	PC(37:4)+H	PC	(37:4)	3885530	2134865	267676	22000044	14351369	3687400	17928177	10324880	2300350
PC(15:0/22:5)+H	PC(37:5)+H	PC	(15:0/22:5)	1610591	786405	319731	10709917	7968334	4210574	8464698	5426397	2278810
PC(37:5)+H	PC(37:5)+H	PC	(37:5)	989292	736530	12639	2600731	1474460	76215	3896763	3172358	49238
PC(37:6)+H	PC(37:6)+H	PC	(37:6)	1445085	1130607	687467	6992369	4823141	3961830	9639965	10234779	1459770
PC(38:1)+H	PC(38:1)+H	PC	(38:1)	1232660	2539576	946684	3607588	2224295	6353098	3320273	3067032	2726034
PC(16:0/22:2)+H	PC(38:2)+H	PC	(16:0/22:2)	13294815	12628607	5409361	41586519	28925428	62622778	52094191	40718260	45362110
PC(38:2e)+H	PC(38:2e)+H	PC	(38:2e)	229490	1436307	225156	887315	840060	3813085	2284921	1804058	1155137
PC(28:1/10:2)+H	PC(38:3)+H	PC	(28:1/10:2)	4842513	4871395	2342254	17875436	12136912	24497522	16650719	13501242	15299840
PC(18:0/20:3)+H	PC(38:3)+H	PC	(18:0/20:3)	22234637	18139485	10100957	79821396	47675284	98004247	63535991	43335502	53327351
PC(38:3e)+H	PC(38:3e)+H	PC	(38:3e)	430882	1130273	1348078	3737602	2261771	6064759	2587927	1215354	2914242
PC(24:0/14:4)+H	PC(38:4)+H	PC	(24:0/14:4)	13612374	9039205	2249372	91093010	74001720	28057296	76972831	51227131	15860636
PC(18:0/20:4)+H	PC(38:4)+H	PC	(18:0/20:4)	95037046	63141328	41694806	563602124	335891571	651523426	469371087	261533147	339631844
PC(38:4e)+H	PC(38:4e)+H	PC	(38:4e)	7490326	7804870	1804445	42622033	42789404	30023761	27705185	26192846	9812738
PC(38:4p)+H	PC(38:4p)+H	PC	(38:4p)	8854264	6385455	5170890	61602988	60679872	88333270	47928163	34973778	40139805
PC(18:1/20:4)+H	PC(38:5)+H	PC	(18:1/20:4)	123488251	67188448	49637662	1026829551	699701692	603445846	785955557	494762868	328329122
PC(16:0/22:6)+H	PC(38:6)+H	PC	(16:0/22:6)	316250	205733	36226	3975732	3952772	1218213	4854762	4271139	1408910
PC(18:1/20:5)+H	PC(38:6)+H	PC	(18:1/20:5)	45908597	23103640	27490795	377976992	216516974	306444819	241072102	151122766	163755763
PC(38:6)+H	PC(38:6)+H	PC	(38:6)	1136205	717162	244153	12683883	10655358	3742676	11676613	10470000	2703754
PC(38:6e)+H	PC(38:6e)+H	PC	(38:6e)	13361334	10387237	7623356	111905865	106674264	102556065	71620916	57715219	44168763
PC(38:6p)+H	PC(38:6p)+H	PC	(38:6p)	15483207	11921944	2986285	186474003	148946710	42082868	109651586	83889829	19230427
PC(38:7)+H	PC(38:7)+H	PC	(38:7)	6194027	2910349	3775199	57266682	41523027	76273809	50017867	32127604	47411750
PC(39:3)+H	PC(39:3)+H	PC	(39:3)	623402	546156	149834	2065681	1141695	2377725	1849315	1089868	1295349
PC(39:4)+H	PC(39:4)+H	PC	(39:4)	758049	583494	17751	2942978	1588346	615509	3026951	1468087	496479
PC(39:5)+H	PC(39:5)+H	PC	(39:5)	2461024	1436182	351073	14635835	9264798	3285667	10916171	6029141	1899999
PC(39:6)+H	PC(39:6)+H	PC	(39:6)	2084165	1100748	136833	15968364	9660793	1536896	12472067	7024924	1006954
PC(39:7)+H	PC(39:7)+H	PC	(39:7)	114469	81599	28040	1373730	1290344	566521	1018109	751688	296566
PC(40:1)+H	PC(40:1)+H	PC	(40:1)	19628	548649	10711	320505	144391	649078	336368	440931	306415
PC(40:2)+H	PC(40:2)+H	PC	(40:2)	1316565	1898315	483061	3032979	2104152	5781091	3826304	3613235	3786897
PC(40:3)+H	PC(40:3)+H	PC	(40:3)	977860	857262	521195	3088227	2227626	4794615	3579280	2717439	3029600
PC(40:3p)+H	PC(40:3p)+H	PC	(40:3p)	1011619	1294865	432318	3408918	3007870	4986580	1860751	1954303	1985437
PC(40:4)+H	PC(40:4)+H	PC	(40:4)	8046549	8004170	5328022	23143083	19299996	32926987	19313266	11617315	24852471
PC(40:5)+H	PC(40:5)+H	PC	(18:1/22:4)	35476101	23868537	17154310	202180672	121659424	235623419	170254214	97410757	123354126
PC(40:5e)+H	PC(40:5e)+H	PC	(40:5e)	2307410	2136195	1247130	12360839	10362062	13311635	6702988	5753036	5438160
PC(18:0/22:6)+H	PC(40:6)+H	PC	(18:0/22:6)	16693392	8173425	7854470	145791613	98490232	124072849	113194090	67292012	62729425
PC(20:3/20:3)+H	PC(40:6)+H	PC	(20:3/20:3)	38490349	22084869	18171055	248754570	155666768	299018660	196182329	102666052	138892105
PC(40:6e)+H	PC(40:6e)+H	PC	(40:6e)	6604220	3954315	2997578	32893458	36758887	34131553	22076761	20657338	13658008
PC(40:6p)+H	PC(40:6p)+H	PC	(40:6p)	6254447	5934471	3846468	45809985	41286808	51568294	26209415	20174539	18324035
PC(20:3/20:4)+H	PC(40:7)+H	PC	(20:3/20:4)	11268944	5665070	6826212	89085576	63804657	91042333	67185541	42362806	45131328
PC(40:7)+H	PC(40:7)+H	PC	(40:7)	625562	301078	474227	5913374	4597529	15169575	4476428	3589157	9559417
PC(40:7p)+H	PC(40:7p)+H	PC	(40:7p)	6945861	4196537	958797	68908268	59375419	17057378	40961551	30962137	224930
PC(40:8)+H	PC(40:8)+H	PC	(40:8)	695421	229585	154945	13027015	10686828	17832081	11433796	5046720	9792159
PC(40:9)+H	PC(40:9)+H	PC	(40:9)	243955	14010	18184	21171238	19778222	12056950	12062641	163603	147944
PC(41:5)+H	PC(41:5)+H	PC	(41:5)	274828	215707	8525	1210076	549552	214202	882757	448922	123176
PC(41:6)+H	PC(41:6)+H	PC	(41:6)	416032	195420	126235	2087186	1286584	2068692	1688521	788683	1128524
PC(41:7)+H	PC(41:7)+H	PC	(41:7)	462302	198785	106902	3976977	2204088	159245	2805904	1497294	16894
PC(42:1)+H	PC(42:1)+H	PC	(42:1)	21921	194041	63453	35201	9395	271505	62485	114660	79275
PC(42:10)+H	PC(42:10)+H	PC	(42:10)	370351	246264	19589	2660492	2327177	208777	1759486	1406162	78571
PC(42:2)+H	PC(42:2)+H	PC	(42:2)	253232	738559	8974	510969	361646	1299045	698072	820622	739271
PC(42:3p)+H	PC(42:3p)+H	PC	(42:3p)	363646	741983	78597	1257336	1010573	2117577	610797	536191	452085
PC(42:4p)+H	PC(42:4p)+H	PC	(42:4p)	2326778	2136343	859992	6337502	5303164	10823482	3644317	2929616	3074872
PC(42:6)+H	PC(42:6)+H	PC	(42:6)	697545	351692	13399	6651920	3672753	1500680	5047201	2678595	696402
PC(42:6e)+H	PC(42:6e)+H	PC	(42:6e)	3281320	3528394	1633277	10986222	9522431	15595214	6976105	5637816	5377179
PC(42:7)+H	PC(42:7)+H	PC	(42:7)	1170406	624085	59451	7819357	4605787	6286859	6271700	3287502	3667056
PC(42:7p)+H	PC(42:7p)+H	PC	(42:7p)	304595	143764	17459	2186988	2477986	2982157	937663	1154367	532011
PC(42:8)+H	PC(42:8)+H	PC	(42:8)	559523	158973	469871	2441322	2105502	596625	1789640	1016874	2671616
PC(42:9)+H	PC(42:9)+H	PC	(42:9)	285002	96473	91760	6320647	5106113	47424	4786814	2985749	620202
PC(44:1)+H	PC(44:1)+H	PC	(44:1)	0	0	0	0	0	0	0	0	0
PC(44:2)+H	PC(44:2)+H	PC	(44:2)	0	0	0	31147	0	34726	28312	14838	15622
PE(12:0/14:0)−H	PE(26:0)−H	PE	(12:0/14:0)	877320	765934	1107693	479419	961567	392491	284392	391284	776168
PE(26:0)−H	PE(26:0)−H	PE	(26:0)	10461181	10193378	10101341	11474936	10941789	11531491	12749953	11031927	11394620
PE(32:0p)−H	PE(32:0p)−H	PE	(32:0p)	20315	31271	113871	516675	436257	840690	345402	542576	787825
PE(16:0/16:1)−H	PE(32:1)−H	PE	(16:0/16:1)	1079702	641093	530955	4259541	3407279	4157685	6354298	6636938	7424439
PE(16:0p/16:1)−H	PE(32:1p)−H	PE	(16:0p/16:1)	1407803	664223	631263	6502003	4179502	4350051	6229817	6216071	5535087
PE(16:1/16:1)−H	PE(32:2)−H	PE	(16:1/16:1)	65384	33617	52703	248608	608883	532581	1362998	1044307	864687
PE(17:1/16:0)−H	PE(33:1)−H	PE	(17:1/16:0)	23662	10943	44465	261173	245512	53221	727329	348259	564830
PE(33:1p)−H	PE(33:1p)−H	PE	(33:1p)	284615	207250	119774	974402	649777	781100	1246718	730405	697463
PE(16:0/18:1)−H	PE(34:1)−H	PE	(16:0/18:1)	4670374	3364575	2608414	18480259	12617967	15319787	21791565	26672687	23939326
PE(16:0p/18:1)−H	PE(34:1p)−H	PE	(16:0p/18:1)	7933089	6776006	4301086	29675616	22313983	30783198	20610189	33048166	31675888
PE(16:1/18:1)−H	PE(34:2)−H	PE	(16:1/18:1)	833382	496698	474042	4411219	3883160	4468359	7423287	6579351	6241736
PE(18:1p/16:1)−H	PE(34:2p)−H	PE	(18:1p/16:1)	1128730	747798	516745	5468672	5833115	6912104	6954297	6583106	4628105
PE(16:0p/18:2)−H	PE(34:2p)−H	PE	(16:0p/18:2)	261859	945372	111451	1475239	1079665	988558	7723369	1038837	995103
PE(16:1/18:2)−H	PE(34:3)−H	PE	(16:1/18:2)	0	0	0	290695	348797	313300	595403	520002	403197
PE(16:0p/18:3)−H	PE(34:3p)−H	PE	(16:0p/18:3)	180812	113867	116422	940853	870734	1073238	1207176	1189320	1194719
PE(17:0/18:1)−H	PE(35:1)−H	PE	(17:0/18:1)	160428	104560	69353	558367	463948	554162	750101	631768	725432
PE(17:1/18:1)−H	PE(35:2)−H	PE	(17:1/18:1)	443278	136018	138961	1424928	1047325	1323660	2812265	2239564	1668603
PE(18:0/18:1)−H	PE(36:1)−H	PE	(18:0/18:1)	1421789	1446827	1034206	5399013	4417839	6643745	5813534	8997591	9114083
PE(36:1)−H	PE(36:1)−H	PE	(36:1)	415360	215231	86790	2628405	2225986	1850515	3310956	5264515	491949
PE(18:0p/18:1)−H	PE(36:1p)−H	PE	(18:0p/18:1)	1125675	1376566	837927	3805201	3763497	5404133	2843291	5172753	4825824
PE(18:1/18:1)−H	PE(36:2)−H	PE	(18:1/18:1)	7505580	720840	507799	4539759	3216573	19669952	47228634	36522494	37490323
PE(18:1p/18:1)−H	PE(36:2p)−H	PE	(18:1p/18:1)	4858022	3206616	2390188	19194067	15306858	18719127	17525117	19272819	17619921
PE(16:0/20:3)−H	PE(36:3)−H	PE	(16:0/20:3)	184100	72617	72449	1339098	891690	1260412	1695811	948950	1924390
PE(18:1/18:2)−H	PE(36:3)−H	PE	(18:1/18:2)	997218	467752	489897	5924653	3813311	4451956	7377826	6046072	5674505
PE(16:0p/20:3)−H	PE(36:3p)−H	PE	(16:0p/20:3)	2060112	1513199	1234494	8966460	8777144	10026565	9017780	10548334	10147034
PE(36:3p)−H	PE(36:3p)−H	PE	(36:3p)	601284	267484	154108	2772190	2822090	2837733	2615126	3819494	1702071
PE(16:0/20:4)−H	PE(36:4)−H	PE	(16:0/20:4)	1533862	784905	839695	8299759	6949456	8296843	13349623	12490424	9798806
PE(16:1/20:3)−H	PE(36:4)−H	PE	(16:1/20:3)	0	0	0	63814	37695	135632	441949	371736	246019
PE(16:0p/20:4)−H	PE(36:4p)−H	PE	(16:0p/20:4)	14847809	9949236	7821593	87386040	82142785	64340444	70538600	82997643	61233670
PE(16:0/20:5)−H	PE(36:5)−H	PE	(16:0/20:5)	43235	18442	8097	276286	296682	265733	471119	542475	352664
PE(16:1/20:4)−H	PE(36:5)−H	PE	(16:1/20:4)	62913	18671	28242	620906	664982	244723	1181079	1241076	776997
PE(18:0/20:2)−H	PE(38:2)−H	PE	(18:0/20:2)	67636	158025	123852	487539	428430	617126	588560	807636	866364
PE(16:0p/22:2)−H	PE(38:2p)−H	PE	(16:0p/22:2)	281159	326553	235086	799865	754510	1097689	672784	1068174	954474
PE(18:0/20:3)−H	PE(38:3)−H	PE	(18:0/20:3)	503807	349186	293419	2141607	1641504	2550106	2498788	3067671	3259440
PE(18:1/20:2)−H	PE(38:3)−H	PE	(18:1/20:2)	604967	342597	279993	2858098	1777982	2811003	3207149	2737000	3129624
PE(16:0p/22:3)−H	PE(38:3p)−H	PE	(16:0p/22:3)	811740	700793	459821	2776342	2469757	3028879	2344400	5848869	5584451
PE(38:3p)−H	PE(38:3p)−H	PE	(38:3p)	390224	375759	223331	1299945	1222774	1608561	1180206	1670534	1558562
PE(18:0/20:4)−H	PE(38:4)−H	PE	(18:0/20:4)	2456039	1537488	1269299	12193195	8539222	9832922	13361844	15608337	12626893
PE(18:1/20:3)−H	PE(38:4)−H	PE	(18:1/20:3)	1007186	585209	382659	4607240	2626747	3983617	6232722	4777064	3920822
PE(38:4e)−H	PE(38:4e)−H	PE	(38:4e)	302434	225823	151889	1057487	979312	1186350	1045707	1293714	1225036
PE(16:0p/22:4)−H	PE(38:4p)−H	PE	(16:0p/22:4)	2824362	2134283	1640321	14855295	12909452	14744387	13726763	13926467	13307321
PE(18:0p/20:4)−H	PE(38:4p)−H	PE	(18:0p/20:4)	4212506	3217026	1968005	17286317	15134250	16677488	14756618	17797914	15074675
PE(38:4p)−H	PE(38:4p)−H	PE	(38:4p)	1275250	603755	418564	7674815	4959208	4517960	5477709	5621069	5548156
PE(18:0/20:5)−H	PE(38:5)−H	PE	(18:0/20:5)	139250	46613	61625	490866	513134	465201	947568	1239005	703804
PE(18:1/20:4)−H	PE(38:5)−H	PE	(18:1/20:4)	2645119	1239988	1330129	19152712	12845909	15561483	24011724	19394560	16184988
PE(16:0p/22:5)−H	PE(38:5p)−H	PE	(16:0p/22:5)	1020132	750007	685545	5253798	4344806	5508538	5815080	6578847	4144991
PE(18:1p/20:4)−H	PE(38:5p)−H	PE	(18:1p/20:4)	18167898	9992029	9133217	109131302	94797038	93740973	99657388	97189853	78540805
PE(16:0/22:6)−H	PE(38:6)−H	PE	(16:0/22:6)	453256	298791	336584	4356562	2654738	3355473	6670093	5408285	4264692
PE(16:1/22:5)−H	PE(38:6)−H	PE	(16:1/22:5)	0	0	0	63696	40670	39605	185213	85136	49828
PE(18:0/22:3)−H	PE(40:3)−H	PE	(18:0/22:3)	10960	32758	5181	90268	86436	103615	98373	164864	157645
PE(18:0p/22:3)−H	PE(40:3p)−H	PE	(18:0p/22:3)	105813	100584	54648	222433	228302	279612	128884	274839	311913
PE(18:0/22:4)−H	PE(40:4)−H	PE	(18:0/22:4)	197285	133407	90504	722815	608975	751146	964835	1176101	1129595
PE(18:0p/22:4)−H	PE(40:4p)−H	PE	(18:0p/22:4)	341756	315309	166741	999742	1024226	1041341	772417	1158364	933855
PE(40:4p)−H	PE(40:4p)−H	PE	(40:4p)	313649	199013	137246	932144	892047	1081932	841184	1130327	866241
PE(18:0/22:5)−H	PE(40:5)−H	PE	(18:0/22:5)	527006	243609	209621	1983469	1550990	1452927	2619422	2904516	2123746
PE(18:1/22:4)−H	PE(40:5)−H	PE	(18:1/22:4)	163699	144849	203727	367469	1494992	2661305	3285923	1513453	259669
PE(18:0p/22:5)−H	PE(40:5p)−H	PE	(18:0p/22:5)	2041532	1449484	937204	9420474	8270899	8781302	8000425	8017983	7306739
PE(40:5p)−H	PE(40:5p)−H	PE	(40:5p)	898601	567161	378189	4555131	4354962	4492408	3859235	4315532	3492115
PE(18:0/22:6)−H	PE(40:6)−H	PE	(18:0/22:6)	456020	320955	351867	3453274	2887356	2770119	4424900	5133451	4233378
PE(18:1/22:5)−H	PE(40:6)−H	PE	(18:1/22:5)	244484	51197	79945	2122305	1002298	1681511	3048169	1158483	1137637
PE(18:0p/22:6)−H	PE(40:6p)−H	PE	(18:0p/22:6)	1800305	1170822	832720	9062998	7123449	7785257	7858181	8270007	6518608
PE(18:1p/22:5)−H	PE(40:6p)−H	PE	(18:1p/22:5)	3161341	1499520	1390375	18729479	16246040	15946927	17515537	14942159	12335392
PE(18:1/22:6)−H	PE(40:7)−H	PE	(18:1/22:6)	941525	355493	371498	3831991	4065602	3598662	8154213	6652706	4374268
PE(18:1p/22:6)−H	PE(40:7p)−H	PE	(18:1p/22:6)	3150208	1584213	1628730	23608459	18768736	19054191	21417700	18405634	15062359
PEt(16:0/16:1)−H	PEt(32:1)−H	PEt	(16:0/16:1)	64720	1344878	93787	1405930	546333	3045089	2114122	3800890	3855758
PEt(32:4)−H	PEt(32:4)−H	PEt	(32:4)	265879817	260099010	208850921	301831955	277220807	266517451	286189223	295454582	230243440
PEt(18:0/16:1)−H	PEt(34:1)−H	PEt	(18:0/16:1)	247830	545581	452038	913504	1187126	1405160	824157	1810344	1678593
PEt(18:2/18:2)−H	PEt(36:4)−H	PEt	(18:2/18:2)	1976185	1909483	752042	5211107	6353043	5173947	5470924	8408667	5567100
PG(12:0/14:0)−H	PG(26:0)−H	PG	(12:0/14:0)	3291637	2997687	3007356	3085481	2843118	3106016	3129105	3256623	3154080
PG(16:0/14:0)−H	PG(30:0)−H	PG	(16:0/14:0)	352443	336195	292120	328874	397200	394890	419035	471553	504201
PG(16:0/16:1)−H	PG(32:1)−H	PG	(16:0/16:1)	631058	418905	401284	4095250	5590127	4226058	3085287	3792768	2368527
PG(17:1/16:0)−H	PG(33:1)−H	PG	(17:1/16:0)	89999	63767	62562	719959	833553	483528	501273	357480	280114
PG(16:0/18:1)−H	PG(34:1)−H	PG	(16:0/18:1)	12340035	3640873	2211621	19828140	34567636	24318862	11385031	21286743	14349189
PG(16:1/18:1)−H	PG(34:2)−H	PG	(16:1/18:1)	10231708	1799465	2152385	74557729	67479992	50699450	45455230	43657072	31137299
PG(17:0/18:1)−H	PG(35:1)−H	PG	(17:0/18:1)	615295	394993	352068	1930499	1593585	1927138	2297579	2264977	2563620
PG(17:1/18:1)−H	PG(35:2)−H	PG	(17:1/18:1)	633193	1433325	1055848	11187622	5669506	4382943	2326950	2856001	4736977
PG(18:0/18:1)−H	PG(36:1)−H	PG	(18:0/18:1)	2700682	2248244	1388609	15456940	18792438	13941546	11082399	13481106	10600139
PG(18:1/18:1)−H	PG(36:2)−H	PG	(18:1/18:1)	6228328	5062662	3249408	80685389	79915316	70401269	37346626	48603600	32344109
PG(18:1/18:2)−H	PG(36:3)−H	PG	(18:1/18:2)	10305443	7427137	6063942	73899862	95911164	65157688	45814873	54018692	33232855
PG(20:1/18:1)−H	PG(38:2)−H	PG	(20:1/18:1)	1282635	1541031	2363373	23928820	14996281	10694865	13223606	17577282	5116248
PG(18:1/20:2)−H	PG(38:3)−H	PG	(18:1/20:2)	7711014	5596874	4185118	69259272	79775353	59832706	36801707	48658730	29691872
PG(18:0/20:4)−H	PG(38:4)−H	PG	(18:0/20:4)	107276	57097	47148	916269	601225	881476	1139932	867436	489397
PG(18:1/20:3)−H	PG(38:4)−H	PG	(18:1/20:3)	1677516	1689625	1307103	17350069	21925654	14982464	10423762	11158839	6163298
PG(18:1/20:4)−H	PG(38:5)−H	PG	(18:1/20:4)	247645	23761	44001	1569165	3109323	1470528	615768	1148114	428740
PG(16:0/22:6)−H	PG(38:6)−H	PG	(16:0/22:6)	343275	184531	177784	3384787	3202373	2231793	1852009	1870197	1111921
PG(18:1/22:4)−H	PG(40:5)−H	PG	(18:1/22:4)	851725	510407	341038	6050912	11603163	4860092	2647128	2217423	1912825
PG(18:0/22:6)−H	PG(40:6)−H	PG	(18:0/22:6)	294653	161602	101326	2756435	2810825	1916883	1142241	1241307	645921
PG(18:1/22:5)−H	PG(40:6)−H	PG	(18:1/22:5)	1056786	773603	596156	9313613	8612813	6405516	4518879	1540214	2326406
PG(18:1/22:6)−H	PG(40:7)−H	PG	(18:1/22:6)	5487140	3216550	2820380	55289513	56888693	39963703	29060838	29322371	17762536
PG(20:1/22:6)−H	PG(42:7)−H	PG	(20:1/22:6)	218470	169244	102094	1569794	2147954	1865542	956297	768939	756347
PG(20:2/22:6)−H	PG(42:8)−H	PG	(20:2/22:6)	46021	24702	7242	1425256	1696390	988736	549617	335515	176670
PI(16:0/18:1)−H	PI(34:1)−H	PI	(16:0/18:1)	5572244	3228984	3578165	28139255	15983205	16247138	23732089	21248245	24374785
PI(16:1/18:1)−H	PI(34:2)−H	PI	(16:1/18:1)	80636	29805	382819	6076637	6375888	5651124	6700759	6249597	5678897
PI(18:0/18:1)−H	PI(36:1)−H	PI	(18:0/18:1)	1353621	1328199	1118896	4262146	3103471	6042663	3657919	3354109	4531768
PI(16:0/20:3)−H	PI(36:3)−H	PI	(16:0/20:3)	54556	24544	37397	2047115	131263	487763	1224725	1109445	1350953
PI(18:1/18:2)−H	PI(36:3)−H	PI	(18:1/18:2)	444587	182945	282025	3105247	2107990	3027684	2622737	2039418	2258793
PI(16:0/20:4)−H	PI(36:4)−H	PI	(16:0/20:4)	598914	0	279422	7055333	4886231	5866008	6599973	6020985	5100684
PI(17:0/20:4)−H	PI(37:4)−H	PI	(17:0/20:4)	126712	42475	62953	833203	374643	743226	559695	389566	471825
PI(20:1/18:1)−H	PI(38:2)−H	PI	(20:1/18:1)	175259	86079	50923	439277	480013	581942	435423	360639	412422
PI(18:0/20:3)−H	PI(38:3)−H	PI	(18:0/20:3)	1217639	840910	794515	6808681	4892684	7298507	4936747	4987182	5263259
PI(18:1/20:3)−H	PI(38:4)−H	PI	(18:1/20:3)	1528781	834554	1015566	9297027	7322788	9009607	7010900	6338816	6206770
PI(18:0/20:5)−H	PI(38:5)−H	PI	(18:0/20:5)	105336	37219	42397	846382	586060	791803	770636	618613	509848
PI(18:1/20:4)−H	PI(38:5)−H	PI	(18:1/20:4)	3401763	567106	1327226	21639659	13688994	16648897	18552681	13090246	11532472
PI(18:0/22:4)−H	PI(40:4)−H	PI	(18:0/22:4)	70300	10342	44277	311169	606438	601805	311001	92442	302088
PMe(14:0/14:0)−H	PMe(28:0)−H	PMe	(14:0/14:0)	171922	180428	199438	130882	139796	213640	129113	162779	138701
PMe(34:5)−H	PMe(34:5)−H	PMe	(34:5)	168686234	186690542	176316357	200012780	179283160	213223306	159526553	188673115	187834497
PMe(42:6)−H	PMe(42:6)−H	PMe	(42:6)	5405393	4557433	4292756	23101077	15239469	28664591	20099893	26906924	25140106
PS(12:0/14:0)−H	PS(26:0)−H	PS	(12:0/14:0)	1747977	1622250	1682897	1716105	1618233	1662372	1717501	1719096	1615557
PS(16:0/16:1)−H	PS(32:1)−H	PS	(16:0/16:1)	728060	235649	568002	3565582	3195954	3075828	4942848	6561468	5130558
PS(33:1)−H	PS(33:1)−H	PS	(33:1)	499826	405674	283691	4401113	5524904	2387274	2958538	3100594	1489335
PS(18:0/16:1)−H	PS(34:1)−H	PS	(18:0/16:1)	1055749	548738	684137	3216582	2835107	3319316	3827580	3654067	4668131
PS(34:3p)−H	PS(34:3p)−H	PS	(34:3p)	153507	21797	41263	198436	98626	367469	722045	457006	312790
PS(35:0)−H	PS(35:0)−H	PS	(35:0)	11891104	7835215	7431554	59353871	68314802	52272315	85687569	86949801	81080831
PS(17:0/18:1)−H	PS(35:1)−H	PS	(17:0/18:1)	1307641	275623	497854	7336355	5700796	2616582	7552859	1167330	1726211
PS(35:1)−H	PS(35:1)−H	PS	(35:1)	848351	757585	414645	4454321	4979022	3822279	3571141	4806042	2964179
PS(35:2)−H	PS(35:2)−H	PS	(35:2)	3415928	2242422	1538013	34883266	36475561	24156246	17252639	19458459	11929743
PS(18:0/18:2)−H	PS(36:2)−H	PS	(18:0/18:2)	2201906	1098564	1372590	8751952	5914013	10784726	11546559	9446142	10329044
PS(16:0/20:3)−H	PS(36:3)−H	PS	(16:0/20:3)	334321	165257	202550	1772472	1390975	1484428	2026197	1917175	1847899
PS(18:1/18:2)−H	PS(36:3)−H	PS	(18:1/18:2)	285503	38926	96427	1161619	883620	1197986	2051319	1271760	1100368
PS(36:3)−H	PS(36:3)−H	PS	(36:3)	856597	537999	391845	2349840	2065038	2484307	4013435	3709721	3775290
PS(36:3p)−H	PS(36:3p)−H	PS	(36:3p)	1785308	961455	880839	4932449	3706720	4723479	4655192	5670664	5512313
PS(16:0/20:4)−H	PS(36:4)−H	PS	(16:0/20:4)	336875	75210	152969	1793993	1503983	1231651	2493720	1831652	1787382
PS(36:4)−H	PS(36:4)−H	PS	(36:4)	2097741	1103090	1282076	6739204	3862077	6358071	5643547	4649414	8285398
PS(37:1)−H	PS(37:1)−H	PS	(37:1)	4922567	2295692	2782683	28551029	24264289	19901449	30741768	29723358	25818331
PS(37:2)−H	PS(37:2)−H	PS	(37:2)	581557	461162	307952	4041114	4606071	2742459	2628164	3564788	1791980
PS(38:1)−H	PS(38:1)−H	PS	(38:1)	119029	88780	159751	636909	486060	845525	553894	730622	913435
PS(20:1/18:1)−H	PS(38:2)−H	PS	(20:1/18:1)	682040	256270	365044	1439149	1314610	2090937	2117005	930228	2366094
PS(18:0/20:3)−H	PS(38:3)−H	PS	(18:0/20:3)	2132736	1403886	1679340	8519606	7565340	9227315	12239549	11672688	9127945
PS(38:3)−H	PS(38:3)−H	PS	(38:3)	303414	264363	212408	1012883	908749	1120841	1194372	1746140	1675591
PS(18:0/20:4)−H	PS(38:4)−H	PS	(18:0/20:4)	5071910	2766460	3001663	15129170	10456168	13919549	18462765	17593909	9290622
PS(18:1/20:3)−H	PS(38:4)−H	PS	(18:1/20:3)	298626	79786	145696	1957206	1133429	1494316	2248184	1564366	1492944
PS(16:0/22:5)−H	PS(38:5)−H	PS	(16:0/22:5)	631783	264480	374479	3800992	2890287	2781598	4853679	3717065	3093176
PS(18:0/20:5)−H	PS(38:5)−H	PS	(18:0/20:5)	94722	20015	40398	562024	452681	479599	749689	610233	520897
PS(18:1/20:4)−H	PS(38:5)−H	PS	(18:1/20:4)	838980	263611	422542	3422104	1488738	2276767	2961791	2468318	3377401
PS(38:5p)−H	PS(38:5p)−H	PS	(38:5p)	439142	327919	234062	1530658	1573139	1511334	1625238	1865422	1770846
PS(16:0/22:6)−H	PS(38:6)−H	PS	(16:0/22:6)	501663	107194	37689	2985399	3010170	2363829	5145432	4645671	2908666
PS(38:6)−H	PS(38:6)−H	PS	(38:6)	190530	116195	73015	1150058	737999	556878	2223577	1819388	743138
PS(38:6p)−H	PS(38:6p)−H	PS	(38:6p)	3058936	1743299	1491375	11124637	10406271	9116870	12830113	12351643	9161699
PS(39:1)−H	PS(39:1)−H	PS	(39:1)	21472680	7500910	8866920	95030677	55056388	80746806	113680900	77726111	88499517
PS(39:2)−H	PS(39:2)−H	PS	(39:2)	1701928	574271	809532	7955388	7889742	9098386	9503874	8905526	8072334
PS(39:3)−H	PS(39:3)−H	PS	(39:3)	7562192	2893484	4314539	39378240	26194377	30330579	34724630	20359292	19099215
PS(39:4)−H	PS(39:4)−H	PS	(39:4)	174727	137788	52371	751861	993695	787691	923285	1390819	853288
PS(18:1/22:1)−H	PS(40:2)−H	PS	(18:1/22:1)	211765	131163	105038	906808	629577	1053062	1004361	1014675	1314302
PS(18:0/22:3)−H	PS(40:3)−H	PS	(18:0/22:3)	117790	97259	86093	319668	380740	347482	556349	545886	711212
PS(18:0/22:4)−H	PS(40:4)−H	PS	(18:0/22:4)	1059849	759626	644472	4483430	3452818	3693518	5363200	4875192	3881488
PS(18:0/22:5)−H	PS(40:5)−H	PS	(18:0/22:5)	4009613	1637162	2078516	18696559	12150436	15059529	21740311	17737498	16812537
PS(18:1/22:5)−H	PS(40:6)−H	PS	(18:1/22:5)	400712	160981	244435	2799589	2059668	2503137	3013450	2259542	2186611
PS(20:3/20:3)−H	PS(40:6)−H	PS	(20:3/20:3)	389400	191995	232757	1710663	883861	1305603	1812219	2230930	1801707
PS(40:6)−H	PS(40:6)−H	PS	(40:6)	494268	248494	234205	1966696	1411806	1424032	2098029	2298978	1936260
PS(40:6p)−H	PS(40:6p)−H	PS	(40:6p)	707637	454905	358939	2629775	2390393	2262508	2398545	2804321	2134177
PS(40:7)−H	PS(40:7)−H	PS	(40:7)	713739	257615	320733	3279013	2087894	2629185	3657706	3494664	3004160
PS(40:7p)−H	PS(40:7p)−H	PS	(40:7p)	3404289	1681403	1688420	14272302	12030497	11620051	14088569	13169645	10420450
PS(40:8p)−H	PS(40:8p)−H	PS	(40:8p)	1158661	586133	600767	6251230	5583727	4875480	5065880	5810101	4726658
PS(41:3)−H	PS(41:3)−H	PS	(41:3)	951865	448118	576995	6334791	4490267	4931429	5021684	4904387	3341606
PS(41:6)−H	PS(41:6)−H	PS	(41:6)	22049	50058	32814	595444	859340	309582	1355926	1290654	348166
PS(18:1/24:0)−H	PS(42:1)−H	PS	(18:1/24:0)	0	0	0	78009	92103	135517	62221	168266	125115
PS(42:8)−H	PS(42:8)−H	PS	(42:8)	528806	186309	250649	2764970	1593369	2156639	2975047	2475408	2258198
PS(42:9)−H	PS(42:9)−H	PS	(42:9)	655042	310004	341129	3220608	465873	777565	3104689	4127216	2719050
PS(43:5)−H	PS(43:5)−H	PS	(43:5)	399617	80990	126503	6515173	2668871	686037	2153097	1749608	2124165
SM(d30:1)+H	SM(d30:1)+H	SM	(d30:1)	184761	5382	14779	866346	781539	209443	1130358	416195	4283
SM(d31:1)+H	SM(d31:1)+H	SM	(d31:1)	427008	150855	466	2270811	2533152	943006	2538922	2311377	668695
SM(d32:0)+H	SM(d32:0)+H	SM	(d32:0)	473741	370664	111987	1735887	1305533	32890	1208742	946996	274128
SM(d18:1/14:0)+H	SM(d32:1)+H	SM	(d18:1/14:0)	7818523	20504	6262040	32366470	27041416	27014979	173448	21930898	23043602
SM(d32:2)+H	SM(d32:2)+H	SM	(d32:2)	600410	243608	168444	3212485	2749113	656289	3608219	2578234	586473
SM(d33:0)+H	SM(d33:0)+H	SM	(d33:0)	12151	16349	0	206379	121547	0	70774	72621	0
SM(d33:1)+H	SM(d33:1)+H	SM	(d33:1)	5596548	5691769	2079406	21071331	17330654	6221359	13635154	13910253	4879777
SM(d33:2)+H	SM(d33:2)+H	SM	(d33:2)	119774	27949	129727	92682	121048	913068	67553	39101	954329
SM(d34:0)+H	SM(d34:0)+H	SM	(d34:0)	531968	1060649	191806	2776677	1677349	462776	1387235	1737559	140360
SM(d18:1/16:0)+H	SM(d34:1)+H	SM	(d18:1/16:0)	35824469	959523	40846729	210739888	159212544	283052883	111241569	116668495	91200619
SM(d34:1)+H	SM(d34:1)+H	SM	(d34:1)	4014190	5945051	871296	22959034	16691791	3042523	14311776	14729234	1573077
SM(d16:1/18:1)+H	SM(d34:2)+H	SM	(d16:1/18:1)	42333904	22114632	17789107	190952103	166808400	162476304	175300054	131907407	132613919
SM(d34:3)+H	SM(d34:3)+H	SM	(d34:3)	232532	60609	136666	388860	346762	703869	508118	255949	698658
SM(d34:4)+H	SM(d34:4)+H	SM	(d34:4)	559956	376400	154329	1521461	1760238	1100388	1117816	1644385	746223
SM(d35:1)+H	SM(d35:1)+H	SM	(d35:1)	180977	391534	136733	1481697	726060	489660	442998	811736	241385
SM(d35:2)+H	SM(d35:2)+H	SM	(d35:2)	798449	449549	75756	3315470	2435349	475610	2569022	1768462	1291097
SM(d35:4)+H	SM(d35:4)+H	SM	(d35:4)	247304	248559	55237	1113039	1198834	576193	650921	907564	201684
SM(d18:1/18:0)+H	SM(d36:1)+H	SM	(d18:1/18:0)	330872	1193973	1424333	22431	2204929	5098132	1453730	11115	941319
SM(d36:2)+H	SM(d36:2)+H	SM	(d18:1/18:1)	16207446	10582364	9355930	70591061	52734427	99978698	50039781	34951530	21926216
SM(d36:4)+H	SM(d36:4)+H	SM	(d36:4)	1746122	3294399	588684	21429659	21737861	3800110	7934140	12147626	1774108
SM(d36:5)+H	SM(d36:5)+H	SM	(d36:5)	1394701	744751	158246	8490499	11206161	648707	6617846	7894908	1119755
SM(d38:2)+H	SM(d38:2)+H	SM	(d38:2)	548044	647588	8751	1338183	1034372	323671	1030639	321318	33557
SM(d39:7)+H	SM(d39:7)+H	SM	(d39:7)	927702	33649	0	187082	25244	0	727460	301859	0
SM(d40:1)+H	SM(d40:1)+H	SM	(d40:1)	10422	20394	130448	150910	118922	15393	179294	132116	44245
SM(d40:2)+H	SM(d40:2)+H	SM	(d40:2)	783199	1207674	643999	893349	680653	1587991	509401	449195	520464
SM(d41:2)+H	SM(d41:2)+H	SM	(d41:2)	101925	449159	58179	247523	178194	138603	113268	128450	16764
SM(d18:1/24:1)+H	SM(d42:2)+H	SM	(d18:1/24:1)	810831	2310964	1211292	2221901	975598	2461500	1085621	1514922	895862
SM(d42:2)+H	SM(d42:2)+H	SM	(d42:2)	413380	1375280	320873	926115	372649	692412	698855	657467	231704
SM(d22:0/20:3)+H	SM(d42:3)+H	SM	(d22:0/20:3)	4379800	5429055	2431167	5152508	4866803	7141033	3732288	3045049	3307694
SM(d42:5)+H	SM(d42:5)+H	SM	(d42:5)	22257	114243	0	0	0	0	0	0	0
SM(d43:3)+H	SM(d43:3)+H	SM	(d43:3)	9359	27348	4137	167426	25219	71679	46618	17132	8872
SM(d44:2)+H	SM(d44:2)+H	SM	(d44:2)	0	0	0	0	0	0	0	0	0
SM(d44:3)+H	SM(d44:3)+H	SM	(d44:3)	0	0	0	0	0	0	0	0	0
SM(d44:5)+H	SM(d44:5)+H	SM	(d44:5)	135954	100436	6363	153592	52620	6770	0	0	0
SM(d44:6)+H	SM(d44:6)+H	SM	(d44:6)	657626	487421	148152	565599	267374	15924	232068	212798	318887
TG(8:0/8:0/8:0)+NH4	TG(24:0)+NH4	TG	(8:0/8:0/8:0)	1242314	3209402	276628	1668652	2213903	145003	949093	5710411	23079
TG(8:0/8:0/10:0)+NH4	TG(26:0)+NH4	TG	(8:0/8:0/10:0)	1318270	4557273	176287	2382874	3207039	379462	1175973	10295214	53577
TG(8:0/10:0/10:0)+NH4	TG(28:0)+NH4	TG	(8:0/10:0/10:0)	980514	5169872	41175	2380979	3560819	53139	343546	12759725	38818
TG(10:0/10:0/10:0)+NH4	TG(30:0)+NH4	TG	(10:0/10:0/10:0)	55030	651568	112134	81597	108823	30295	86425	1053795	8762
TG(16:0/8:0/8:0)+NH4	TG(32:0)+NH4	TG	(16:0/8:0/8:0)	96036	486150	104161	117254	179479	150507	93300	403362	136769
TG(16:0/9:0/9:0)+NH4	TG(34:0)+NH4	TG	(16:0/9:0/9:0)	604664	901783	82022	683116	536194	33185	649749	1187702	246282
TG(8:0/8:0/18:1)+NH4	TG(34:1)+NH4	TG	(8:0/8:0/18:1)	324371	672784	36583	348459	396874	152647	259133	765937	56563
TG(15:0/14:0/15:0)+NH4	TG(44:0)+NH4	TG	(15:0/14:0/15:0)	1618712	1135131	286008	1525824	1395121	268843	1619431	1331287	279788
TG(44:5p)+NH4	TG(44:5p)+NH4	TG	(44:5p)	25543551	3986465	0	12597022	2446009	0	16349391	18607921	0
TG(15:0/14:0/16:0)+NH4	TG(45:0)+NH4	TG	(15:0/14:0/16:0)	4118381	3720750	2541428	3930797	1343349	1475096	3565070	4164813	1675552
TG(16:0/14:0/16:0)+NH4	TG(46:0)+NH4	TG	(16:0/14:0/16:0)	6795037	5052665	2878349	6022438	6242181	2016110	5937839	5877263	4336876
TG(46:1)+NH4	TG(46:1)+NH4	TG	(46:1)	4768439	4079835	1473258	4479206	6299514	1142516	5959002	7292691	1431422
TG(15:0/16:0/16:0)+NH4	TG(47:0)+NH4	TG	(15:0/16:0/16:0)	6090749	8838655	3231545	6335618	7647977	2156161	5507524	5972182	2732225
TG(16:0/16:0/16:0)+NH4	TG(48:0)+NH4	TG	(16:0/16:0/16:0)	8140830	5972302	2771992	7304466	7413730	4021792	7229802	6299631	3277730
TG(16:0/16:0/16:1)+NH4	TG(48:1)+NH4	TG	(16:0/16:0/16:1)	6248878	5074336	2682659	5508672	5837385	1721460	5661303	11138979	2297653
TG(18:0/16:0/16:0)+NH4	TG(50:0)+NH4	TG	(18:0/16:0/16:0)	11403968	9758925	1882255	10674644	11493263	1366220	10672750	11306892	1668406
TG(16:0/16:0/18:1)+NH4	TG(50:1)+NH4	TG	(16:0/16:0/18:1)	12279240	8130914	2969720	11354171	6912034	1356077	6611148	5494692	3212002
TG(18:0/16:0/18:1)+NH4	TG(52:1)+NH4	TG	(18:0/16:0/18:1)	5819358	4864513	1634976	5557327	5378670	1096755	5506420	5373435	1335435
TG(16:0/18:1/18:1)+NH4	TG(52:2)+NH4	TG	(16:0/18:1/18:1)	9061242	10072579	2244070	8249171	8874213	1748260	10205374	11992060	1732428
TG(16:1/18:1/18:1)+NH4	TG(52:3)+NH4	TG	(16:1/18:1/18:1)	4725098	2738900	643761	2902454	4511770	452722	2861650	3029603	581059
TG(18:0/18:1/18:1)+NH4	TG(54:2)+NH4	TG	(18:0/18:1/18:1)	5654594	4489151	979908	5202267	5196027	709183	5389810	5130435	810488
TG(18:1/18:1/18:1)+NH4	TG(54:3)+NH4	TG	(18:1/18:1/18:1)	11724150	8431628	1404204	14224033	14670275	991967	10211213	9201560	1610674
TG(18:1/18:1/18:2)+NH4	TG(54:4)+NH4	TG	(18:1/18:1/18:2)	4680624	3917537	621665	4058915	4707413	505091	3930835	4407047	590330
TG(18:1/18:2/18:2)+NH4	TG(54:5)+NH4	TG	(18:1/18:2/18:2)	2507239	3032444	297703	2273173	2380920	261082	2921273	2359582	259348
TG(18:2/18:2/18:2)+NH4	TG(54:6)+NH4	TG	(18:2/18:2/18:2)	1746477	1389870	74698	1991526	1796868	49971	1894205	1709018	44805
			TOTAL	3.829E+09	3.116E+09	2.416E+09	2.059E+10	1.748E+10	2.482E+10	2.240E+10	1.886E+10	1.810E+10

The values in the tale are relative signal response (signal's peak area count is normalized to sample weight and peak area count of the internal standard signal)

	TABLE 7

	MDA-MB-4175 Exomere	MDA-MB-4175 Exo-S

Lipid

Fatty Acid

Exomere

Exo-S

Exo-L

Lipid Ion

Group

Class

Chain

replicate 1

replicate 2

replicate 3

replicate 1

replicate 2

replicate 3

replicate 1

replicate 2

replicate 3

AcCa(14:0)+H	AcCa(14:0)+H	AcCa	(14:0)	236921	60280	40369	126480	157838	152846	1834630	284936	421408
AcCa(16:0)+H	AcCa(16:0)+H	AcCa	(16:0)	173132	115487	99632	290548	540773	602220	2904828	842170	1601012
AcCa(18:0)+H	AcCa(18:0)+H	AcCa	(18:0)	130334	141303	32252	253192	333198	458510	3139944	678326	1301103
AcCa(18:1)+H	AcCa(18:1)+H	AcCa	(18:1)	262216	178530	181058	384403	477005	418190	3551080	722892	775428
Cer(d18:1/10:0)+H	Cer(d28:1)+H	Cer	(d18:1/10:0)	2655983	2717474	3068942	2245269	3685342	3417381	43785352	7453770	10821114
Cer(d17:1/12:0)+H	Cer(d29:1)+H	Cer	(d17:1/12:0)	2207505	1985339	2087242	2100964	2174159	2042473	33378548	4186857	7247456
Cer(d18:0/12:0)+H	Cer(d30:0)+H	Cer	(d18:0/12:0)	5662075	4936509	5593158	4329811	4729504	5127001	91609934	12484096	19337026
Cer(d18:1/13:0)+H	Cer(d31:1)+H	Cer	(d18:1/13:0)	2422764	2096279	2465362	2107474	2288184	2188704	43488083	6183718	9429952
Cer(d18:1/14:0)+H	Cer(d32:1)+H	Cer	(d18:1/14:0)	11477831	12948297	13238862	20733298	19005688	24257680	552120875	103925661	151641948
Cer(d17:1/16:0)+H	Cer(d33:1)+H	Cer	(d17:1/16:0)	7509	21555	78579	105088	78637	118214	3460630	595755	714338
Cer(d18:1/16:0)+H	Cer(d34:1)+H	Cer	(d18:1/16:0)	1117353	955878	661319	1287571	834189	1375069	105933055	20713069	12389874
Cer(d18:2/16:0)+H	Cer(d34:2)+H	Cer	(d18:2/16:0)	52597	95208	87456	722863	663076	1096538	8685089	2242095	4531503
Cer(d18:1/18:0)+H	Cer(d36:1)+H	Cer	(d18:1/18:0)	0	0	0	0	0	0	0	0	0
Cer(d18:2/18:0)+H	Cer(d36:2)+H	Cer	(d18:2/18:0)	0	0	0	132962	135786	231728	1894280	654766	816443
Cer(d18:2/22:0)+H	Cer(d40:2)+H	Cer	(d18:2/22:0)	0	0	0	30277	18349	77779	3095047	813500	480540
Cer(d18:1/24:1)+H	Cer(d42:2)+H	Cer	(d18:1/24:1)	8852	7438	10947	29671	40745	44239	14047924	4203440	659553
Cer(d18:2/24:0)+H	Cer(d42:2)+H	Cer	(d18:2/24:0)	0	0	0	6450	32143	62391	16020443	3580326	1286726
Cer(d18:2/24:1)+H	Cer(d42:3)+H	Cer	(d18:2/24:1)	0	0	155166	692323	276902	427271	21410849	2070555	2387340
CerG1(d18:1/16:0)+H	CerG1(d34:1)+H	CerG1	(d18:1/16:0)	698553	626375	657261	1463831	1176889	1258903	82287509	18156168	8437858
CerG1(d18:2/16:0)+H	CerG1(d34:2)+H	CerG1	(d18:2/16:0)	3242	3156	11881	483819	412033	377232	4663727	1388424	1738013
CerG1(d42:2)+H	CerG1(d42:2)+H	CerG1	(d42:2)	19156	38663	63825	199891	124949	159067	23002018	6801232	2282490
CerG1(d42:3)+H	CerG1(d42:3)+H	CerG1	(d42:3)	8267	28320	19495	312700	408677	349795	8310956	2537848	2515903
CerG2(d42:2)+H	CerG2(d42:2)+H	CerG2	(d42:2)	194539	104065	132010	164952	0	52049	4688460	1084666	642286
CerG3(d34:1)+H	CerG3(d34:1)+H	CerG3	(d34:1)	0	0	0	0	0	0	6648833	1077241	342845
CL(14:0/14:0/14:0/14:0)−H	CL(56:0)−H	CL	(14:0/14:0/14:0/14:0)	82694	93825	71653	89390	81669	186557	281420	135313	346310
CL(18:2/14:0/14:0/14:0)−H	CL(60:2)−H	CL	(18:2/14:0/14:0/14:0)	2113997	2158943	2979313	1583175	1872591	2024747	49191055	6044383	13647980
CL(63:3)−H	CL(63:3)−H	CL	(63:3)	26567573	21638964	23018821	22393188	15475284	19590970	400846638	64364027	87152106
cPA(18:0)−H	cPA(18:0)−H	cPA	(18:0)	0	0	0	0	0	0	0	0	0
DG(9:0/9:0)+NH4	DG(18:0)+NH4	DG	(9:0/9:0)	220351	708397	661717	384894	2224405	1359115	3427027	4147519	7516632
DG(10:0/10:0)+NH4	DG(20:0)+NH4	DG	(10:0/10:0)	71305	204318	238931	163071	549574	429392	1582819	1280889	2030563
DG(16:0/14:0)+NH4	DG(30:0)+NH4	DG	(16:0/14:0)	98618	116865	122307	190236	29748	75232	12666456	10303377	662173
DG(16:0/16:0)+NH4	DG(32:0)+NH4	DG	(16:0/16:0)	514614	516721	656759	1139209	275352	405462	344533948	281069758	2244319
DG(18:0/16:0)+NH4	DG(34:0)+NH4	DG	(18:0/16:0)	523401	401524	547006	1034357	749309	472075	282519224	197608752	2857014
DG(16:0/18:1)+NH4	DG(34:1)+NH4	DG	(16:0/18:1)	503615	210946	434667	610589	252986	290973	32916503	10441702	664591
DG(16:0/18:2)+NH4	DG(34:2)+NH4	DG	(16:0/18:2)	459485	142139	292571	374868	117960	263493	10967709	3507411	498989
DG(16:1/18:1)+NH4	DG(34:2)+NH4	DG	(16:1/18:1)	116274	14653	51648	244903	80962	113728	4036728	690883	230487
DG(18:0/18:0)+NH4	DG(36:0)+NH4	DG	(18:0/18:0)	423894	417786	374555	362393	534468	357479	36801516	21585066	1702063
DG(18:0/18:1)+NH4	DG(36:1)+NH4	DG	(18:0/18:1)	178324	112775	162706	212795	145109	187325	15156412	3941484	458267
DG(18:1/18:1)+NH4	DG(36:2)+NH4	DG	(18:1/18:1)	920441	325775	574502	836183	394846	392394	32608125	11438424	1054671
DG(18:0/20:4)+NH4	DG(38:4)+NH4	DG	(18:0/20:4)	7822	18131	84842	797163	56090	66050	21574088	4067784	106538
LPA(16:0)−H	LPA(16:0)−H	LPA	(16:0)	0	0	0	67179	98416	73714	313125	111830	377480
LPA(18:0)−H	LPA(18:0)−H	LPA	(18:0)	0	0	0	45473	25422	17533	0	0	0
LPC(12:0)+H	LPC(12:0)+H	LPC	(12:0)	23790298	22254886	24392408	29863151	27665994	29303338	361686129	44332937	75970404
LPC(14:0)+H	LPC(14:0)+H	LPC	(14:0)	258413	269648	257370	640244	613158	604231	8167030	1968034	2947532
LPC(15:0)+H	LPC(15:0)+H	LPC	(15:0)	0	0	0	145644	162622	131006	945377	287920	557726
LPC(16:0)+H	LPC(16:0)+H	LPC	(16:0)	511142	1092870	1012735	11212410	1799189	12355716	17922295	4817545	7379930
LPC(16:0e)+H	LPC(16:0e)+H	LPC	(16:0e)	0	0	0	874804	908642	859413	7751597	2571194	4345836
LPC(16:0p)+H	LPC(16:0p)+H	LPC	(16:0p)	0	0	0	392879	421352	451399	2760657	909709	1887498
LPC(16:1)+H	LPC(16:1)+H	LPC	(16:1)	19507	33259	27441	549700	675184	315464	4116434	983238	1243586
LPC(17:0)+H	LPC(17:0)+H	LPC	(17:0)	0	0	0	346485	295634	350623	2818005	786339	1280303
LPC(17:1)+H	LPC(17:1)+H	LPC	(17:1)	231456	223932	251852	184051	244014	283861	2109653	63059	722448
LPC(18:0)+H	LPC(18:0)+H	LPC	(18:0)	19387	50326	49929	1201301	892073	924000	9518868	2775978	4052148
LPC(18:0e)+H	LPC(18:0e)+H	LPC	(18:0e)	15738	5452	26816	817225	668857	717703	6971758	2424788	3234886
LPC(18:0p)+H	LPC(18:0p)+H	LPC	(18:0p)	0	0	0	227445	197809	273952	2086704	421383	1034629
LPC(18:1)+H	LPC(18:1)+H	LPC	(18:1)	78091	682864	127626	721046	4981691	5413606	6909206	1415971	2577564
LPC(18:1p)+H	LPC(18:1p)+H	LPC	(18:1p)	0	0	0	67857	74595	66462	587746	39423	164326
LPC(18:3)+H	LPC(18:3)+H	LPC	(18:3)	13452	19986	26017	557398	539676	653872	3604766	831154	2385132
LPC(19:0)+H	LPC(19:0)+H	LPC	(19:0)	0	0	0	57278	36508	56391	170840	127841	228556
LPC(19:1)+H	LPC(19:1)+H	LPC	(19:1)	0	0	0	162478	53902	73923	1910635	138093	313977
LPC(20:0)+H	LPC(20:0)+H	LPC	(20:0)	0	0	0	293411	211624	240786	2453147	715073	743929
LPC(20:0e)+H	LPC(20:0e)+H	LPC	(20:0e)	0	0	0	234902	111226	78719	1168978	566868	429543
LPC(20:1)+H	LPC(20:1)+H	LPC	(20:1)	0	0	0	335575	286950	340239	3264208	819246	1361028
LPC(20:2)+H	LPC(20:2)+H	LPC	(20:2)	0	0	0	36382	13892	29190	168153	59093	171380
LPC(20:3)+H	LPC(20:3)+H	LPC	(20:3)	0	0	0	328124	309797	384235	2021632	745731	1282454
LPC(20:4)+H	LPC(20:4)+H	LPC	(20:4)	34037	58518	13333	338945	247493	292033	1318615	238225	417465
LPC(22:0)+H	LPC(22:0)+H	LPC	(22:0)	11590	15703	64811	223020	442232	167871	2068227	245694	376881
LPC(22:3)+H	LPC(22:3)+H	LPC	(22:3)	0	0	0	0	0	0	0	0	0
LPC(22:4)+H	LPC(22:4)+H	LPC	(22:4)	0	0	0	80883	49136	74257	232824	62682	142827
LPC(22:5)+H	LPC(22:5)+H	LPC	(22:5)	65136	81601	116317	250836	124532	197416	1004195	180428	615367
LPC(22:6)+H	LPC(22:6)+H	LPC	(22:6)	18278	57941	24093	200822	131690	137944	668379	126365	210287
LPC(24:0)+H	LPC(24:0)+H	LPC	(24:0)	0	0	0	295991	79870	102461	4693934	832912	215834
LPC(24:1)+H	LPC(24:1)+H	LPC	(24:1)	31659	31216	41679	173287	174716	123822	1326615	233447	479517
LPC(26:1)+H	LPC(26:1)+H	LPC	(26:1)	0	0	0	549956	257896	326456	3384573	828026	966603
LPC(28:0)+H	LPC(28:0)+H	LPC	(28:0)	157867	164827	158659	277434	285209	257965	4174619	532864	792395
LPE(16:0p)−H	LPE(16:0p)−H	LPE	(16:0p)	0	0	0	305679	283786	221726	2656494	966481	1258679
LPE(18:0)−H	LPE(18:0)−H	LPE	(18:0)	0	0	0	94595	80165	48211	587544	266583	302159
LPE(20:1)−H	LPE(20:1)−H	LPE	(20:1)	0	0	0	0	0	0	0	0	0
LPE(20:4)−H	LPE(20:4)−H	LPE	(20:4)	0	0	0	109524	0	227902	686252	143917	92179
LPG(14:0)−H	LPG(14:0)−H	LPG	(14:0)	841382	533465	723427	781822	969793	697483	9507672	1709038	1682271
LPG(16:0)−H	LPG(16:0)−H	LPG	(16:0)	0	0	0	0	0	0	0	0	0
LPG(18:0)−H	LPG(18:0)−H	LPG	(18:0)	0	0	0	0	0	0	0	0	0
LPI(16:0)−H	LPI(16:0)−H	LPI	(16:0)	0	0	0	0	0	0	0	0	0
LPI(18:0)−H	LPI(18:0)−H	LPI	(18:0)	0	0	0	181523	120996	114373	922878	249061	286788
LPI(18:1)−H	LPI(18:1)−H	LPI	(18:1)	0	0	0	41923	58531	37030	45212	29766	77178
MG(14:0)+H	MG(14:0)+H	MG	(14:0)	80142	261874	286521	242666	1084836	793557	1650270	2004628	3872438
MG(16:0)+H	MG(16:0)+H	MG	(16:0)	12869069	31337664	31311812	34428216	112543180	89476091	302124878	207870963	391306257
MG(18:0)+H	MG(18:0)+H	MG	(18:0)	26759010	56344973	50203262	58402624	149507750	119903599	569548694	299981609	543660516
MG(18:1)+H	MG(18:1)+H	MG	(18:1)	84627	419296	473167	329588	1072455	1095516	2286684	2207664	4669714
MG(18:2)+H	MG(18:2)+H	MG	(18:2)	1228472	3913705	4429676	2302455	9738060	7108632	23436433	14762001	40048238
MG(20:0)+H	MG(20:0)+H	MG	(20:0)	159813	369824	444858	417350	1360358	985683	5947641	4715563	5555721
PA(16:0/18:1)−H	PA(34:1)−H	PA	(16:0/18:1)	0	0	0	128304	44768	22112	11676449	3075195	822160
PA(18:0/18:1)−H	PA(36:1)−H	PA	(18:0/18:1)	0	0	0	0	0	0	5255963	836174	41319
PA(18:1/18:1)−H	PA(36:2)−H	PA	(18:1/18:1)	0	0	0	24916	6261	18636	495065	203516	31337
PA(18:0/20:3)−H	PA(38:3)−H	PA	(18:0/20:3)	0	0	0	0	0	0	0	0	0
PA(18:0/20:4)−H	PA(38:4)−H	PA	(18:0/20:4)	0	0	0	116156	236214	150452	3350436	1066561	462363
PC(16:1)+H	PC(16:1)+H	PC	(16:1)	19591	5480	35757	0	0	0	830071	129709	424885
PC(19:1)+H	PC(19:1)+H	PC	(19:1)	66872	44171	63718	70629	48755	50862	737794	28089	480361
PC(19:3)+H	PC(19:3)+H	PC	(19:3)	0	0	0	131408	143675	157187	406932	200393	346421
PC(22:0)+H	PC(22:0)+H	PC	(22:0)	1209573	1094999	1354462	1437283	1984186	2284747	22643890	5455915	10269555
PC(23:0)+H	PC(23:0)+H	PC	(23:0)	5339351	4669007	4487087	4517013	5053785	5620807	98412130	14078728	24316430
PC(14:0e/10:1)+H	PC(24:1e)+H	PC	(14:0e/10:1)	18927	34312	17817	254420	175844	149650	397945	331104	495352
PC(25:0)+H	PC(25:0)+H	PC	(25:0)	4795944	4771963	4689231	4646389	5384415	5397486	82455880	14723232	24993078
PC(26:0)+H	PC(26:0)+H	PC	(26:0)	8275985	9665322	9366029	10007300	10956431	11269836	178564980	31653172	51208899
PC(28:0)+H	PC(28:0)+H	PC	(28:0)	1106432	1564248	1283876	10343330	9318637	10191677	120894036	36202735	40883521
PC(28:1)+H	PC(28:1)+H	PC	(28:1)	0	0	0	99913	237367	276974	1513235	845586	744729
PC(29:0)+H	PC(29:0)+H	PC	(29:0)	97319	156901	123794	1855491	1088262	1528313	26081981	8107306	8222932
PC(29:0e)+H	PC(29:0e)+H	PC	(29:0e)	0	0	0	710799	54443	105836	13485470	4110747	769984
PC(11:0/18:1)+H	PC(29:1)+H	PC	(11:0/18:1)	0	0	0	437930	287908	436551	11237279	4296388	3999681
PC(29:1)+H	PC(29:1)+H	PC	(29:1)	62112	48118	51039	835680	638711	785531	18447079	6478900	7145525
PC(29:2)+H	PC(29:2)+H	PC	(29:2)	0	0	0	80540	44239	15226	691827	139225	281986
PC(30:0)+H	PC(30:0)+H	PC	(16:0/14:0)	4602771	5817623	5767028	79234042	32584150	42966990	1508181733	424951699	322438613
PC(30:0e)+H	PC(30:0e)+H	PC	(30:0e)	304701	316988	226846	3779659	1251994	1715938	118766002	26689414	15498310
PC(14:0p/16:0)+H	PC(30:0p)+H	PC	(14:0p/16:0)	93264	143555	110028	2345472	971311	1283197	52865186	16180389	10403201
PC(30:1)+H	PC(30:1)+H	PC	(16:1/14:0)	407718	849085	1082984	10532736	9075411	11430108	102121238	32513960	45150121
PC(30:1e)+H	PC(30:1e)+H	PC	(30:1e)	0	0	0	194714	44585	30466	538629	133688	391370
PC(30:2)+H	PC(30:2)+H	PC	(30:2)	0	0	0	91216	72114	11601	100994	82363	99437
PC(30:3)+H	PC(30:3)+H	PC	(30:3)	0	0	0	411483	241471	318855	3584986	979753	984458
PC(31:0)+H	PC(31:0)+H	PC	(31:0)	562500	627388	559937	7354254	3551647	4512756	208041413	47825353	32844338
PC(31:0e)+H	PC(31:0e)+H	PC	(31:0e)	0	0	0	49923	88484	128105	9698835	2094762	514492
PC(31:0p)+H	PC(31:0p)+H	PC	(31:0p)	18256	28763	21665	444680	169613	327796	21283193	6617157	3517941
PC(31:1)+H	PC(31:1)+H	PC	(31:1)	593255	949941	862657	6727970	6636680	4282044	17179788	6946787	15305665
PC(31:2)+H	PC(31:2)+H	PC	(31:2)	243432	226001	203058	2400815	2395224	2729234	29484643	9514034	13869594
PC(31:3)+H	PC(31:3)+H	PC	(31:3)	0	0	0	55491	101513	122586	960142	388584	807060
PC(32:0)+H	PC(32:0)+H	PC	(16:0/16:0)	4452214	2889246	2375948	41137504	10567863	16394058	2087207816	347790235	169800028
PC(32:0e)+H	PC(32:0e)+H	PC	(32:0e)	307077	147749	118830	1737991	483328	487339	139262430	19947233	9190125
PC(32:1)+H	PC(32:1)+H	PC	(16:0/16:1)	14353042	28328769	26110626	529609443	433380858	542241915	5381706408	2063475962	3055296955
PC(32:1e)+H	PC(32:1e)+H	PC	(16:0e/16:1)	1078010	1712709	1981262	25413205	6266129	24022660	345203508	106814263	136122307
PC(14:0p/18:1)+H	PC(32:1p)+H	PC	(14:0p/18:1)	583540	789904	785394	2906868	2478844	3294616	27864431	11846874	15056673
PC(16:1/16:1)+H	PC(32:2)+H	PC	(16:1/16:1)	0	0	0	628969	185957	1198006	6309128	1354212	3441281
PC(18:1/14:1)+H	PC(32:2)+H	PC	(18:1/14:1)	235289	894714	789383	5724137	6037716	11659556	54210287	22696729	44333826
PC(21:1/11:1)+H	PC(32:2)+H	PC	(21:1/11:1)	265285	403424	382575	4643474	3896380	4635801	37184531	11008614	18631916
PC(32:2)+H	PC(32:2)+H	PC	(32:2)	72433	115745	80056	918661	988226	1104863	14177352	4586534	7158142
PC(32:3)+H	PC(32:3)+H	PC	(32:3)	150303	208901	176005	4872181	1947270	3036516	99696800	34888565	22920925
PC(33:0)+H	PC(33:0)+H	PC	(33:0)	299515	449912	377067	3892036	2881155	4444185	130248419	40153652	41041615
PC(33:0e)+H	PC(33:0e)+H	PC	(33:0e)	0	0	0	0	0	0	8841852	2396748	908284
PC(33:0p)+H	PC(33:0p)+H	PC	(33:0p)	18466	108597	230880	2084485	1917217	2388961	28996703	14237415	9002347
PC(17:1/16:0)+H	PC(33:1)+H	PC	(17:1/16:0)	3413139	5449602	5653190	32372315	43970587	34366470	384339956	125506096	267487377
PC(33:1)+H	PC(33:1)+H	PC	(33:1)	314910	460149	390434	2541014	2769235	3468141	42665807	15482314	19700029
PC(33:2)+H	PC(33:2)+H	PC	(33:2)	761232	2090402	1559426	18403227	20292111	24424599	322583076	130677739	152994104
PC(33:3)+H	PC(33:3)+H	PC	(33:3)	72710	194838	177564	3107071	3361698	3945206	43629291	15720761	21568498
PC(33:5)+H	PC(33:5)+H	PC	(33:5)	0	0	0	298512	308865	334620	3269444	1121079	1639091
PC(34:0)+H	PC(34:0)+H	PC	(18:0/16:0)	359178	138684	116188	2875745	379151	455950	164192583	23179515	10404795
PC(34:0e)+H	PC(34:0e)+H	PC	(34:0e)	0	0	0	258427	0	0	128258	2004318	974019
PC(34:1)+H	PC(34:1)+H	PC	(16:0/18:1)	57137534	98672207	107106161	795131990	572739671	756829498	11789012881	4335104749	3983319960
PC(16:1/18:1)+H	PC(34:2)+H	PC	(16:1/18:1)	10629527	19254471	18577229	171372963	149702675	191866057	1476657263	559485361	919564135
PC(34:2e)+H	PC(34:2e)+H	PC	(34:2e)	471190	661956	796266	5739580	4765008	6466545	61371776	19953333	30353824
PC(16:1p/18:1)+H	PC(34:2p)+H	PC	(16:1p/18:1)	348115	445260	450255	1314342	1782390	2065504	16487697	4256585	8290040
PC(12:0/22:3)+H	PC(34:3)+H	PC	(12:0/22:3)	207158	391727	370405	3588907	3207816	4182560	33314570	9789081	16389868
PC(16:1/18:2)+H	PC(34:3)+H	PC	(16:1/18:2)	303230	592858	570302	3533522	3247461	3848673	28631133	8520327	14523682
PC(34:3)+H	PC(34:3)+H	PC	(34:3)	80962	88993	72423	2591499	502094	1155065	105092248	26409680	9652776
PC(34:3p)+H	PC(34:3p)+H	PC	(34:3p)	87579	58778	12971	45418	35989	26470	6555518	212250	1794458
PC(34:4)+H	PC(34:4)+H	PC	(34:4)	421603	1081928	1026392	24732541	17463581	24723821	242010300	126604844	126424875
PC(34:4p)+H	PC(34:4p)+H	PC	(34:4p)	0	0	0	192902	202640	230415	556467	317251	716708
PC(35:0)+H	PC(35:0)+H	PC	(35:0)	0	0	0	196324	9486	61320	19014704	5747836	2095895
PC(35:0p)+H	PC(35:0p)+H	PC	(35:0p)	0	0	0	418255	354606	853138	14203794	4259540	3307349
PC(19:1/16:0)+H	PC(35:1)+H	PC	(19:1/16:0)	3133288	3555178	3893050	13215816	9084072	11886536	260862746	89727407	68046608
PC(17:0/18:1)+H	PC(35:1)+H	PC	(17:0/18:1)	2782400	1047864	4954808	5958611	5798727	8972067	95375582	34107661	44071513
PC(35:1p)+H	PC(35:1p)+H	PC	(35:1p)	139966	33374	127542	369383	214242	469772	4355549	1415727	2022928
PC(19:1/16:1)+H	PC(35:2)+H	PC	(19:1/16:1)	747200	2278320	1495286	7672831	6006169	6978198	60291353	21889342	31838641
PC(24:1/11:1)+H	PC(35:2)+H	PC	(24:1/11:1)	1292254	3034693	3387131	8385675	9318449	11591575	91610347	25851589	45034606
PC(35:2)+H	PC(35:2)+H	PC	(35:2)	141960	130151	141313	305530	224042	278553	9095747	1881723	1732148
PC(35:2p)+H	PC(35:2p)+H	PC	(35:2p)	0	0	0	134779	90719	68046	869637	47608	217366
PC(35:3)+H	PC(35:3)+H	PC	(35:3)	35879	134764	136385	765021	709713	883674	5599691	1713948	3176495
PC(35:4)+H	PC(35:4)+H	PC	(35:4)	806222	2113943	1934974	25307595	24489607	30362405	530262258	185381102	208972036
PC(35:5)+H	PC(35:5)+H	PC	(35:5)	612374	965134	858495	11032586	11108646	14167399	133092399	42870576	69639718
PC(35:6)+H	PC(35:6)+H	PC	(35:6)	69916	137340	135718	3344582	2825816	8996555	36094208	14513254	58654585
PC(36:1)+H	PC(36:1)+H	PC	(18:0/18:1)	14354613	23074082	21222962	106232599	52856440	79634742	2676365291	877117929	526965795
PC(36:1e)+H	PC(36:1e)+H	PC	(36:1e)	142639	268436	289657	2872654	942616	1605243	135322709	35375263	16657216
PC(20:1p/16:0)+H	PC(36:1p)+H	PC	(20:1p/16:0)	1149625	1349211	1178245	5773630	4422143	6031619	82452717	31737314	35955504
PC(36:2)+H	PC(36:2)+H	PC	(18:1/18:1)	12347664	21045862	22975327	147346375	137578739	174502375	1425739272	536108764	733903650
PC(36:2e)+H	PC(36:2e)+H	PC	(36:2e)	935383	379451	316803	1002762	962875	1250333	48894919	15312247	7197236
PC(18:2p/18:0)+H	PC(36:2p)+H	PC	(18:2p/18:0)	37748	143970	116727	830809	986409	1259345	4209807	2265577	4214776
PC(36:2p)+H	PC(36:2p)+H	PC	(36:2p)	351335	638617	847840	3237954	2583674	3249846	46290306	11480192	13324388
PC(36:3)+H	PC(36:3)+H	PC	(16:0/20:3)	1690448	3191987	3015638	17836489	15541315	21796250	143920100	51635572	88458918
PC(18:2p/18:1)+H	PC(36:3p)+H	PC	(18:2p/18:1)	691197	1451070	1251471	6962543	6461936	8420711	68087446	23446875	42920697
PC(36:4)+H	PC(36:4)+H	PC	(16:0/20:4)	9394271	20097905	19564301	152665192	129101686	165501686	1209501671	477832927	789596316
PC(36:4e)+H	PC(36:4e)+H	PC	(36:4e)	219433	254367	351759	1569224	1206603	1500749	34148260	9926038	8671422
PC(36:4p)+H	PC(36:4p)+H	PC	(36:4p)	9437	61315	46368	342578	341984	391212	1614369	749413	1101011
PC(18:4/18:1)+H	PC(36:5)+H	PC	(18:4/18:1)	53274	145234	154703	2725178	1945785	2328049	18298553	6366793	13937647
PC(36:5)+H	PC(36:5)+H	PC	(36:5)	177514	410206	493593	8713623	6888474	8218716	63955426	22288016	55888048
PC(16:0e/20:5)+H	PC(36:5e)+H	PC	(16:0e/20:5)	834024	1544292	1633714	6935415	6895680	7951932	59953983	21782986	36822222
PC(36:5p)+H	PC(36:5p)+H	PC	(36:5p)	1320	26285	11034	299606	296764	387818	1253127	657998	982420
PC(36:6)+H	PC(36:6)+H	PC	(36:6)	0	0	0	287913	289054	264226	1514897	471056	1197797
PC(36:6p)+H	PC(36:6p)+H	PC	(36:6p)	0	0	0	522516	529248	588457	1372048	911732	666167
PC(37:1)+H	PC(37:1)+H	PC	(37:1)	326333	641380	621942	1088926	1129462	1495669	35558474	11785222	5959811
PC(37:2)+H	PC(37:2)+H	PC	(37:2)	1113462	1742791	1816241	6255028	4749550	7379115	92279827	26237587	32341794
PC(37:3)+H	PC(37:3)+H	PC	(37:3)	397112	804738	786513	2556994	2071053	2753874	24785467	8123931	10947225
PC(37:4)+H	PC(37:4)+H	PC	(37:4)	676997	1381302	1391308	3766460	3736291	4733406	32341740	10867211	15524541
PC(15:0/22:5)+H	PC(37:5)+H	PC	(15:0/22:5)	156900	344526	342675	1523155	1484416	1617145	9727396	3101276	5148944
PC(37:5)+H	PC(37:5)+H	PC	(37:5)	318105	423021	409321	4047138	3850702	4738137	75095817	28598880	31554457
PC(37:6)+H	PC(37:6)+H	PC	(37:6)	136541	390953	363517	7394508	6542390	7276105	134391160	43437543	50721848
PC(38:1)+H	PC(38:1)+H	PC	(38:1)	541951	374505	1068409	2008647	1392397	1447825	94746198	29174352	11662458
PC(16:0/22:2)+H	PC(38:2)+H	PC	(16:0/22:2)	1023062	3746088	2833811	15860475	13910180	17935763	254078121	94809394	100011137
PC(38:2e)+H	PC(38:2e)+H	PC	(38:2e)	0	0	0	565336	178521	364565	9718490	5538982	2886988
PC(28:1/10:2)+H	PC(38:3)+H	PC	(28:1/10:2)	1217945	2009628	2634703	8205689	6275382	7778534	120878165	42671315	39116391
PC(18:0/20:3)+H	PC(38:3)+H	PC	(18:0/20:3)	5608615	10179956	11780551	24550013	19834404	25375969	249200358	92388362	89148939
PC(38:3e)+H	PC(38:3e)+H	PC	(38:3e)	36942	69567	74682	1135142	699972	832972	20927578	5425967	6070033
PC(24:0/14:4)+H	PC(38:4)+H	PC	(24:0/14:4)	2221230	4362980	4738731	14991589	14373161	17962577	119704693	41215604	66433405
PC(18:0/20:4)+H	PC(38:4)+H	PC	(18:0/20:4)	17483958	34897014	39831332	110336870	103353537	126598098	912804948	367123332	456219389
PC(38:4e)+H	PC(38:4e)+H	PC	(38:4e)	455091	426209	482502	6176603	4995887	7130835	90639464	25480228	36083224
PC(38:4p)+H	PC(38:4p)+H	PC	(38:4p)	713157	1404646	1298517	5623064	5918103	7547131	46811765	16829415	33283941
PC(18:1/20:4)+H	PC(38:5)+H	PC	(18:1/20:4)	11449628	24490329	23673925	155745407	138651176	178269826	1079638231	443320655	707512020
PC(16:0/22:6)+H	PC(38:6)+H	PC	(16:0/22:6)	0	0	0	334908	344430	431665	3301071	1396051	2013185
PC(18:1/20:5)+H	PC(38:6)+H	PC	(18:1/20:5)	6779923	12734088	12128090	40965770	41881252	49101290	299592871	96385480	147209871
PC(38:6)+H	PC(38:6)+H	PC	(38:6)	168670	302465	337779	1306540	1244633	1528541	9693004	4337657	6028592
PC(38:6e)+H	PC(38:6e)+H	PC	(38:6e)	995876	2511086	2389073	8313063	5999773	8096177	48793626	19073302	32073670
PC(38:6p)+H	PC(38:6p)+H	PC	(38:6p)	1106556	2335929	2559792	11264696	9874598	14138543	68137931	31562996	50812002
PC(38:7)+H	PC(38:7)+H	PC	(38:7)	469376	787255	762519	7278466	5528504	7748374	48359785	18183225	33060843
PC(39:3)+H	PC(39:3)+H	PC	(39:3)	60048	253599	236247	825124	664776	857006	10074118	4042324	3599834
PC(39:4)+H	PC(39:4)+H	PC	(39:4)	106623	311653	363814	583769	831288	1181466	3764668	2681453	4488329
PC(39:5)+H	PC(39:5)+H	PC	(39:5)	460907	847895	846817	2199359	2246591	2734654	14849064	5472785	8094852
PC(39:6)+H	PC(39:6)+H	PC	(39:6)	363776	688682	690252	2120943	2091226	2458762	13242595	5115345	7263732
PC(39:7)+H	PC(39:7)+H	PC	(39:7)	56257	18932	57154	245885	159847	114030	1740294	275171	460596
PC(40:1)+H	PC(40:1)+H	PC	(40:1)	0	0	0	223423	12228	122727	24758649	8035527	2770742
PC(40:2)+H	PC(40:2)+H	PC	(40:2)	11471	58462	98252	1628851	1362984	1752107	37550967	14319920	11884426
PC(40:3)+H	PC(40:3)+H	PC	(40:3)	95201	157022	297187	1240209	1228392	1520557	17158437	5881270	7057461
PC(40:3p)+H	PC(40:3p)+H	PC	(40:3p)	13777	45386	99017	608956	383896	658993	11065763	2675669	3684100
PC(40:4)+H	PC(40:4)+H	PC	(40:4)	2038465	4097217	5521298	13079879	8503255	14253766	115203954	40789419	55358204
PC(40:5)+H	PC(40:5)+H	PC	(18:1/22:4)	7147724	14103713	15191429	34009226	32683347	41313293	248040907	101126845	119844944
PC(40:5e)+H	PC(40:5e)+H	PC	(40:5e)	105343	320868	410181	1292640	1589237	1339751	16333641	4669949	5591935
PC(18:0/22:6)+H	PC(40:6)+H	PC	(18:0/22:6)	1156960	2571737	2677364	13913706	13529273	22356511	104912863	52151880	81789080
PC(20:3/20:3)+H	PC(40:6)+H	PC	(20:3/20:3)	7293565	14518867	14889706	36636027	35270303	41922413	245243158	94456000	118046565
PC(40:6e)+H	PC(40:6e)+H	PC	(40:6e)	315472	724752	836623	4084783	2781592	1567767	28123198	10080132	12676041
PC(40:6p)+H	PC(40:6p)+H	PC	(40:6p)	223556	772277	734407	2371860	1954178	2622667	17136802	6721238	7738071
PC(20:3/20:4)+H	PC(40:7)+H	PC	(20:3/20:4)	1282401	2591495	2408340	8872549	8657668	10811380	55079478	19577905	31892380
PC(40:7)+H	PC(40:7)+H	PC	(40:7)	57115	139379	162919	475980	579759	704352	3751728	1296351	2219531
PC(40:7p)+H	PC(40:7p)+H	PC	(40:7p)	373074	864902	783967	3833467	3640745	4767614	19587132	8605721	14529135
PC(40:8)+H	PC(40:8)+H	PC	(40:8)	18393	32255	17394	308734	469715	469742	866539	1113470	606524
PC(40:9)+H	PC(40:9)+H	PC	(40:9)	250728	494056	437201	118333	140654	2212508	12556276	1169429	76071
PC(41:5)+H	PC(41:5)+H	PC	(41:5)	5389	48601	99717	223947	184873	279181	465955	611325	541978
PC(41:6)+H	PC(41:6)+H	PC	(41:6)	40175	177451	194621	484153	300102	320600	1584945	1116418	796995
PC(41:7)+H	PC(41:7)+H	PC	(41:7)	16601	68343	127215	327811	347996	297400	1446872	574323	460063
PC(42:1)+H	PC(42:1)+H	PC	(42:1)	0	0	0	104336	60000	12157	12214976	5859261	1589612
PC(42:10)+H	PC(42:10)+H	PC	(42:10)	75400	91965	102256	324751	244027	371237	2711854	597427	1111573
PC(42:2)+H	PC(42:2)+H	PC	(42:2)	8057	9377	27272	775882	666964	809519	27301630	12167560	7553855
PC(42:3p)+H	PC(42:3p)+H	PC	(42:3p)	0	0	0	71738	21793	55290	688883	516737	225527
PC(42:4p)+H	PC(42:4p)+H	PC	(42:4p)	43717	215862	205533	539284	390398	485673	4705712	1897518	1503785
PC(42:6)+H	PC(42:6)+H	PC	(42:6)	63862	177219	265448	827589	526664	1053065	2148818	2324189	2943310
PC(42:6e)+H	PC(42:6e)+H	PC	(42:6e)	922337	712128	557875	1133591	1180121	2608847	11602850	2922275	2228431
PC(42:7)+H	PC(42:7)+H	PC	(42:7)	148736	288245	310230	826301	887737	1177085	6471063	2309885	8580354
PC(42:7p)+H	PC(42:7p)+H	PC	(42:7p)	0	0	0	0	0	0	0	0	0
PC(42:8)+H	PC(42:8)+H	PC	(42:8)	13084	44531	64376	363119	276813	535165	1175356	669385	1410597
PC(42:9)+H	PC(42:9)+H	PC	(42:9)	0	0	0	529808	97790	425505	531716	253268	437388
PC(44:1)+H	PC(44:1)+H	PC	(44:1)	0	0	0	0	0	0	7277079	3453354	132204
PC(44:2)+H	PC(44:2)+H	PC	(44:2)	0	0	0	216407	176688	339912	13242236	7068657	2775961
PE(12:0/14:0)−H	PE(26:0)−H	PE	(12:0/14:0)	835307	539707	679586	252112	998694	279666	9383820	1583508	3187973
PE(26:0)−H	PE(26:0)−H	PE	(26:0)	11173607	10466733	11364016	15551617	16650782	11779956	197251294	35467547	40448099
PE(32:0p)−H	PE(32:0p)−H	PE	(32:0p)	0	0	0	0	0	0	0	0	0
PE(16:0/16:1)−H	PE(32:1)−H	PE	(16:0/16:1)	94951	37124	17572	1065144	660921	558596	10308810	3058037	3321271
PE(16:0p/16:1)−H	PE(32:1p)−H	PE	(16:0p/16:1)	0	0	0	290319	201459	198653	4399204	1633435	1276485
PE(16:1/16:1)−H	PE(32:2)−H	PE	(16:1/16:1)	0	0	0	0	0	0	0	0	0
PE(17:1/16:0)−H	PE(33:1)−H	PE	(17:1/16:0)	0	0	0	23380	36474	78191	1133848	212068	1105410
PE(33:1p)−H	PE(33:1p)−H	PE	(33:1p)	0	0	0	0	0	0	318858	323710	144975
PE(16:0/18:1)−H	PE(34:1)−H	PE	(16:0/18:1)	206504	334455	310634	5617754	2995755	3694359	113349997	32998453	23003406
PE(16:0p/18:1)−H	PE(34:1p)−H	PE	(16:0p/18:1)	142407	254608	224241	2690549	1668689	2211092	79765754	24986491	15759256
PE(16:1/18:1)−H	PE(34:2)−H	PE	(16:1/18:1)	30506	58033	40849	1587297	1142572	1186081	15456377	3769218	5161718
PE(18:1p/16:1)−H	PE(34:2p)−H	PE	(18:1p/16:1)	0	0	0	139831	144147	165536	1782364	543231	860529
PE(16:0p/18:2)−H	PE(34:2p)−H	PE	(16:0p/18:2)	0	0	0	33554	16533	15392	1223756	487117	320193
PE(16:1/18:2)−H	PE(34:3)−H	PE	(16:1/18:2)	0	0	0	0	0	0	0	0	0
PE(16:0p/18:3)−H	PE(34:3p)−H	PE	(16:0p/18:3)	0	0	0	40999	24227	19109	273614	165267	188813
PE(17:0/18:1)−H	PE(35:1)−H	PE	(17:0/18:1)	2209	20007	14312	262875	109011	142165	8120137	2433136	1169734
PE(17:1/18:1)−H	PE(35:2)−H	PE	(17:1/18:1)	0	0	0	586753	433919	397708	3719476	1400061	1829147
PE(18:0/18:1)−H	PE(36:1)−H	PE	(18:0/18:1)	191345	351458	310031	3036954	1174787	1895087	152971684	36247881	13477931
PE(36:1)−H	PE(36:1)−H	PE	(36:1)	0	0	0	500440	99027	104474	374526	718588	387728
PE(18:0p/18:1)−H	PE(36:1p)−H	PE	(18:0p/18:1)	41360	86530	51250	776959	325626	527579	54566522	14237459	4369348
PE(18:1/18:1)−H	PE(36:2)−H	PE	(18:1/18:1)	79739	204486	160651	2758963	2118188	2188103	45653047	11981057	12619870
PE(18:1p/18:1)−H	PE(36:2p)−H	PE	(18:1p/18:1)	34733	87099	85100	1597350	1410722	1699456	24484446	8227246	9609538
PE(16:0/20:3)−H	PE(36:3)−H	PE	(16:0/20:3)	0	0	0	328447	303902	232609	4341687	1797352	1447398
PE(18:1/18:2)−H	PE(36:3)−H	PE	(18:1/18:2)	7100	46368	35709	1490445	1196964	1111499	11136679	3198549	4513279
PE(16:0p/20:3)−H	PE(36:3p)−H	PE	(16:0p/20:3)	19304	44949	42032	1268423	870438	1020219	20589211	5949373	6328041
PE(36:3p)−H	PE(36:3p)−H	PE	(36:3p)	0	0	0	572319	729990	576361	6870467	2004004	3275495
PE(16:0/20:4)−H	PE(36:4)−H	PE	(16:0/20:4)	50380	119492	134119	4151507	3321396	3055142	30639960	11435475	15410484
PE(16:1/20:3)−H	PE(36:4)−H	PE	(16:1/20:3)	0	0	0	0	0	0	0	0	0
PE(16:0p/20:4)−H	PE(36:4p)−H	PE	(16:0p/20:4)	449635	942082	748609	28906215	21933749	21043073	274931065	84010230	109932723
PE(16:0/20:5)−H	PE(36:5)−H	PE	(16:0/20:5)	0	0	0	126255	112308	92195	810145	276599	407791
PE(16:1/20:4)−H	PE(36:5)−H	PE	(16:1/20:4)	0	0	0	68330	37926	48663	31917	65750	75139
PE(18:0/20:2)−H	PE(38:2)−H	PE	(18:0/20:2)	0	0	0	232491	69821	117203	7924892	1739209	1172649
PE(16:0p/22:2)−H	PE(38:2p)−H	PE	(16:0p/22:2)	9179	3624	8871	82701	65923	96853	3443986	3591328	2518852
PE(18:0/20:3)−H	PE(38:3)−H	PE	(18:0/20:3)	18783	42202	34658	1044458	449921	608800	28252412	6814471	4377476
PE(18:1/20:2)−H	PE(38:3)−H	PE	(18:1/20:2)	0	0	0	610637	511182	451475	6939566	2406010	2393191
PE(16:0p/22:3)−H	PE(38:3p)−H	PE	(16:0p/22:3)	39875	90868	63938	1387337	930503	1070084	34481880	9303204	7516530
PE(38:3p)−H	PE(38:3p)−H	PE	(38:3p)	19051	29963	30405	630524	301635	387666	19467240	5106644	3168267
PE(18:0/20:4)−H	PE(38:4)−H	PE	(18:0/20:4)	405290	885331	777260	19075195	14174694	15495000	267184606	74663348	76291857
PE(18:1/20:3)−H	PE(38:4)−H	PE	(18:1/20:3)	0	0	0	2108646	2060060	1889938	35755830	5695213	7573573
PE(38:4e)−H	PE(38:4e)−H	PE	(38:4e)	12581	44768	48122	499125	427049	496021	8606627	2422792	2636873
PE(16:0p/22:4)−H	PE(38:4p)−H	PE	(16:0p/22:4)	151270	294349	257827	6786173	5264637	5345856	106540361	28801033	33997114
PE(18:0p/20:4)−H	PE(38:4p)−H	PE	(18:0p/20:4)	479623	952310	840565	17498934	13054619	14200003	262698265	84197752	88532563
PE(38:4p)−H	PE(38:4p)−H	PE	(38:4p)	0	0	0	490390	1202551	774262	3944790	2024931	2731247
PE(18:0/20:5)−H	PE(38:5)−H	PE	(18:0/20:5)	0	0	0	1107104	755449	917099	9752576	2991200	3408702
PE(18:1/20:4)−H	PE(38:5)−H	PE	(18:1/20:4)	123816	228908	183672	6641324	4927459	5540034	54661096	16512000	26062282
PE(16:0p/22:5)−H	PE(38:5p)−H	PE	(16:0p/22:5)	50814	121783	97024	3372903	2325193	2416725	34245607	9331589	11576069
PE(18:1p/20:4)−H	PE(38:5p)−H	PE	(18:1p/20:4)	381469	819881	630017	24437101	18622450	18857868	191478808	56117605	82006414
PE(16:0/22:6)−H	PE(38:6)−H	PE	(16:0/22:6)	13771	54424	41714	2116853	1468262	1602887	16018589	4647496	6464006
PE(16:1/22:5)−H	PE(38:6)−H	PE	(16:1/22:5)	0	0	0	0	0	0	0	0	0
PE(18:0/22:3)−H	PE(40:3)−H	PE	(18:0/22:3)	0	0	0	272443	88099	177541	10417001	2484548	1206377
PE(18:0p/22:3)−H	PE(40:3p)−H	PE	(18:0p/22:3)	24540	63366	92585	455346	212622	292005	18035611	4014990	2343446
PE(18:0/22:4)−H	PE(40:4)−H	PE	(18:0/22:4)	35382	121013	91387	2004137	1562228	1803772	39779114	12796498	10745717
PE(18:0p/22:4)−H	PE(40:4p)−H	PE	(18:0p/22:4)	61500	206627	205977	2758411	1944484	2289797	64276841	19859786	16876343
PE(40:4p)−H	PE(40:4p)−H	PE	(40:4p)	92698	59537	50475	548298	368519	433752	9157475	2145907	2592482
PE(18:0/22:5)−H	PE(40:5)−H	PE	(18:0/22:5)	45634	118396	95194	3869623	2888128	2869796	37421032	13739126	12316101
PE(18:1/22:4)−H	PE(40:5)−H	PE	(18:1/22:4)	0	0	0	1569018	1627205	1278158	14695125	4102260	5804913
PE(18:0p/22:5)−H	PE(40:5p)−H	PE	(18:0p/22:5)	157299	337815	277143	6790956	4972075	5471298	91297210	28760281	31407034
PE(40:5p)−H	PE(40:5p)−H	PE	(40:5p)	25753	108558	79567	2462095	2273855	2408565	29093596	8725776	11101220
PE(18:0/22:6)−H	PE(40:6)−H	PE	(18:0/22:6)	59238	140868	117954	5238826	3569772	3883856	56312579	14746047	17878948
PE(18:1/22:5)−H	PE(40:6)−H	PE	(18:1/22:5)	0	0	0	889236	633031	251098	3642796	2001033	2376216
PE(18:0p/22:6)−H	PE(40:6p)−H	PE	(18:0p/22:6)	166678	345040	287739	8309820	5910984	6060502	103751941	30759033	30452499
PE(18:1p/22:5)−H	PE(40:6p)−H	PE	(18:1p/22:5)	8409	53482	67277	2314717	1952777	2123023	17862011	5902815	9449441
PE(18:1/22:6)−H	PE(40:7)−H	PE	(18:1/22:6)	11085	72340	28437	2185913	1607630	1761904	12967591	4420414	6910775
PE(18:1p/22:6)−H	PE(40:7p)−H	PE	(18:1p/22:6)	32343	110093	93609	3689468	2948804	3075709	27896860	7085740	11040470
PEt(16:0/16:1)−H	PEt(32:1)−H	PEt	(16:0/16:1)	0	0	0	128304	39382	35309	1107104	3401896	115566
PEt(32:4)−H	PEt(32:4)−H	PEt	(32:4)	279069077	278343754	271228360	396595593	398286346	297179039	5018214562	759502356	1098284317
PEt(18:0/16:1)−H	PEt(34:1)−H	PEt	(18:0/16:1)	0	0	0	0	0	0	5241098	871252	52438
PEt(18:2/18:2)−H	PEt(36:4)−H	PEt	(18:2/18:2)	2500	12347	20658	116156	236214	150452	3350436	1066561	462363
PG(12:0/14:0)−H	PG(26:0)−H	PG	(12:0/14:0)	2996776	3034456	3078805	3509108	3360390	3108790	52042151	7349663	12209703
PG(16:0/14:0)−H	PG(30:0)−H	PG	(16:0/14:0)	331753	313756	323116	197723	174778	271719	6298537	620232	1265644
PG(16:0/16:1)−H	PG(32:1)−H	PG	(16:0/16:1)	0	0	0	0	0	0	0	0	0
PG(17:1/16:0)−H	PG(33:1)−H	PG	(17:1/16:0)	0	0	0	0	0	0	0	0	0
PG(16:0/18:1)−H	PG(34:1)−H	PG	(16:0/18:1)	0	0	0	159437	16537	208936	411800	325227	455332
PG(16:1/18:1)−H	PG(34:2)−H	PG	(16:1/18:1)	0	0	0	0	0	0	0	0	0
PG(17:0/18:1)−H	PG(35:1)−H	PG	(17:0/18:1)	21746	37325	27360	430603	313435	339642	8614846	2313954	1858741
PG(17:1/18:1)−H	PG(35:2)−H	PG	(17:1/18:1)	0	0	0	56595	37085	42525	582042	143033	236520
PG(18:0/18:1)−H	PG(36:1)−H	PG	(18:0/18:1)	0	0	0	1016441	964046	1046676	6363266	2399116	4766762
PG(18:1/18:1)−H	PG(36:2)−H	PG	(18:1/18:1)	0	0	0	163448	223055	139385	2448112	727509	2369852
PG(18:1/18:2)−H	PG(36:3)−H	PG	(18:1/18:2)	0	0	0	12323	15255	16153	0	0	0
PG(20:1/18:1)−H	PG(38:2)−H	PG	(20:1/18:1)	0	0	0	0	0	0	0	0	0
PG(18:1/20:2)−H	PG(38:3)−H	PG	(18:1/20:2)	21801	25235	21324	151709	71063	68036	7717501	1554686	570911
PG(18:0/20:4)−H	PG(38:4)−H	PG	(18:0/20:4)	0	0	0	0	0	0	0	0	0
PG(18:1/20:3)−H	PG(38:4)−H	PG	(18:1/20:3)	0	0	0	0	0	0	0	0	0
PG(18:1/20:4)−H	PG(38:5)−H	PG	(18:1/20:4)	0	0	0	0	0	0	0	0	0
PG(16:0/22:6)−H	PG(38:6)−H	PG	(16:0/22:6)	0	0	0	97613	99608	76825	276833	20025	25349
PG(18:1/22:4)−H	PG(40:5)−H	PG	(18:1/22:4)	0	0	0	18159	12885	17817	0	0	0
PG(18:0/22:6)−H	PG(40:6)−H	PG	(18:0/22:6)	0	0	0	80771	72399	66510	0	0	0
PG(18:1/22:5)−H	PG(40:6)−H	PG	(18:1/22:5)	0	0	0	18976	6070	8874	0	0	0
PG(18:1/22:6)−H	PG(40:7)−H	PG	(18:1/22:6)	0	0	0	862230	778194	782003	973979	347757	591283
PG(20:1/22:6)−H	PG(42:7)−H	PG	(20:1/22:6)	0	0	0	0	0	0	0	0	0
PG(20:2/22:6)−H	PG(42:8)−H	PG	(20:2/22:6)	0	0	0	0	0	0	0	0	0
PI(16:0/18:1)−H	PI(34:1)−H	PI	(16:0/18:1)	207265	403469	326785	1666070	5377187	6216683	107496050	31734088	25126566
PI(16:1/18:1)−H	PI(34:2)−H	PI	(16:1/18:1)	0	0	0	34339	24300	11368	59477	76990	129444
PI(18:0/18:1)−H	PI(36:1)−H	PI	(18:0/18:1)	123836	155434	144386	2550655	852089	1197680	73101178	13569781	5409439
PI(16:0/20:3)−H	PI(36:3)−H	PI	(16:0/20:3)	0	0	0	125921	178738	111964	972536	313168	444576
PI(18:1/18:2)−H	PI(36:3)−H	PI	(18:1/18:2)	0	0	0	166755	75972	152945	657662	167481	290916
PI(16:0/20:4)−H	PI(36:4)−H	PI	(16:0/20:4)	0	0	0	150229	0	100424	0	684790	704467
PI(17:0/20:4)−H	PI(37:4)−H	PI	(17:0/20:4)	0	0	0	94240	77828	80481	227158	182549	293712
PI(20:1/18:1)−H	PI(38:2)−H	PI	(20:1/18:1)	0	0	0	0	0	0	278494	132307	81096
PI(18:0/20:3)−H	PI(38:3)−H	PI	(18:0/20:3)	76367	100481	97880	2117265	837601	1015849	28117369	6105706	4678508
PI(18:1/20:3)−H	PI(38:4)−H	PI	(18:1/20:3)	5237	31011	16173	593889	492634	465764	10496166	2294116	2055798
PI(18:0/20:5)−H	PI(38:5)−H	PI	(18:0/20:5)	0	0	0	162798	119010	160884	781145	382314	467393
PI(18:1/20:4)−H	PI(38:5)−H	PI	(18:1/20:4)	0	0	0	1066944	1133842	709902	440501	1267290	981722
PI(18:0/22:4)−H	PI(40:4)−H	PI	(18:0/22:4)	0	0	0	938473	539919	655085	10271169	3056243	3723958
PMe(14:0/14:0)−H	PMe(28:0)−H	PMe	(14:0/14:0)	197233	186704	197921	233689	185238	197160	3102192	415794	650827
PMe(34:5)−H	PMe(34:5)−H	PMe	(34:5)	217296466	220166027	191306985	206143596	192082882	182843648	3509160915	489928236	692043430
PMe(42:6)−H	PMe(42:6)−H	PMe	(42:6)	445198	687639	616907	9188019	3439530	4622705	307712685	55136066	24483243
PS(12:0/14:0)−H	PS(26:0)−H	PS	(12:0/14:0)	1614543	1496858	1622028	2032182	1992144	1914011	28345507	3516088	6713480
PS(16:0/16:1)−H	PS(32:1)−H	PS	(16:0/16:1)	0	0	0	709341	440595	1551648	4883070	1785540	2550206
PS(33:1)−H	PS(33:1)−H	PS	(33:1)	0	0	0	0	0	0	0	0	0
PS(18:0/16:1)−H	PS(34:1)−H	PS	(18:0/16:1)	97442	177311	158102	2907496	1967089	2337056	64986325	16745343	12735657
PS(34:3p)−H	PS(34:3p)−H	PS	(34:3p)	0	0	0	0	0	0	0	0	0
PS(35:0)−H	PS(35:0)−H	PS	(35:0)	538666	997297	827237	17896669	16069304	15753705	120092087	34487043	53222088
PS(17:0/18:1)−H	PS(35:1)−H	PS	(17:0/18:1)	31744	72837	67051	1172966	1168309	741564	22190294	6860227	1750383
PS(35:1)−H	PS(35:1)−H	PS	(35:1)	0	0	0	56732	40007	142128	1062434	396446	948516
PS(35:2)−H	PS(35:2)−H	PS	(35:2)	0	0	0	0	0	0	0	0	0
PS(18:0/18:2)−H	PS(36:2)−H	PS	(18:0/18:2)	159698	297725	309362	5530936	3866723	4266397	66482481	18498648	22120371
PS(16:0/20:3)−H	PS(36:3)−H	PS	(16:0/20:3)	0	0	0	433637	394704	451184	3608732	959942	1466983
PS(18:1/18:2)−H	PS(36:3)−H	PS	(18:1/18:2)	0	0	0	457286	369805	414584	3219733	837026	1521022
PS(36:3)−H	PS(36:3)−H	PS	(36:3)	12858	39203	37556	695047	554855	475378	18794108	4205650	3460278
PS(36:3p)−H	PS(36:3p)−H	PS	(36:3p)	5987	32847	28797	401345	273130	306407	12147615	3764508	2312891
PS(16:0/20:4)−H	PS(36:4)−H	PS	(16:0/20:4)	0	0	0	504066	518995	539917	3728026	1291304	2254419
PS(36:4)−H	PS(36:4)−H	PS	(36:4)	25147	57619	40780	1546353	1123863	1205590	22348538	5254145	5302937
PS(37:1)−H	PS(37:1)−H	PS	(37:1)	103925	202624	279931	2153303	1878319	1896115	14650703	3433156	7796187
PS(37:2)−H	PS(37:2)−H	PS	(37:2)	0	0	0	38551	6857	23932	93052	68361	202167
PS(38:1)−H	PS(38:1)−H	PS	(38:1)	0	0	0	131682	38421	87348	9647588	1900465	606454
PS(20:1/18:1)−H	PS(38:2)−H	PS	(20:1/18:1)	0	0	0	619467	710257	822693	11620755	2317125	3558461
PS(18:0/20:3)−H	PS(38:3)−H	PS	(18:0/20:3)	124012	227219	216689	4604588	3285498	3016987	71377509	20263566	20164143
PS(38:3)−H	PS(38:3)−H	PS	(38:3)	18158	50225	47308	526136	255058	382293	28584312	6181587	2638371
PS(18:0/20:4)−H	PS(38:4)−H	PS	(18:0/20:4)	80422	337987	305279	3498645	2639132	2196641	124069072	19908644	24987487
PS(18:1/20:3)−H	PS(38:4)−H	PS	(18:1/20:3)	0	0	0	493488	441954	460899	3498990	937986	1731247
PS(16:0/22:5)−H	PS(38:5)−H	PS	(16:0/22:5)	0	0	0	1003868	974808	848464	6483253	1514751	3327651
PS(18:0/20:5)−H	PS(38:5)−H	PS	(18:0/20:5)	0	0	0	538951	431797	506824	4234705	1134025	2139227
PS(18:1/20:4)−H	PS(38:5)−H	PS	(18:1/20:4)	0	0	0	1170057	942290	972128	8853041	2651403	4753546
PS(38:5p)−H	PS(38:5p)−H	PS	(38:5p)	0	0	0	236524	164874	161078	4083243	980936	1287805
PS(16:0/22:6)−H	PS(38:6)−H	PS	(16:0/22:6)	0	0	0	270747	359955	311336	1601519	755752	1053010
PS(38:6)−H	PS(38:6)−H	PS	(38:6)	1400	2242	1751	625536	497739	538392	6058304	1405830	2302998
PS(38:6p)−H	PS(38:6p)−H	PS	(38:6p)	46150	115567	100964	4545355	3712660	3847072	44270610	12373809	21403878
PS(39:1)−H	PS(39:1)−H	PS	(39:1)	632737	1169704	1237962	13169394	11288740	13799910	96013550	25615901	42391171
PS(39:2)−H	PS(39:2)−H	PS	(39:2)	48913	180273	168408	1827523	1366200	1466328	9475552	2265026	4469531
PS(39:3)−H	PS(39:3)−H	PS	(39:3)	455644	1035957	936332	8581795	6720518	7079635	36227939	11577047	21463136
PS(39:4)−H	PS(39:4)−H	PS	(39:4)	0	0	0	325492	249540	390643	3228058	1330179	2173109
PS(18:1/22:1)−H	PS(40:2)−H	PS	(18:1/22:1)	0	0	0	399045	278443	412582	12622421	3528113	3159688
PS(18:0/22:3)−H	PS(40:3)−H	PS	(18:0/22:3)	0	0	0	515829	190865	326068	15123603	2782564	2036074
PS(18:0/22:4)−H	PS(40:4)−H	PS	(18:0/22:4)	105924	249253	213426	5380454	4319323	4997892	74157592	23847958	25983626
PS(18:0/22:5)−H	PS(40:5)−H	PS	(18:0/22:5)	146606	332309	312213	10408121	8221687	8462113	98727320	27994819	37323768
PS(18:1/22:5)−H	PS(40:6)−H	PS	(18:1/22:5)	0	0	0	472766	500458	375232	3453673	872529	1990946
PS(20:3/20:3)−H	PS(40:6)−H	PS	(20:3/20:3)	0	0	0	762938	497252	628108	10402954	3179957	2786238
PS(40:6)−H	PS(40:6)−H	PS	(40:6)	61297	120647	131804	3380755	2766894	2842966	43140475	11633277	15731032
PS(40:6p)−H	PS(40:6p)−H	PS	(40:6p)	4899	39528	36501	1247587	920284	1258763	17653104	5071904	7497451
PS(40:7)−H	PS(40:7)−H	PS	(40:7)	30049	103106	84358	2473682	2240180	2439584	26615648	6819779	10425179
PS(40:7p)−H	PS(40:7p)−H	PS	(40:7p)	36328	102896	92165	3598766	3152882	3094159	28421479	8287972	14217323
PS(40:8p)−H	PS(40:8p)−H	PS	(40:8p)	11019	47088	43527	1889102	1678944	1718199	17592428	4535674	7409624
PS(41:3)−H	PS(41:3)−H	PS	(41:3)	63894	168426	166946	1023016	990598	1006077	5369528	1270858	3264903
PS(41:6)−H	PS(41:6)−H	PS	(41:6)	0	0	0	85844	62171	78130	235119	525658	227692
PS(18:1/24:0)−H	PS(42:1)−H	PS	(18:1/24:0)	0	0	0	0	0	0	1225623	295963	0
PS(42:8)−H	PS(42:8)−H	PS	(42:8)	0	0	0	1464832	1257884	1518317	14565124	3699847	5231179
PS(42:9)−H	PS(42:9)−H	PS	(42:9)	18485	59321	66432	2183237	1799535	1710184	20608914	4278020	7010451
PS(43:5)−H	PS(43:5)−H	PS	(43:5)	7144	78406	120590	473049	192901	447790	372283	437908	163301
SM(d30:1)+H	SM(d30:1)+H	SM	(d30:1)	0	0	0	615719	631109	706314	3874423	1510201	2740728
SM(d31:1)+H	SM(d31:1)+H	SM	(d31:1)	0	0	0	1347188	837142	1012924	14414899	2724618	3640860
SM(d32:0)+H	SM(d32:0)+H	SM	(d32:0)	59798	147569	122434	869251	580700	773451	23835547	4601133	4964988
SM(d18:1/14:0)+H	SM(d32:1)+H	SM	(d18:1/14:0)	1108812	1843673	1730073	13790596	10293325	38165	827380	38866734	48372637
SM(d32:2)+H	SM(d32:2)+H	SM	(d32:2)	88181	199227	188339	1845386	1607297	1712799	12711829	2468275	4436539
SM(d33:0)+H	SM(d33:0)+H	SM	(d33:0)	0	0	0	0	0	0	4282150	852481	648658
SM(d33:1)+H	SM(d33:1)+H	SM	(d33:1)	1265735	1658083	1491116	8515714	5175910	6484024	214903255	41780873	34028177
SM(d33:2)+H	SM(d33:2)+H	SM	(d33:2)	90044	19403	53726	76483	58515	35559	1172869	62959	120133
SM(d34:0)+H	SM(d34:0)+H	SM	(d34:0)	836958	692120	449173	2178600	899902	1467732	253094120	34639587	13974017
SM(d18:1/16:0)+H	SM(d34:1)+H	SM	(d18:1/16:0)	18307271	523327	596877	86186422	1574211	50388936	4274137129	761578752	379912703
SM(d34:1)+H	SM(d34:1)+H	SM	(d34:1)	1307895	1582630	1278876	15109596	6342518	9158968	665606026	146205846	86089783
SM(d16:1/18:1)+H	SM(d34:2)+H	SM	(d16:1/18:1)	25065	7050024	6809144	66462167	57496045	62349821	671063320	149729965	220490415
SM(d34:3)+H	SM(d34:3)+H	SM	(d34:3)	0	0	0	105094	235189	302699	880943	341502	840518
SM(d34:4)+H	SM(d34:4)+H	SM	(d34:4)	34521	134939	154759	794716	638889	656125	11690213	2568360	2945863
SM(d35:1)+H	SM(d35:1)+H	SM	(d35:1)	247968	184095	114466	506334	214959	225096	39671655	6028091	1974718
SM(d35:2)+H	SM(d35:2)+H	SM	(d35:2)	48150	106033	120968	1050795	937930	994361	11489940	2772515	4023233
SM(d35:4)+H	SM(d35:4)+H	SM	(d35:4)	43594	79926	27139	491640	258966	304051	12156988	2319433	1850319
SM(d18:1/18:0)+H	SM(d36:1)+H	SM	(d18:1/18:0)	629637	12300	195989	615028	111917	93344	2565988	220310	3411614
SM(d36:2)+H	SM(d36:2)+H	SM	(d18:1/18:1)	1765732	3344235	3379071	21409555	5364100	7847110	303348283	92989524	115325271
SM(d36:4)+H	SM(d36:4)+H	SM	(d36:4)	994807	1005077	839513	4586442	1678318	2342663	294109613	61473943	18141979
SM(d36:5)+H	SM(d36:5)+H	SM	(d36:5)	297164	295012	319403	2163920	1717689	1990882	18367470	4400627	6902944
SM(d38:2)+H	SM(d38:2)+H	SM	(d38:2)	54214	146906	297379	522298	328078	495458	6403489	3337412	2148728
SM(d39:7)+H	SM(d39:7)+H	SM	(d39:7)	0	0	0	0	270310	884517	0	681309	1750601
SM(d40:1)+H	SM(d40:1)+H	SM	(d40:1)	0	0	0	79805	23955	0	26547071	3398222	722860
SM(d40:2)+H	SM(d40:2)+H	SM	(d40:2)	753188	561975	887099	893095	720769	778773	48584542	11488146	7168405
SM(d41:2)+H	SM(d41:2)+H	SM	(d41:2)	215875	294435	350164	276787	129588	227992	32278155	6183741	2228561
SM(d18:1/24:1)+H	SM(d42:2)+H	SM	(d18:1/24:1)	1625404	1629174	1821544	2188269	1410509	1368023	442748344	85798660	20143857
SM(d42:2)+H	SM(d42:2)+H	SM	(d42:2)	1477469	2347652	2528121	4200414	3059142	3681665	360071978	93337240	34821089
SM(d22:0/20:3)+H	SM(d42:3)+H	SM	(d22:0/20:3)	2310406	5232438	5683793	11679202	12661396	12795722	235300942	84914349	63611365
SM(d42:5)+H	SM(d42:5)+H	SM	(d42:5)	30185	63739	132957	153193	60884	115125	6166808	2526148	1151755
SM(d43:3)+H	SM(d43:3)+H	SM	(d43:3)	19195	107388	126659	210701	181102	363854	5998722	2459182	1694580
SM(d44:2)+H	SM(d44:2)+H	SM	(d44:2)	0	0	0	155768	39339	27387	14665416	4883880	1036712
SM(d44:3)+H	SM(d44:3)+H	SM	(d44:3)	232986	486935	384438	801404	996177	1334532	52172136	15175888	8646236
SM(d44:5)+H	SM(d44:5)+H	SM	(d44:5)	98025	306566	328238	653603	714749	810959	2877078	1336496	1357844
SM(d44:6)+H	SM(d44:6)+H	SM	(d44:6)	125004	342163	431832	873569	1222537	1100198	26141105	7759675	6006034
TG(8:0/8:0/8:0)+NH4	TG(24:0)+NH4	TG	(8:0/8:0/8:0)	3965811	797256	1476350	1896834	2535680	14707218	60690014	23530427	18048952
TG(8:0/8:0/10:0)+NH4	TG(26:0)+NH4	TG	(8:0/8:0/10:0)	6559548	573706	2153442	2932146	1997854	16154403	70751794	36506807	12530641
TG(8:0/10:0/10:0)+NH4	TG(28:0)+NH4	TG	(8:0/10:0/10:0)	2342790	432826	1314501	3392028	941790	8740681	77221691	55368425	6075965
TG(10:0/10:0/10:0)+NH4	TG(30:0)+NH4	TG	(10:0/10:0/10:0)	266416	47127	79600	182075	31480	41853	6743274	3168680	122003
TG(16:0/8:0/8:0)+NH4	TG(32:0)+NH4	TG	(16:0/8:0/8:0)	167145	73331	141206	123244	102925	71211	3755974	1433263	282992
TG(16:0/9:0/9:0)+NH4	TG(34:0)+NH4	TG	(16:0/9:0/9:0)	381580	466967	731092	838300	459850	667295	14125951	8628510	2357855
TG(8:0/8:0/18:1)+NH4	TG(34:1)+NH4	TG	(8:0/8:0/18:1)	251726	288316	368907	273793	313742	234995	7130788	3378372	1289724
TG(15:0/14:0/15:0)+NH4	TG(44:0)+NH4	TG	(15:0/14:0/15:0)	1469265	1351220	1511143	1219858	1652197	1338090	18523461	2777117	4691112
TG(44:5p)+NH4	TG(44:5p)+NH4	TG	(44:5p)	4535937	15262064	18607564	16764338	11357362	23614638	105316939	57814262	95944906
TG(15:0/14:0/16:0)+NH4	TG(45:0)+NH4	TG	(15:0/14:0/16:0)	4242570	3545451	4058101	4398708	4761167	4015965	57774139	7439883	11538391
TG(16:0/14:0/16:0)+NH4	TG(46:0)+NH4	TG	(16:0/14:0/16:0)	5598020	5491304	5740841	12961236	12433442	6066611	91201311	14934132	17853551
TG(46:1)+NH4	TG(46:1)+NH4	TG	(46:1)	4535191	3566419	3822725	3849498	6655554	4088181	59982626	7728002	12991948
TG(15:0/16:0/16:0)+NH4	TG(47:0)+NH4	TG	(15:0/16:0/16:0)	6014432	5475226	3911054	7677815	8650709	6115645	94128965	10564714	16907993
TG(16:0/16:0/16:0)+NH4	TG(48:0)+NH4	TG	(16:0/16:0/16:0)	13215849	7342566	9280311	9210184	10219013	11944126	126876794	17516384	23203629
TG(16:0/16:0/16:1)+NH4	TG(48:1)+NH4	TG	(16:0/16:0/16:1)	7167337	9384379	9164578	6488288	7035808	5850627	113045336	10346750	31796586
TG(18:0/16:0/16:0)+NH4	TG(50:0)+NH4	TG	(18:0/16:0/16:0)	10886254	9827643	10291485	9795707	12440452	10621227	155760042	19004297	30876653
TG(16:0/16:0/18:1)+NH4	TG(50:1)+NH4	TG	(16:0/16:0/18:1)	12797301	11948320	11868663	10238770	15749855	13016257	175512959	20929309	37775864
TG(18:0/16:0/18:1)+NH4	TG(52:1)+NH4	TG	(18:0/16:0/18:1)	5543436	4792621	4939364	4904924	6207580	5483708	81536378	9679591	15175364
TG(16:0/18:1/18:1)+NH4	TG(52:2)+NH4	TG	(16:0/18:1/18:1)	11155612	9475346	10146783	9652307	11401260	10267952	169800206	17824084	32126335
TG(16:1/18:1/18:1)+NH4	TG(52:3)+NH4	TG	(16:1/18:1/18:1)	3426569	3780301	2696411	4518607	3413526	3080318	39668886	6703732	8320377
TG(18:0/18:1/18:1)+NH4	TG(54:2)+NH4	TG	(18:0/18:1/18:1)	5548113	4798377	5110804	4945345	6065474	5205137	80452150	10110102	15282425
TG(18:1/18:1/18:1)+NH4	TG(54:3)+NH4	TG	(18:1/18:1/18:1)	13490074	10791203	12914132	12217457	13097620	9435764	178846144	23630828	38109977
TG(18:1/18:1/18:2)+NH4	TG(54:4)+NH4	TG	(18:1/18:1/18:2)	4370798	3844782	4120196	4044172	4899610	4325095	64491431	7551802	11839565
TG(18:1/18:2/18:2)+NH4	TG(54:5)+NH4	TG	(18:1/18:2/18:2)	2129330	2134385	2098042	1890230	2384790	2093424	31554468	4111277	6377519
TG(18:2/18:2/18:2)+NH4	TG(54:6)+NH4	TG	(18:2/18:2/18:2)	1634749	1308316	1632192	1291751	1805935	1569949	25320674	3118308	4816398
			TOTAL	1.067E+09	1.286E+09	1.278E+09	4.515E+09	3.858E+09	4.478E+09	6.485E+10	1.922E+10	2.134E+10

	TABLE 8

	AsPC-1 Exomere

		Lipid	Fatty Acid	Exomere	Exomere	Exomere
Lipid Ion	Lipid Group	Class	Chain	replicate 1	replicate 2	replicate 3

Cer(d18:1/10:0) + H	Cer(d28:1) + H	Cer	(d18:1/10:0)	922714	731280	588136
Cer(d18:0/12:0) + H	Cer(d30:0) + H	Cer	(d18:0/12:0)	433299	379876	642277
Cer(d18:1/13:0) + H	Cer(d31:1) + H	Cer	(d18:1/13:0)	739433	804300	724615
Cer(d18:1/14:0) + H	Cer(d32:1) + H	Cer	(d18:1/14:0)	3355483	1035085	3512941
Cer(d17:1/16:0) + H	Cer(d33:1) + H	Cer	(d17:1/16:0)	111097	86765	101162
Cer(d18:0/16:0) + H	Cer(d34:0) + H	Cer	(d18:0/16:0)	475984	215732	127355
Cer(d18:1/16:0) + H	Cer(d34:1) + H	Cer	(d18:1/16:0)	1884993	1962653	2259029
Cer(d18:2/16:0) + H	Cer(d34:2) + H	Cer	(d18:2/16:0)	0	0	0
Cer(d35:4) + H	Cer(d35:4) + H	Cer	(d35:4)	30075019	22790952	18212913
Cer(d18:1/18:0) + H	Cer(d36:1) + H	Cer	(d18:1/18:0)	723102	876411	741514
Cer(d36:4) + H	Cer(d36:4) + H	Cer	(d36:4)	1739037	1282484	1030974
Cer(d18:0/20:0) + H	Cer(d38:0) + H	Cer	(d18:0/20:0)	1024850	396750	688157
Cer(d18:1/20:0) + H	Cer(d38:1) + H	Cer	(d18:1/20:0)	782329	603911	456780
Cer(d18:0/22:0) + H	Cer(d40:0) + H	Cer	(d18:0/22:0)	1145918	799985	654517
Cer(d18:1/22:0) + H	Cer(d40:1) + H	Cer	(d18:1/22:0)	1272049	832318	801555
Cer(d18:2/22:0) + H	Cer(d40:2) + H	Cer	(d18:2/22:0)	790415	139469	61759
Cer(d40:2) + H	Cer(d40:2) + H	Cer	(d40:2)	73301	109610	36883
Cer(d18:1/23:0) + H	Cer(d41:1) + H	Cer	(d18:1/23:0)	1344840	815327	677163
Cer(d18:1/23:1) + H	Cer(d41:2) + H	Cer	(d18:1/23:1)	442846	460976	361945
Cer(d18:0/24:0) + H	Cer(d42:0) + H	Cer	(d18:0/24:0)	1807119	1314270	930309
Cer(d18:1/24:0) + H	Cer(d42:1) + H	Cer	(d18:1/24:0)	2697504	2278243	1370820
Cer(d42:2) + H	Cer(d42:2) + H	Cer	(d42:2)	256524	219449	157660
Cer(d18:1/24:1) + H	Cer(d42:2) + H	Cer	(d18:1/24:1)	876326	1125299	1167028
Cer(d18:2/24:1) + H	Cer(d42:3) + H	Cer	(d18:2/24:1)	568391	643582	672065
Cer(d18:1/24:2) + H	Cer(d42:3) + H	Cer	(d18:1/24:2)	0	0	0
Cer(d18:1/25:1) + H	Cer(d43:2) + H	Cer	(d18:1/25:1)	488430	362169	225244
Cer(d18:1/26:0) + H	Cer(d44:1) + H	Cer	(d18:1/26:0)	1136492	862823	743784
Cer(d18:1/26:1) + H	Cer(d44:2) + H	Cer	(d18:1/26:1)	242162	185654	156839
Cer(d20:0/26:0) + H	Cer(d46:0) + H	Cer	(d20:0/26:0)	1635975	1712563	1476686
CerG1(d18:0/16:0) + H	CerG1(d34:0) + H	CerG1	(d18:0/16:0)	0	0	0
CerG1(d34:1) + H	CerG1(d34:1) + H	CerG1	(d34:1)	1351687	1012105	257971
CerG1(d18:1/16:1) + H	CerG1(d34:2) + H	CerG1	(d18:1/16:1)	0	0	0
CerG1(d18:0/22:0) + H	CerG1(d40:0) + H	CerG1	(d18:0/22:0)	364522	47776	34953
CerG1(d40:1) + H	CerG1(d40:1) + H	CerG1	(d40:1)	823991	744770	372644
CerG1(d40:2) + H	CerG1(d40:2) + H	CerG1	(d40:2)	2839228	1492021	1193354
CerG1(d41:1) + H	CerG1(d41:1) + H	CerG1	(d41:1)	764164	806908	542801
CerG1(d41:2) + H	CerG1(d41:2) + H	CerG1	(d41:2)	89410	276065	109846
CerG1(d18:0/24:0) + H	CerG1(d42:0) + H	CerG1	(d18:0/24:0)	0	0	0
CerG1(d18:0/24:1) + H	CerG1(d42:1) + H	CerG1	(d18:0/24:1)	788807	631057	464415
CerG1(d18:1/24:0) + H	CerG1(d42:1) + H	CerG1	(d18:1/24:0)	1557377	1337767	1049977
CerG1(d18:1/24:1) + H	CerG1(d42:2) + H	CerG1	(d18:1/24:1)	925349	672135	566679
CerG1(d18:1/24:2) + H	CerG1(d42:3) + H	CerG1	(d18:1/24:2)	801368	459387	393780
CerG1(d42:3) + H	CerG1(d42:3) + H	CerG1	(d42:3)	226214	176624	108803
CerG1(d43:1) + H	CerG1(d43:1) + H	CerG1	(d43:1)	5239817	4204259	3347078
CerG2(d34:1) + H	CerG2(d34:1) + H	CerG2	(d34:1)	0	0	0
CerG2(d42:1) + H	CerG2(d42:1) + H	CerG2	(d42:1)	0	0	0
CerG2(d42:2) + H	CerG2(d42:2) + H	CerG2	(d42:2)	96147	16943	24405
CerG3(d18:1/16:0) + H	CerG3(d34:1) + H	CerG3	(d18:1/16:0)	0	0	0
CerG3(d40:1) + H	CerG3(d40:1) + H	CerG3	(d40:1)	0	0	0
CerG3(d18:1/24:0) + H	CerG3(d42:1) + H	CerG3	(d18:1/24:0)	101864	57543	85153
CerG3(d18:1/24:1) + H	CerG3(d42:2) + H	CerG3	(d18:1/24:1)	0	0	0
ChE(18:1) + NH4	ChE(18:1) + NH4	ChE	(18:1)	983008	946225	868231
ChE(20:4) + NH4	ChE(20:4) + NH4	ChE	(20:4)	3982391	3632500	2741401
CL(65:6) − H	CL(65:6) − H	CL	(65:6)	0	0	0
DG(16:0/14:0) + NH4	DG(30:0) + NH4	DG	(16:0/14:0)	584979	556763	475330
DG(16:0/16:0) + NH4	DG(32:0) + NH4	DG	(16:0/16:0)	28029721	27287300	24519117
DG(18:0/16:0) + NH4	DG(34:0) + NH4	DG	(18:0/16:0)	160330793	150546977	121630016
DG(16:0/18:1) + NH4	DG(34:1) + NH4	DG	(16:0/18:1)	3632172	2822252	2470031
DG(18:0/18:0) + NH4	DG(36:0) + NH4	DG	(18:0/18:0)	113870656	102936251	85807125
DG(18:0/18:1) + NH4	DG(36:1) + NH4	DG	(18:0/18:1)	1063008	1652304	1230277
DG(18:1/18:1) + NH4	DG(36:2) + NH4	DG	(18:1/18:1)	2184985	1723649	2463802
DG(38:4) + NH4	DG(38:4) + NH4	DG	(38:4)	0	0	0
LPC(12:0) + H	LPC(12:0) + H	LPC	(12:0)	30343631	23220962	18423149
LPC(14:0) + H	LPC(14:0) + H	LPC	(14:0)	312936	153702	124001
LPC(16:0) + H	LPC(16:0) + H	LPC	(16:0)	795149	691995	25605
LPC(16:0e) + H	LPC(16:0e) + H	LPC	(16:0e)	0	0	0
LPC(16:1) + H	LPC(16:1) + H	LPC	(16:1)	0	0	0
LPC(17:0) + H	LPC(17:0) + H	LPC	(17:0)	0	0	0
LPC(18:0) + H	LPC(18:0) + H	LPC	(18:0)	400591	288773	267154
LPC(18:0p) + H	LPC(18:0p) + H	LPC	(18:0p)	0	0	0
LPC(18:1) + H	LPC(18:1) + H	LPC	(18:1)	113490	94404	51393
LPC(18:2) + H	LPC(18:2) + H	LPC	(18:2)	426612	372635	277702
LPC(20:4) + H	LPC(20:4) + H	LPC	(20:4)	0	0	0
LPC(22:5) + H	LPC(22:5) + H	LPC	(22:5)	0	0	0
LPC(22:6) + H	LPC(22:6) + H	LPC	(22:6)	0	0	0
LPE(18:1) − H	LPE(18:1) − H	LPE	(18:1)	0	0	0
LPE(20:3) − H	LPE(20:3) − H	LPE	(20:3)	0	0	0
LPE(20:4) − H	LPE(20:4) − H	LPE	(20:4)	0	0	0
LPG(14:0) − H	LPG(14:0) − H	LPG	(14:0)	2357456	1668521	608968
LPI(18:1) − H	LPI(18:1) − H	LPI	(18:1)	0	0	0
LPS(18:0) − H	LPS(18:0) − H	LPS	(18:0)	0	0	0
LPS(18:1) − H	LPS(18:1) − H	LPS	(18:1)	0	0	0
LPS(19:1) − H	LPS(19:1) − H	LPS	(19:1)	0	0	0
MG(16:0) + H	MG(16:0) + H	MG	(16:0)	14143724	10172586	5817578
MG(18:0) + H	MG(18:0) + H	MG	(18:0)	4367977	3317133	2317922
PC(19:1) + H	PC(19:1) + H	PC	(19:1)	84259	62866	193858
PC(23:0) + H	PC(23:0) + H	PC	(23:0)	1281742	1045112	1050288
PC(25:0) + H	PC(25:0) + H	PC	(25:0)	653226	869218	815950
PC(26:0) + H	PC(26:0) + H	PC	(26:0)	164278	240402	305841
PC(29:0e) + H	PC(29:0e) + H	PC	(29:0e)	0	0	0
PC(30:0) + H	PC(30:0) + H	PC	(30:0)	0	0	0
PC(30:0e) + H	PC(30:0e) + H	PC	(30:0e)	0	0	0
PC(30:1e) + H	PC(30:1e) + H	PC	(30:1e)	0	0	0
PC(31:0) + H	PC(31:0) + H	PC	(31:0)	32936	91914	136126
PC(31:0e) + H	PC(31:0e) + H	PC	(31:0e)	0	0	0
PC(31:1) + H	PC(31:1) + H	PC	(31:1)	500377	352524	1407330
PC(31:2) + H	PC(31:2) + H	PC	(31:2)	18434	55350	119105
PC(32:0) + H	PC(32:0) + H	PC	(32:0)	4203748	6634160	9559867
PC(32:0e) + H	PC(32:0e) + H	PC	(16:0e/16:0)	4180999	6545060	7882185
PC(32:1) + H	PC(32:1) + H	PC	(32:1)	278801	300378	524287
PC(32:1e) + H	PC(32:1e) + H	PC	(32:1e)	0	0	0
PC(32:1p) + H	PC(32:1p) + H	PC	(32:1p)	0	0	0
PC(32:3) + H	PC(32:3) + H	PC	(32:3)	0	0	0
PC(33:0) + H	PC(33:0) + H	PC	(33:0)	33618	199395	300228
PC(33:0e) + H	PC(33:0e) + H	PC	(33:0e)	63572	186964	237328
PC(33:0p) + H	PC(33:0p) + H	PC	(33:0p)	0	0	0
PC(15:0/18:1) + H	PC(33:1) + H	PC	(15:0/18:1)	209392	322296	506948
PC(33:1p) + H	PC(33:1p) + H	PC	(33:1p)	66058	846230	85808
PC(33:2) + H	PC(33:2) + H	PC	(33:2)	415422	2012692	1805737
PC(11:0/22:2) + H	PC(33:2) + H	PC	(11:0/22:2)	200582	274147	165613
PC(33:3) + H	PC(33:3) + H	PC	(33:3)	0	0	0
PC(34:0) + H	PC(34:0) + H	PC	(34:0)	4121693	6223644	6771436
PC(34:0e) + H	PC(34:0e) + H	PC	(18:0e/16:0)	7150592	8261064	11354670
PC(34:1) + H	PC(34:1) + H	PC	(16:0/18:1)	3730178	5307082	11620462
PC(34:1e) + H	PC(34:1e) + H	PC	(16:0e/18:1)	1212041	2036509	4798753
PC(34:2) + H	PC(34:2) + H	PC	(34:2)	1072380	873593	1129504
PC(34:2e) + H	PC(34:2e) + H	PC	(34:2e)	125308	444773	424157
PC(34:2p) + H	PC(34:2p) + H	PC	(34:2p)	64077	287277	291967
PC(34:3) + H	PC(34:3) + H	PC	(34:3)	0	0	0
PC(34:3p) + H	PC(34:3p) + H	PC	(34:3p)	387366	831234	334851
PC(35:0) + H	PC(35:0) + H	PC	(35:0)	0	0	0
PC(35:0p) + H	PC(35:0p) + H	PC	(35:0p)	35538	27787	36860
PC(17:0/18:1) + H	PC(35:1) + H	PC	(17:0/18:1)	3835758	4031186	4927640
PC(35:1p) + H	PC(35:1p) + H	PC	(35:1p)	0	0	0
PC(35:2) + H	PC(35:2) + H	PC	(35:2)	1675917	2001719	2156091
PC(35:3) + H	PC(35:3) + H	PC	(35:3)	0	0	0
PC(35:4) + H	PC(35:4) + H	PC	(35:4)	0	0	0
PC(35:5) + H	PC(35:5) + H	PC	(35:5)	356462	364034	384169
PC(35:6) + H	PC(35:6) + H	PC	(35:6)	0	0	0
PC(36:0e) + H	PC(36:0e) + H	PC	(36:0e)	79388	149892	194133
PC(36:1) + H	PC(36:1) + H	PC	(18:0/18:1)	5112511	6497046	14742392
PC(36:1e) + H	PC(36:1e) + H	PC	(18:0e/18:1)	1771544	3519641	6392630
PC(36:2) + H	PC(36:2) + H	PC	(18:1/18:1)	9310826	10328068	13038034
PC(36:2e) + H	PC(36:2e) + H	PC	(36:2e)	785677	1391854	375522
PC(36:2p) + H	PC(36:2p) + H	PC	(36:2p)	0	0	0
PC(24:0/12:3) + H	PC(36:3) + H	PC	(24:0/12:3)	164735	139799	256712
PC(36:3) + H	PC(36:3) + H	PC	(36:3)	269683	270909	201161
PC(36:4) + H	PC(36:4) + H	PC	(36:4)	0	0	0
PC(36:4e) + H	PC(36:4e) + H	PC	(36:4e)	0	0	0
PC(36:4p) + H	PC(36:4p) + H	PC	(36:4p)	0	0	0
PC(36:5) + H	PC(36:5) + H	PC	(36:5)	0	0	0
PC(37:1) + H	PC(37:1) + H	PC	(37:1)	50848	144950	220004
PC(19:1/18:1) + H	PC(37:2) + H	PC	(19:1/18:1)	6949122	6440153	8273071
PC(37:3) + H	PC(37:3) + H	PC	(37:3)	0	0	0
PC(37:4) + H	PC(37:4) + H	PC	(37:4)	0	0	0
PC(37:5) + H	PC(37:5) + H	PC	(37:5)	0	0	0
PC(37:6) + H	PC(37:6) + H	PC	(37:6)	0	0	0
PC(38:0e) + H	PC(38:0e) + H	PC	(38:0e)	108496	166253	117810
PC(38:1e) + H	PC(38:1e) + H	PC	(38:1e)	87765	98939	280764
PC(38:2) + H	PC(38:2) + H	PC	(38:2)	342595	893920	1083926
PC(38:2e) + H	PC(38:2e) + H	PC	(38:2e)	156710	180106	182506
PC(38:3) + H	PC(38:3) + H	PC	(38:3)	0	0	0
PC(38:3e) + H	PC(38:3e) + H	PC	(38:3e)	0	0	0
PC(38:4) + H	PC(38:4) + H	PC	(38:4)	149067	90719	379869
PC(38:4e) + H	PC(38:4e) + H	PC	(38:4e)	0	0	0
PC(38:4p) + H	PC(38:4p) + H	PC	(38:4p)	59228	240250	343852
PC(27:1/11:4) + H	PC(38:5) + H	PC	(27:1/11:4)	0	0	0
PC(38:5) + H	PC(38:5) + H	PC	(38:5)	305262	236785	505916
PC(38:6) + H	PC(38:6) + H	PC	(38:6)	0	0	0
PC(38:6e) + H	PC(38:6e) + H	PC	(38:6e)	0	0	0
PC(38:7) + H	PC(38:7) + H	PC	(38:7)	0	0	0
PC(39:5) + H	PC(39:5) + H	PC	(39:5)	0	0	0
PC(39:6) + H	PC(39:6) + H	PC	(39:6)	0	0	0
PC(40:1e) + H	PC(40:1e) + H	PC	(40:1e)	168705	477182	303757
PC(40:2) + H	PC(40:2) + H	PC	(40:2)	0	0	0
PC(40:2e) + H	PC(40:2e) + H	PC	(40:2e)	1056476	1307641	1555495
PC(40:3) + H	PC(40:3) + H	PC	(40:3)	0	0	0
PC(40:4) + H	PC(40:4) + H	PC	(40:4)	2363346	2333289	964621
PC(40:5) + H	PC(40:5) + H	PC	(40:5)	0	0	0
PC(40:5e) + H	PC(40:5e) + H	PC	(40:5e)	0	0	0
PC(18:0/22:6) + H	PC(40:6) + H	PC	(18:0/22:6)	0	0	0
PC(40:6) + H	PC(40:6) + H	PC	(40:6)	0	0	0
PC(40:6e) + H	PC(40:6e) + H	PC	(40:6e)	0	0	0
PC(40:6p) + H	PC(40:6p) + H	PC	(40:6p)	0	0	0
PC(40:7) + H	PC(40:7) + H	PC	(40:7)	0	0	0
PC(42:1) + H	PC(42:1) + H	PC	(42:1)	19564	87575	145919
PC(42:1e) + H	PC(42:1e) + H	PC	(42:1e)	200433	166531	298280
PC(42:2) + H	PC(42:2) + H	PC	(42:2)	14940	20915	13240
PC(42:2e) + H	PC(42:2e) + H	PC	(42:2e)	25323	92239	19366
PC(42:3p) + H	PC(42:3p) + H	PC	(42:3p)	0	0	0
PC(44:1) + H	PC(44:1) + H	PC	(44:1)	0	0	0
PC(44:2) + H	PC(44:2) + H	PC	(44:2)	0	0	0
PE(32:1p) − H	PE(32:1p) − H	PE	(16:0p/16:1)	0	0	0
PE(33:1p) − H	PE(33:1p) − H	PE	(33:1p)	0	0	0
PE(16:0/18:1) − H	PE(34:1) − H	PE	(16:0/18:1)	601756	200276	219181
PE(34:1e) − H	PE(34:1e) − H	PE	(34:1e)	0	0	0
PE(16:0p/18:1) − H	PE(34:1p) − H	PE	(16:0p/18:1)	114895	145950	281241
PE(16:1/18:1) − H	PE(34:2) − H	PE	(16:1/18:1)	0	0	0
PE(34:2p) − H	PE(34:2p) − H	PE	(18:1p/16:1)	0	0	0
PE(16:0p/18:2) − H	PE(34:2p) − H	PE	(16:0p/18:2)	0	0	0
PE(18:0/18:1) − H	PE(36:1) − H	PE	(18:0/18:1)	153814	93203	171083
PE(18:0e/18:1) − H	PE(36:1e) − H	PE	(18:0e/18:1)	28588	33260	43901
PE(18:0p/18:1) − H	PE(36:1p) − H	PE	(18:0p/18:1)	680151	522948	730742
PE(18:1/18:1) − H	PE(36:2) − H	PE	(18:1/18:1)	1173527	447335	395702
PE(18:1p/18:1) − H	PE(36:2p) − H	PE	(18:1p/18:1)	0	0	0
PE(18:0p/18:2) − H	PE(36:2p) − H	PE	(18:0p/18:2)	0	0	0
PE(18:1/18:2) − H	PE(36:3) − H	PE	(18:1/18:2)	0	0	0
PE(16:0p/20:3) − H	PE(36:3p) − H	PE	(16:0p/20:3)	0	0	0
PE(16:0p/20:4) − H	PE(36:4p) − H	PE	(16:0p/20:4)	0	0	0
PE(36:5p) − H	PE(36:5p) − H	PE	(16:0p/20:5)	0	0	0
PE(20:1/18:1) − H	PE(38:2) − H	PE	(20:1/18:1)	0	0	0
PE(18:0/20:2) − H	PE(38:2) − H	PE	(18:0/20:2)	0	0	0
PE(18:0p/20:2) − H	PE(38:2p) − H	PE	(18:0p/20:2)	0	0	0
PE(18:1/20:2) − H	PE(38:3) − H	PE	(18:1/20:2)	0	0	0
PE(18:0/20:3) − H	PE(38:3) − H	PE	(18:0/20:3)	0	0	0
PE(18:0p/20:3) − H	PE(38:3p) − H	PE	(18:0p/20:3)	0	0	0
PE(38:3p) − H	PE(38:3p) − H	PE	(18:0p/20:3)	0	0	0
PE(18:0/20:4) − H	PE(38:4) − H	PE	(18:0/20:4)	0	0	0
PE(18:1p/20:3) − H	PE(38:4p) − H	PE	(18:1p/20:3)	0	0	0
PE(18:0p/20:4) − H	PE(38:4p) − H	PE	(18:0p/20:4)	0	0	0
PE(18:1/20:4) − H	PE(38:5) − H	PE	(18:1/20:4)	0	0	0
PE(18:1p/20:4) − H	PE(38:5p) − H	PE	(18:1p/20:4)	0	0	0
PE(38:5p) − H	PE(38:5p) − H	PE	(38:5p)	0	0	0
PE(16:0p/22:6) − H	PE(38:6p) − H	PE	(16:0p/22:6)	0	0	0
PE(40:4p) − H	PE(40:4p) − H	PE	(18:0p/22:4)	0	0	0
PE(18:0p/22:5) − H	PE(40:5p) − H	PE	(18:0p/22:5)	0	0	0
PE(40:5p) − H	PE(40:5p) − H	PE	(18:0p/22:5)	0	0	0
PE(18:1p/22:5) − H	PE(40:6p) − H	PE	(18:1p/22:5)	0	0	0
PE(18:0p/22:6) − H	PE(40:6p) − H	PE	(18:0p/22:6)	0	0	0
PE(18:1/24:0) − H	PE(42:1) − H	PE	(18:1/24:0)	0	0	0
PE(50:2) − H	PE(50:2) − H	PE	(50:2)	0	0	0
PG(12:0/14:0) − H	PG(26:0) − H	PG	(12:0/14:0)	2289790	1297380	747348
PG(18:1/18:1) − H	PG(36:2) − H	PG	(18:1/18:1)	671672	355189	227011
PI(16:0/18:1) − H	PI(34:1) − H	PI	(16:0/18:1)	0	0	0
PI(18:0/18:1) − H	PI(36:1) − H	PI	(18:0/18:1)	513756	828168	529957
PI(18:1/18:1) − H	PI(36:2) − H	PI	(18:1/18:1)	0	0	0
PI(18:0/20:2) − H	PI(38:2) − H	PI	(18:0/20:2)	0	0	0
PI(18:1/20:2) − H	PI(38:3) − H	PI	(18:1/20:2)	0	0	0
PI(18:0/20:3) − H	PI(38:3) − H	PI	(18:0/20:3)	0	0	0
PI(18:0/20:4) − H	PI(38:4) − H	PI	(18:0/20:4)	0	0	0
PI(18:1/20:4) − H	PI(38:5) − H	PI	(18:1/20:4)	0	0	0
PI(18:0/22:4) − H	PI(40:4) − H	PI	(18:0/22:4)	0	0	0
PI(18:0/22:5) − H	PI(40:5) − H	PI	(18:0/22:5)	0	0	0
PS(12:0/14:0) − H	PS(26:0) − H	PS	(12:0/14:0)	714575	346586	294597
PS(18:0/16:1) − H	PS(34:1) − H	PS	(18:0/16:1)	0	0	0
PS(35:1) − H	PS(35:1) − H	PS	(35:1)	0	0	0
PS(17:1/18:0) − H	PS(35:1) − H	PS	(17:1/18:0)	0	0	0
PS(18:0/18:1) − H	PS(36:1) − H	PS	(18:0/18:1)	5033800	3921496	3947054
PS(18:0e/18:1) − H	PS(36:1e) − H	PS	(18:0e/18:1)	0	0	0
PS(18:1/18:1) − H	PS(36:2) − H	PS	(18:1/18:1)	0	0	0
PS(18:0/18:2) − H	PS(36:2) − H	PS	(18:0/18:2)	0	0	0
PS(36:3p) − H	PS(36:3p) − H	PS	(36:3p)	0	0	0
PS(37:0) − H	PS(37:0) − H	PS	(37:0)	760780	1021200	860070
PS(19:0/18:1) − H	PS(37:1) − H	PS	(19:0/18:1)	759832	604079	391985
PS(37:1) − H	PS(37:1) − H	PS	(37:1)	0	0	0
PS(37:2) − H	PS(37:2) − H	PS	(37:2)	156968	148413	131555
PS(20:0/18:1) − H	PS(38:1) − H	PS	(20:0/18:1)	0	0	0
PS(20:1/18:1) − H	PS(38:2) − H	PS	(20:1/18:1)	0	0	0
PS(18:0/20:2) − H	PS(38:2) − H	PS	(18:0/20:2)	0	0	0
PS(38:2p) − H	PS(38:2p) − H	PS	(38:2p)	0	0	0
PS(18:0/20:3) − H	PS(38:3) − H	PS	(18:0/20:3)	0	0	0
PS(18:0/20:4) − H	PS(38:4) − H	PS	(18:0/20:4)	649164	763006	512449
PS(38:6p) − H	PS(38:6p) − H	PS	(38:6p)	499022	80779	72098
PS(39:1) − H	PS(39:1) − H	PS	(39:1)	1337394	688759	467218
PS(39:2) − H	PS(39:2) − H	PS	(39:2)	0	0	0
PS(39:3) − H	PS(39:3) − H	PS	(39:3)	0	0	0
PS(39:4) − H	PS(39:4) − H	PS	(39:4)	0	0	0
PS(18:1/22:0) − H	PS(40:1) − H	PS	(18:1/22:0)	298959	265951	213382
PS(18:1/22:1) − H	PS(40:2) − H	PS	(18:1/22:1)	0	0	0
PS(18:1/22:2) − H	PS(40:3) − H	PS	(18:1/22:2)	0	0	0
PS(18:0/22:4) − H	PS(40:4) − H	PS	(18:0/22:4)	0	0	0
PS(18:0/22:5) − H	PS(40:5) − H	PS	(18:0/22:5)	0	0	0
PS(40:5) − H	PS(40:5) − H	PS	(40:5)	0	0	0
PS(18:1/24:0) − H	PS(42:1) − H	PS	(18:1/24:0)	304095	175928	112391
PS(18:1/24:1) − H	PS(42:2) − H	PS	(18:1/24:1)	0	0	0
SM(d31:1) + H	SM(d31:1) + H	SM	(d31:1)	0	0	0
SM(d32:0) + H	SM(d32:0) + H	SM	(d32:0)	0	0	0
SM(d32:1) + H	SM(d32:1) + H	SM	(d32:1)	0	0	0
SM(d32:2) + H	SM(d32:2) + H	SM	(d32:2)	0	0	0
SM(d33:1) + H	SM(d33:1) + H	SM	(d33:1)	0	0	0
SM(d33:5) + H	SM(d33:5) + H	SM	(d33:5)	13767217	7353865	5205066
SM(d34:0) + H	SM(d34:0) + H	SM	(d34:0)	968701	762450	1218142
SM(d34:1) + H	SM(d34:1) + H	SM	(d18:1/16:0)	5647889	7474945	9598049
SM(d34:2) + H	SM(d34:2) + H	SM	(d18:1/16:1)	0	0	0
SM(d35:1) + H	SM(d35:1) + H	SM	(d35:1)	0	0	0
SM(d35:2) + H	SM(d35:2) + H	SM	(d35:2)	0	0	0
SM(d35:4) + H	SM(d35:4) + H	SM	(d35:4)	0	0	0
SM(d36:0) + H	SM(d36:0) + H	SM	(d20:0/16:0)	277558	245117	232802
SM(d36:1) + H	SM(d36:1) + H	SM	(d18:0/18:1)	1693696	1847623	1767849
SM(d36:2) + H	SM(d36:2) + H	SM	(d18:1/18:1)	0	0	0
SM(d36:4) + H	SM(d36:4) + H	SM	(d36:4)	144312	410617	402904
SM(d38:1) + H	SM(d38:1) + H	SM	(d38:1)	629163	1171614	641719
SM(d40:0) + H	SM(d40:0) + H	SM	(d40:0)	831110	694245	576019
SM(d40:1) + H	SM(d40:1) + H	SM	(d40:1)	29722	17597	41575
SM(d17:1/23:0) + H	SM(d40:1) + H	SM	(d17:1/23:0)	8002552	7469383	6186988
SM(d40:2) + H	SM(d40:2) + H	SM	(d40:2)	249009	428289	225202
SM(d41:1) + H	SM(d41:1) + H	SM	(d41:1)	2217904	2152799	1219236
SM(d41:2) + H	SM(d41:2) + H	SM	(d41:2)	519481	377538	271016
SM(d18:1/24:0) + H	SM(d42:1) + H	SM	(d18:1/24:0)	1403775	1329722	1375161
SM(d42:1) + H	SM(d42:1) + H	SM	(d42:1)	20063175	15292830	12264345
SM(d18:1/24:1) + H	SM(d42:2) + H	SM	(d18:1/24:1)	10177555	10775373	13484127
SM(d18:1/24:2) + H	SM(d42:3) + H	SM	(d18:1/24:2)	100418	221234	596242
SM(d42:5) + H	SM(d42:5) + H	SM	(d42:5)	0	0	0
SM(d43:1) + H	SM(d43:1) + H	SM	(d43:1)	328738	498963	163191
SM(d18:2/25:0) + H	SM(d43:2) + H	SM	(d18:2/25:0)	432950	328433	217142
SM(d43:3) + H	SM(d43:3) + H	SM	(d43:3)	0	0	0
SM(d43:4) + H	SM(d43:4) + H	SM	(d43:4)	71421	79867	29898
SM(d44:1) + H	SM(d44:1) + H	SM	(d44:1)	99170	26271	53968
SM(d44:2) + H	SM(d44:2) + H	SM	(d16:1/28:1)	408391	331363	542864
SM(d44:3) + H	SM(d44:3) + H	SM	(d44:3)	0	0	0
SM(d44:4) + H	SM(d44:4) + H	SM	(d44:4)	1756037	1561280	1165937
SM(d44:5) + H	SM(d44:5) + H	SM	(d44:5)	397723	404998	1273342
SM(d44:6) + H	SM(d44:6) + H	SM	(d44:6)	0	0	0
TG(16:0/14:0/14:0) + NH4	TG(44:0) + NH4	TG	(16:0/14:0/14:0)	5303660	4167352	3263212
TG(44:5p) + NH4	TG(44:5p) + NH4	TG	(44:5p)	1849254	2918445	1219107
TG(15:0/14:0/16:0) + NH4	TG(45:0) + NH4	TG	(15:0/14:0/16:0)	15559462	10888195	12475767
TG(16:0/14:0/16:0) + NH4	TG(46:0) + NH4	TG	(16:0/14:0/16:0)	12694392	9433598	7845824
TG(15:0/16:0/16:0) + NH4	TG(47:0) + NH4	TG	(15:0/16:0/16:0)	21832045	18085839	12810089
TG(16:0/16:0/16:0) + NH4	TG(48:0) + NH4	TG	(16:0/16:0/16:0)	26521769	20001891	17929239
TG(16:0/16:0/16:1) + NH4	TG(48:1) + NH4	TG	(16:0/16:0/16:1)	12412377	9353767	7694661
TG(16:0/16:0/17:0) + NH4	TG(49:0) + NH4	TG	(16:0/16:0/17:0)	13887670	10159264	8261798
TG(18:0/16:0/16:0) + NH4	TG(50:0) + NH4	TG	(18:0/16:0/16:0)	22287169	19441382	14565244
TG(16:0/16:0/18:1) + NH4	TG(50:1) + NH4	TG	(16:0/16:0/18:1)	14395579	11314658	10220305
TG(18:0/16:0/18:0) + NH4	TG(52:0) + NH4	TG	(18:0/16:0/18:0)	30196361	24602915	18880481
TG(18:0/16:0/18:1) + NH4	TG(52:1) + NH4	TG	(18:0/16:0/18:1)	8767125	6675705	6023037
TG(16:0/18:1/18:1) + NH4	TG(52:2) + NH4	TG	(16:0/18:1/18:1)	14399280	10801174	9022808
TG(18:0/18:0/18:0) + NH4	TG(54:0) + NH4	TG	(18:0/18:0/18:0)	13232227	15433611	9540723
TG(18:0/18:1/18:1) + NH4	TG(54:2) + NH4	TG	(18:0/18:1/18:1)	6466612	4735583	4070103
TG(18:1/18:1/18:1) + NH4	TG(54:3) + NH4	TG	(18:1/18:1/18:1)	10995098	11297342	8798428
TG(18:1/18:1/18:2) + NH4	TG(54:4) + NH4	TG	(18:1/18:1/18:2)	3984880	3595645	4091756
			sum	8.28E+08	7.39E+08	6.62E+08

AsPC-1 Exo-S

	Exo-S	Exo-S	Exo-S	Exo-L	Exo-L	Exo-L
Lipid Ion	replicate 1	replicate 2	replicate 3	replicate 1	replicate 2	replicate 3

Cer(d18:1/10:0) + H	1161226	1104881	1080798	1009806	970265	1019853
Cer(d18:0/12:0) + H	1487628	1400270	1360991	1328204	1348430	1431742
Cer(d18:1/13:0) + H	710091	727948	646497	691943	734274	672348
Cer(d18:1/14:0) + H	15028983	10756351	12788017	11837058	13185164	17142365
Cer(d17:1/16:0) + H	379140	281638	284626	294461	333111	675879
Cer(d18:0/16:0) + H	3199021	2170628	2182341	1908614	2413209	6114505
Cer(d18:1/16:0) + H	30384987	18029692	17991061	16808869	20310534	49842138
Cer(d18:2/16:0) + H	843471	433831	601831	464028	719898	2103409
Cer(d35:4) + H	26319798	39255398	33728028	32453953	32665201	31914933
Cer(d18:1/18:0) + H	1412875	1266895	1230538	948796	1065738	2469847
Cer(d36:4) + H	2490620	3453773	3082102	3179130	3200308	2943881
Cer(d18:0/20:0) + H	558221	750178	724697	477538	650722	593305
Cer(d18:1/20:0) + H	740000	699573	731853	567302	694776	788387
Cer(d18:0/22:0) + H	1121869	1105527	942368	991968	1038308	1156820
Cer(d18:1/22:0) + H	3416578	3333647	3005547	2442580	2996249	5201654
Cer(d18:2/22:0) + H	1069304	923098	858943	770839	965341	1376478
Cer(d40:2) + H	528227	488293	496723	330455	403122	646014
Cer(d18:1/23:0) + H	1502701	1633233	1468462	1376976	1460770	2052176
Cer(d18:1/23:1) + H	1015675	969892	820092	682008	931279	1370667
Cer(d18:0/24:0) + H	3184280	2823858	2154463	2737526	2510675	2760974
Cer(d18:1/24:0) + H	8271652	8929382	7674317	6102411	6961716	11310964
Cer(d42:2) + H	1536223	1036262	955659	1041349	1277473	1516394
Cer(d18:1/24:1) + H	17012350	12558081	13636203	13392651	13806383	21927440
Cer(d18:2/24:1) + H	2506551	2252357	2314338	1654686	1934877	3114050
Cer(d18:1/24:2) + H	481704	451615	492035	310218	427242	714094
Cer(d18:1/25:1) + H	683789	534379	404730	538777	584311	861871
Cer(d18:1/26:0) + H	597931	883146	637774	578080	619969	729993
Cer(d18:1/26:1) + H	1016476	699431	1098185	457425	742659	1061528
Cer(d20:0/26:0) + H	708722	868500	765557	718766	819701	574516
CerG1(d18:0/16:0) + H	864168	506638	609843	553861	832616	1432924
CerG1(d34:1) + H	7021599	3688081	4458304	3677552	5456609	13273493
CerG1(d18:1/16:1) + H	1018845	378416	668961	859960	785304	2817295
CerG1(d18:0/22:0) + H	628338	650900	612148	474909	575159	858877
CerG1(d40:1) + H	3052629	3270582	3204884	2599195	3459299	4077409
CerG1(d40:2) + H	1626853	3387723	1536495	1988983	2509258	2777076
CerG1(d41:1) + H	1692484	1838120	1850641	1652466	1936847	2397855
CerG1(d41:2) + H	930620	993643	1181784	754978	1200394	1394143
CerG1(d18:0/24:0) + H	286005	335772	233098	272549	249176	348811
CerG1(d18:0/24:1) + H	1347692	1371485	1352479	1062879	1364555	1724422
CerG1(d18:1/24:0) + H	11027272	11234757	10945107	8532979	9834946	14096107
CerG1(d18:1/24:1) + H	10337681	10431503	10034066	7372647	7652274	11279492
CerG1(d18:1/24:2) + H	2792122	2494495	2245846	2027476	2376374	3649090
CerG1(d42:3) + H	763634	754435	672905	490811	646572	907412
CerG1(d43:1) + H	1744932	3094670	1778420	2204296	2159863	2140945
CerG2(d34:1) + H	642664	394912	547028	210517	329480	997227
CerG2(d42:1) + H	444004	559695	522116	224790	312103	529451
CerG2(d42:2) + H	363978	386598	391621	364937	522104	635200
CerG3(d18:1/16:0) + H	1373647	1156975	1202804	871826	1506591	3563338
CerG3(d40:1) + H	614765	752731	728724	523409	893840	1312598
CerG3(d18:1/24:0) + H	1065963	1287090	1345598	970774	1487985	1822581
CerG3(d18:1/24:1) + H	1649962	1982943	1952014	1287818	2043444	3161386
ChE(18:1) + NH4	244470	334002	466805	4256	26349	21028
ChE(20:4) + NH4	916434	1627716	1352287	218337	315787	374468
CL(65:6) − H	702956	552776	339214	421191	247487	424130
DG(16:0/14:0) + NH4	973603	1158921	1174666	679157	616636	1121200
DG(16:0/16:0) + NH4	61921185	68407427	76083764	24020977	22806431	38878778
DG(18:0/16:0) + NH4	230841669	280004469	270615121	86899288	71714323	119350648
DG(16:0/18:1) + NH4	5330915	5580228	5683906	2482229	3806795	5541844
DG(18:0/18:0) + NH4	148057137	181162446	202969267	53698985	48750456	69243389
DG(18:0/18:1) + NH4	13092850	7393824	7041699	3960275	4899353	11105058
DG(18:1/18:1) + NH4	3398696	3475896	3375059	1848013	2473660	5223812
DG(38:4) + NH4	2710444	2416677	2784049	1019162	1626647	4373355
LPC(12:0) + H	6875148	15237499	11335390	14641381	14103549	10268177
LPC(14:0) + H	64246	180518	79870	378594	305649	127215
LPC(16:0) + H	601713	3672654	3446154	5882972	4860845	3275044
LPC(16:0e) + H	243209	292626	414534	958148	742665	456908
LPC(16:1) + H	5785	396438	298862	668287	1013889	1032464
LPC(17:0) + H	70542	69075	63959	44774	60125	49923
LPC(18:0) + H	1927402	2299656	2103428	1827748	1489634	1855024
LPC(18:0p) + H	207965	581189	448823	1212508	1085489	577963
LPC(18:1) + H	395707	643028	828394	1931504	1011218	1224900
LPC(18:2) + H	179810	283973	196460	406567	363529	411191
LPC(20:4) + H	49823	225843	255573	689835	486941	632133
LPC(22:5) + H	80675	143231	178404	316426	293360	293421
LPC(22:6) + H	0	0	0	86510	232105	208305
LPE(18:1) − H	139448	325069	344966	367619	394221	250340
LPE(20:3) − H	111547	222401	208878	370133	364009	246116
LPE(20:4) − H	216557	274736	267947	628875	568699	494027
LPG(14:0) − H	274890	153355	180713	311235	243986	138531
LPI(18:1) − H	87027	181641	99964	246331	223662	144567
LPS(18:0) − H	169089	134254	166002	201245	164475	166604
LPS(18:1) − H	98760	213048	177159	729058	573819	375246
LPS(19:1) − H	68019	113820	70263	394183	217347	131259
MG(16:0) + H	5899502	13145688	12085848	10489087	10194685	9503516
MG(18:0) + H	12732909	10535058	11683794	9733769	8411484	14989788
PC(19:1) + H	122926	555836	486515	353299	632150	454546
PC(23:0) + H	496786	263827	326499	376003	316583	274788
PC(25:0) + H	38200	310715	298325	291393	536801	301964
PC(26:0) + H	998129	825300	807520	1199487	1147117	1418367
PC(29:0e) + H	1328872	250142	389003	260507	365739	2364802
PC(30:0) + H	63943542	23370942	34507465	21699367	26794604	93096918
PC(30:0e) + H	10114487	3331109	5098548	3783590	4224563	12140025
PC(30:1e) + H	1025759	429715	578419	378541	629557	1730399
PC(31:0) + H	5492878	2611494	3358798	1673558	2388076	7241760
PC(31:0e) + H	4080802	1568609	1948575	1311610	1663829	6970217
PC(31:1) + H	6011892	3305120	2855380	3426604	3830843	10886160
PC(31:2) + H	1276901	777935	857771	771724	910353	2150286
PC(32:0) + H	222463088	73885024	103573737	54969035	72787383	299526790
PC(32:0e) + H	157924876	66726205	83052912	52829339	71874784	265148202
PC(32:1) + H	163964056	62072430	95532316	65253343	86138840	277506868
PC(32:1e) + H	47258395	18758432	27062064	20165414	29575036	85804351
PC(32:1p) + H	3616382	2130448	3237822	1995523	2466831	8302859
PC(32:3) + H	3043031	624002	1562553	974509	1007768	2506956
PC(33:0) + H	5008317	2448474	3276320	2097658	2556923	8080469
PC(33:0e) + H	8189510	2678103	4082465	2067407	2869997	9251867
PC(33:0p) + H	6397448	3614457	5354263	3315323	5213227	13839753
PC(15:0/18:1) + H	13728709	8024142	10367415	6489996	7833478	23435505
PC(33:1p) + H	1601956	919262	506235	761065	455790	4502564
PC(33:2) + H	18052045	12291802	13081911	10187182	13373854	30000402
PC(11:0/22:2) + H	2267403	1862264	2225879	1889345	1931937	5281977
PC(33:3) + H	1027039	723191	925158	739632	904388	2124102
PC(34:0) + H	83132810	44647806	52253530	29513711	37605970	120581424
PC(34:0e) + H	148548027	79146970	94349458	62492086	78166918	262828420
PC(34:1) + H	879077540	342271124	515629960	271405044	374571523	1222348694
PC(34:1e) + H	429736059	213607792	294219626	182594040	259325783	819615632
PC(34:2) + H	103666480	27739950	41819777	47595261	62731171	207685629
PC(34:2e) + H	32855363	17844060	28402773	16462324	23447065	78892513
PC(34:2p) + H	10792235	3406653	5366602	2977466	4086870	20524193
PC(34:3) + H	15792697	5429109	8623407	4658898	6390042	20994718
PC(34:3p) + H	3582997	920913	1979741	1407326	2005150	6383460
PC(35:0) + H	1734535	1270945	1224689	603898	1099443	3302983
PC(35:0p) + H	12025612	6316554	10202029	4259749	8172861	27656575
PC(17:0/18:1) + H	31280319	17547139	22758164	13605271	16063303	47602977
PC(35:1p) + H	2945064	1901246	2534803	1761623	2427471	8412815
PC(35:2) + H	9468768	7036386	8657409	5322675	7203421	18903749
PC(35:3) + H	1036383	480327	483676	455052	957129	1529499
PC(35:4) + H	5013103	3145516	4224496	2622638	2878708	8523382
PC(35:5) + H	1201973	926920	1187038	822205	1013364	2508421
PC(35:6) + H	1573743	1751103	876983	574233	432246	1841804
PC(36:0e) + H	3902025	2912032	3624892	2099787	2860792	7737745
PC(36:1) + H	474400334	237173016	317062368	154373907	212240303	715131037
PC(36:1e) + H	169591121	91320975	121158842	67761706	96009736	356456609
PC(36:2) + H	324666678	166775194	242131812	141046062	192670417	628039448
PC(36:2e) + H	97167630	64719454	86759273	57974614	85255206	258144537
PC(36:2p) + H	17222343	8050609	13465033	8143532	12746039	42746990
PC(24:0/12:3) + H	27092292	15772942	21768455	11076757	15766577	44703253
PC(36:3) + H	22911621	13362379	11646157	10181077	9496648	52059855
PC(36:4) + H	110189529	49588882	78576258	35415317	50297707	169256945
PC(36:4e) + H	38069521	15576770	24673031	13316096	22319102	73379638
PC(36:4p) + H	2756595	1621421	2048603	1182737	1741625	5461028
PC(36:5) + H	4857625	1164977	1738355	1194229	1649032	9973998
PC(37:1) + H	5304541	3115622	3885020	2035884	2456141	7811462
PC(19:1/18:1) + H	10817575	8676144	10426351	7418812	8565635	20790315
PC(37:3) + H	1317492	436963	738382	770785	671578	2512843
PC(37:4) + H	1793297	1081559	1438550	513421	881156	2902676
PC(37:5) + H	438600	333449	379787	112158	211023	685896
PC(37:6) + H	11368778	864920	6598599	4309772	9144234	14451472
PC(38:0e) + H	962497	828723	1192211	548438	821886	2314833
PC(38:1e) + H	4748876	2261374	5431443	2278222	3264407	12824060
PC(38:2) + H	34125965	21280872	27187219	15335018	18679914	57870214
PC(38:2e) + H	3818221	4501763	5303514	2094512	3911259	15442890
PC(38:3) + H	24010539	15620616	20829185	8215146	12265718	39116728
PC(38:3e) + H	3587391	2515178	3466725	1942637	3010984	9560628
PC(38:4) + H	75591644	39154589	49192201	22831255	31486336	109458253
PC(38:4e) + H	13218272	5783361	6021199	4736207	7437737	33065247
PC(38:4p) + H	5819383	2721201	4471814	2815866	4860563	17618545
PC(27:1/11:4) + H	97295656	52924138	71975188	31312601	45819149	146213524
PC(38:5) + H	23628994	12356472	19405208	10808692	16371303	46459934
PC(38:6) + H	18810629	9749474	13899646	5722454	8560641	25258911
PC(38:6e) + H	2427286	2079964	2721481	1354677	1977902	5503233
PC(38:7) + H	3087577	1540326	2842895	1255838	1853276	6723691
PC(39:5) + H	1020470	1082619	871351	445031	840162	2093480
PC(39:6) + H	747698	424200	426959	215079	367974	864086
PC(40:1e) + H	3884757	3006051	3225138	2089874	2866312	7385477
PC(40:2) + H	3684092	2396213	2789114	1530716	2002747	5797455
PC(40:2e) + H	21597550	15602090	15705062	14040184	14623827	30631508
PC(40:3) + H	1365862	1009399	1290362	770109	881906	2508288
PC(40:4) + H	7279839	4805786	6050261	2919457	4100939	9004000
PC(40:5) + H	24484856	12247208	16650322	7234538	9777885	31592139
PC(40:5e) + H	1350707	687572	1713199	869781	878835	2174102
PC(18:0/22:6) + H	9863904	4518368	8459785	3770019	4383755	16982380
PC(40:6) + H	15959856	8768453	12263365	4851447	6729448	21167408
PC(40:6e) + H	1533314	1013363	1247103	640465	1006167	3681620
PC(40:6p) + H	455558	475649	398468	237876	704771	2287725
PC(40:7) + H	3139185	2870450	3222294	1346363	1744917	5947286
PC(42:1) + H	2690383	1705651	1913714	1330900	1393883	2948525
PC(42:1e) + H	1098437	887955	1060519	832552	1064504	1929601
PC(42:2) + H	2255144	1444606	2317659	1326718	1604104	3559083
PC(42:2e) + H	1855766	1700702	3051695	1324034	1575294	3915372
PC(42:3p) + H	381684	341452	384422	301856	371096	631483
PC(44:1) + H	1742298	861037	1098095	653008	825915	1534289
PC(44:2) + H	2528570	1283888	1096267	829378	1064444	2243455
PE(32:1p) − H	2618424	1659248	1315766	2321072	2099928	3372313
PE(33:1p) − H	903743	496539	705154	679063	710887	1119170
PE(16:0/18:1) − H	3674815	2620161	2406102	2484273	2912718	4582132
PE(34:1e) − H	3930789	2940532	2474440	3132362	3487901	6300910
PE(16:0p/18:1) − H	55642480	39253682	35950078	40694139	41661111	72631371
PE(16:1/18:1) − H	1079465	582126	547000	749387	762330	1180379
PE(34:2p) − H	1839730	1207904	1354935	1652512	1924765	2984746
PE(16:0p/18:2) − H	4227446	2685483	3085951	3395463	3886736	5547793
PE(18:0/18:1) − H	11340890	7811024	7961603	6938323	7334625	13314806
PE(18:0e/18:1) − H	4316902	3518076	2792821	3240904	3735407	6075437
PE(18:0p/18:1) − H	54066906	36260521	32597299	35285393	40334232	70748309
PE(18:1/18:1) − H	11670295	9231838	8898946	8511974	9914551	15091442
PE(18:1p/18:1) − H	26808491	22917708	18908008	21821759	28678381	41213892
PE(18:0p/18:2) − H	10135135	7837039	7172352	7760336	9253548	14886682
PE(18:1/18:2) − H	605983	353804	358398	464863	528073	728493
PE(16:0p/20:3) − H	7055940	6380755	5433122	5443765	6793168	12925825
PE(16:0p/20:4) − H	8750192	7231959	8800934	7424227	8717467	14671467
PE(36:5p) − H	583346	147901	427459	214610	434422	1391145
PE(20:1/18:1) − H	843611	845936	829466	613446	470787	1084624
PE(18:0/20:2) − H	977179	1001059	705111	661540	774746	1227265
PE(18:0p/20:2) − H	3147747	3298923	3096878	3134453	3328251	5755997
PE(18:1/20:2) − H	644606	831645	729447	571635	741194	1087995
PE(18:0/20:3) − H	822655	704189	1094886	575801	981705	1080121
PE(18:0p/20:3) − H	8459059	7275978	7770000	6440927	7674115	12883313
PE(38:3p) − H	1941455	1832068	1455014	1569157	1749962	3371718
PE(18:0/20:4) − H	3101405	2666193	2834578	2173039	2086309	5223388
PE(18:1p/20:3) − H	3923194	3515701	3275846	2736430	4721849	5885507
PE(18:0p/20:4) − H	16780935	13978386	15402870	13548809	14239309	31132615
PE(18:1/20:4) − H	689015	670541	695704	639850	678666	1251839
PE(18:1p/20:4) − H	8309135	8501259	8196929	7289834	9364385	15176212
PE(38:5p) − H	1997401	1394232	2059438	1523582	1680179	3412340
PE(16:0p/22:6) − H	3008957	2368636	2575277	2259033	2822695	5717519
PE(40:4p) − H	1285881	1175993	1075705	963090	1237635	2112065
PE(18:0p/22:5) − H	6341812	3949646	5236486	3722745	4914949	10481870
PE(40:5p) − H	1099314	984994	913846	647611	884381	1577766
PE(18:1p/22:5) − H	1338473	1488843	1684049	1200405	1353040	2516287
PE(18:0p/22:6) − H	3972438	3382531	4093363	2825394	3236022	7810302
PE(18:1/24:0) − H	311901	238088	228092	232206	215615	365493
PE(50:2) − H	1005524	954836	600499	840041	829035	954214
PG(12:0/14:0) − H	778862	751787	786034	886930	722802	684809
PG(18:1/18:1) − H	679359	639578	1624713	2197066	3214381	1367647
PI(16:0/18:1) − H	9424925	6626824	8703877	4371912	6004310	10841649
PI(18:0/18:1) − H	18101102	16364444	19666540	9088733	13020178	18097026
PI(18:1/18:1) − H	13751746	10787284	14167932	7131297	11284150	19000924
PI(18:0/20:2) − H	4946120	3287562	4387715	1912738	4403415	2778679
PI(18:1/20:2) − H	2618637	2212748	2484554	1781400	2169038	3190556
PI(18:0/20:3) − H	6943837	6228542	8075780	4483756	5798218	10751576
PI(18:0/20:4) − H	17035253	10708750	16861168	8148968	13076195	24840850
PI(18:1/20:4) − H	287961	1266010	607255	733593	1006391	1894491
PI(18:0/22:4) − H	1080406	1115417	1299789	630077	811852	1337280
PI(18:0/22:5) − H	1120507	764545	942205	465617	779390	1137390
PS(12:0/14:0) − H	446810	362690	365026	407321	374932	359106
PS(18:0/16:1) − H	8268715	4949134	6550191	4779166	7728863	10374329
PS(35:1) − H	2924351	1496904	1470006	1339749	1516361	1964229
PS(17:1/18:0) − H	2471947	2147249	2440076	1451199	1576367	3397779
PS(18:0/18:1) − H	167102689	119649760	143690425	90644014	116354851	197535824
PS(18:0e/18:1) − H	4853460	3820314	4839820	2919212	3519713	6837569
PS(18:1/18:1) − H	9194266	6754491	8027641	5640061	5875839	9680330
PS(18:0/18:2) − H	7880013	5801947	7222412	5220068	5742639	10402688
PS(36:3p) − H	7633078	6703107	7416310	6323698	6492904	10986258
PS(37:0) − H	66650314	48250113	70052193	35212504	46133049	80874243
PS(19:0/18:1) − H	36339253	21526885	18947113	16597452	19390253	26717642
PS(37:1) − H	1490285	1230200	1235925	1091459	1106377	1765735
PS(37:2) − H	2442618	1570682	1294178	1322160	1543685	1712773
PS(20:0/18:1) − H	4404058	3438353	3842166	2467943	3232377	5076280
PS(20:1/18:1) − H	4447972	3647879	3171644	1468351	2896624	4625348
PS(18:0/20:2) − H	7301485	6132980	6804179	4283064	6532851	10844537
PS(38:2p) − H	868292	557395	539300	591168	509697	1082727
PS(18:0/20:3) − H	7291099	5913557	6277321	3075230	4133649	8275825
PS(18:0/20:4) − H	16230081	13766920	15571875	7992582	13196623	12118691
PS(38:6p) − H	1128932	979481	1380585	1009252	1217181	2419377
PS(39:1) − H	24120671	22985681	29804384	17316401	24036311	39411959
PS(39:2) − H	2186666	1356194	1539822	1142790	1149598	1675487
PS(39:3) − H	347503	2594189	3858644	1551287	103153	1098989
PS(39:4) − H	5368112	2963524	3154200	2495422	3334581	4466299
PS(18:1/22:0) − H	4064467	3910947	3839662	2517450	3511624	5597790
PS(18:1/22:1) − H	3408770	3588086	3990233	2412607	3464760	3787527
PS(18:1/22:2) − H	1900591	1419299	1770545	1001745	1410861	1663921
PS(18:0/22:4) − H	439453	24376	1323084	733550	83798	235578
PS(18:0/22:5) − H	2505071	1868756	1987230	1119427	1352943	2677997
PS(40:5) − H	918647	782875	799605	438104	636444	1227835
PS(18:1/24:0) − H	1779094	1588676	1707118	1285165	1539504	2224250
PS(18:1/24:1) − H	1309327	1699668	1528903	817297	1296234	2146994
SM(d31:1) + H	661321	39442	320879	242393	432634	801486
SM(d32:0) + H	2126244	1150012	1461213	1264922	1557618	2827620
SM(d32:1) + H	20813576	9887653	13209613	9420803	13501642	26642009
SM(d32:2) + H	458904	420542	479445	322512	460479	1013558
SM(d33:1) + H	20176171	12082191	15165424	9881287	13816429	22692799
SM(d33:5) + H	6655681	10365948	5994543	5789838	6753033	6067349
SM(d34:0) + H	74348703	38343858	42519960	32268555	43803065	99339303
SM(d34:1) + H	720958003	316727747	485339516	278121991	391114186	1145239538
SM(d34:2) + H	46429259	24835983	33744528	22937733	34010155	62982988
SM(d35:1) + H	9805746	5698520	7181681	4112566	5511952	13770024
SM(d35:2) + H	1022318	469336	783310	366076	438517	1396268
SM(d35:4) + H	1219039	605454	847635	521359	700116	1618402
SM(d36:0) + H	7328963	4292407	5027560	3365397	4533780	9924313
SM(d36:1) + H	54907427	27142651	32516465	17480858	26319513	73609304
SM(d36:2) + H	73447874	36400065	51621875	29077068	41004005	92640203
SM(d36:4) + H	129364326	36073210	64720825	25862014	44697675	189484155
SM(d38:1) + H	10928751	8448664	8192737	4663660	5920079	14480605
SM(d40:0) + H	6777251	6240481	6467063	4250276	5850613	7784881
SM(d40:1) + H	5321195	3301180	3456980	2931700	2960577	5389633
SM(d17:1/23:0) + H	77647588	57935569	56954593	36365164	48256698	96592883
SM(d40:2) + H	20148273	12043372	16079145	9871827	10367643	24131468
SM(d41:1) + H	29502586	21030836	20205698	20637081	23624592	29878221
SM(d41:2) + H	24595173	17713926	20309327	9317081	16938431	18494247
SM(d18:1/24:0) + H	32414981	25623789	29154664	20909991	23987512	34003673
SM(d42:1) + H	166038798	165707986	149865103	125824271	138966875	238445675
SM(d18:1/24:1) + H	506599392	378706422	418698134	249048472	323643458	726366870
SM(d18:1/24:2) + H	72319308	51039170	59775725	31725892	40856511	81325582
SM(d42:5) + H	3304635	1559076	2157023	827924	1384270	4405363
SM(d43:1) + H	6820186	4921753	4122241	5040782	5337809	7556288
SM(d18:2/25:0) + H	17119825	11991050	12580046	10240012	12843064	16158271
SM(d43:3) + H	2864266	2367593	2485033	1532869	1866262	3096500
SM(d43:4) + H	2486363	2400248	2112193	1914779	2254701	4635707
SM(d44:1) + H	4289607	3697676	3704597	2665198	2898206	4652818
SM(d44:2) + H	18273013	14139423	14425244	10407028	11349422	21240496
SM(d44:3) + H	10419505	7020260	8225159	4954560	5508703	16922243
SM(d44:4) + H	22148591	18165758	17943939	14457717	16811064	27270177
SM(d44:5) + H	76471759	47687528	58666392	33031699	49433776	118380147
SM(d44:6) + H	11507445	6706391	7295208	3601424	5492228	12759843
TG(16:0/14:0/14:0) + NH4	1520012	2642041	2166017	2045191	1966742	1871033
TG(44:5p) + NH4	10866658	11424737	12368285	7563479	10164188	14413003
TG(15:0/14:0/16:0) + NH4	4376313	6379941	6680392	5753912	5320679	4772871
TG(16:0/14:0/16:0) + NH4	3287947	5879047	4773183	4417473	4360536	3798349
TG(15:0/16:0/16:0) + NH4	5817155	9760490	7353934	6660437	7230331	7362384
TG(16:0/16:0/16:0) + NH4	8610155	13960240	11763798	9318170	9654277	8493829
TG(16:0/16:0/16:1) + NH4	3634548	5632293	4785307	4086280	4105906	3643224
TG(16:0/16:0/17:0) + NH4	3563345	6092571	5302713	4686056	4723196	4083586
TG(18:0/16:0/16:0) + NH4	12997842	21828186	19306947	7698317	6909395	8532186
TG(16:0/16:0/18:1) + NH4	4723975	6972172	6228199	5303522	5453287	4920543
TG(18:0/16:0/18:0) + NH4	24098293	29381643	29176733	10602096	8673417	11135253
TG(18:0/16:0/18:1) + NH4	3041252	4863162	3889854	2613999	2985202	2793863
TG(16:0/18:1/18:1) + NH4	4355829	8429628	5769497	5502647	4773535	5666641
TG(18:0/18:0/18:0) + NH4	10143089	17446411	14178580	6393429	5741955	6201394
TG(18:0/18:1/18:1) + NH4	2453354	3672295	3070653	2003892	2259814	2201605
TG(18:1/18:1/18:1) + NH4	3815697	5370092	4497566	5050375	5521008	4612804
TG(18:1/18:1/18:2) + NH4	1817698	1933037	1684654	2111086	1645265	1672020
	8.12E+09	4.90E+09	6.03E+09	3.57E+09	4.61E+09	1.20E+10

Example 1—Identification of a Distinct Nanoparticle Population and Subsets of Exosomes

B16-F10 Melanoma-Derived sEVs were First Fractionated by AF4 (See Methods). A linear separation of the sEV mixture was achieved based on the hydrodynamic radius (black dots, Y axis) along the time course (X axis) (FIG. 1A). The online QELS monitor for real-time dynamic light scattering (DLS) measurement (red trace) determined the hydrodynamic radius of particles. UV absorbance (blue trace) measured protein concentration and abundance of particles at specific time points for corresponding particle sizes. Particles with a 35-150 nm diameter were successfully separated by AF4 (FIG. 1A). Five peaks (P1-P5) were identified, corresponding to the time and particle size, at which most abundant particles were detected. P1 represented the void peak, a mixture of all types of nanoparticles. P5 was composed of individual or aggregated particles and protein aggregates with much larger sizes, which are outside the separation range of the current AF4 protocol, and eluted when crossflow dropped to zero (FIG. 2A). The hydrodynamic diameters of peaks P2, P3 and P4 were 47 nm, 62 nm and 101 nm, respectively. To infer the hydrodynamic radius, correlation functions were fitted to single exponentials (FIG. 1B, representative P3 fraction graph).
Individual fractions were measured using Nanosight Tracking Analysis (NTA), validating consistent particle size for each fraction between 60 nm and 140 nm (FIG. 2B). DLS combined with AF4 showed a broader dynamic range than NTA for those particles with a smaller (˜70 nm) or larger (˜160 nm) particle size (FIG. 2C). Moreover, NTA of each individual fraction in the range of 60-160 nm revealed a monomodal profile with a peak of ˜77 nm (FIG. 2D).
Transmission electron microscopy (TEM) with negative staining of AF4 input and representative fractions across the full dynamic range revealed three populations of particles (P2, P3, P4; FIG. 1A) with distinct morphology and size (FIG. 1C). P2 represented a distinct population of nanoparticles not previously described, which were smaller than 50 nm (˜35 nm) and clearly lacked an external membrane structure (FIG. 1C); these structures were therefore named “exomeres”. The other two nanoparticle subpopulations are referred to as small exosomes (Exo-S; 60-80 nm [P3]) and large exosomes (Exo-L; 90-120 nm [P4]) (FIG. 1C). All three particle types were readily detected in the input TEM image (FIG. 1C). Western blot analysis confirmed exosome markers Tsg101 and Alix for Exo-S and Exo-L, and heat shock protein 90 (Hsp90) for exomeres (FIG. 1D). The sizes of each particle type measured in batch mode showed consistent results (FIG. 1E).
In summary, a single run of AF4 can efficiently discern exomeres and two distinct exosome subpopulations in a robust and highly reproducible manner (FIG. 2E, 2F). Freeze-thawing of samples led to inconsequential differences (FIG. 2G). However, changes in culture conditions led to differences in relative abundance of each particle type (FIG. 2H-2I).
Importantly, only a minor peak eluted in the time range similar to exomeres in a blank media control compared to CM of B16-F10 and MDA-MB-4175 when processed in parallel (FIG. 2J, 2K), thereby confirming that exomeres are indeed actively secreted by cultured cells and not mere aggregates present in media.
Using AF4, distinct particles were detected with diameters corresponding to exomeres and Exo-S/L in more than 20 cell lines analyzed (Table 9, FIG. 3A), findings confirmed by TEM analysis of pooled fractions from selected cell lines (FIG. 38).

TABLE 9

Cancer	Cell lines	Species

Melanoma	B16-F10	m
	B16-F1	m
	SK-Mel113	h
	A375M	h
	A375P	h
Pancreatic Cancer	AsPC-1	h
	Pan02	m
	PANC1	h
	HPAPII	h
	BxPC3	h
CRC	HCT116	h
	SW620	h
NSCLC	PC9	h
	LLC	m
Prostate Cancer	DU145	h
	PC3	h
Breast Cancer	4TI	m
	MDA-MB-231	h
	MDA-MB-1833	h
	MDA-MB-4175	h
	MDA-MB-831	h
Leukemia	K562	h
	NB4	h
Transformed Non-cancer cells	NIH3T3	m
	ET2B	h

Based on UV absorbance and TEM analysis, all cells secreted higher amounts of exomeres relative to Exo-S/L, except for B16-F10 and B16-F1 where Exo-S were relatively more abundant (FIG. 3A and FIG. 1A, 1B). Measurement of the hydrodynamic diameter of each of these particles using Zetasizer showed sizes similar to the B16-F10 preparations (FIG. 1E).

Exomeres and Exo-S/L were also detected in AF4-fractionated sEVs from CM of human melanoma tumor explants by TEM (FIG. 1F, arrows; FIG. 4A). Exomere and Exo-S size, measured in batch mode using Zetasizer, was comparable to results from tumor cell lines (FIG. 1G). AF4 profiling and TEM imaging analysis showed that the normal mouse tissue explants (mammary fat pad and lung) also secreted exomeres, and Exo-S/L nanoparticles (FIG. 4B).

Example 2—Biophysical Characterization of Exomeres and Exosome Subpopulations

Given the structural differences between exomeres and Exo-S/L, their biophysical properties, such as zeta potential and stiffness, were examined. Measuring zeta potential, an average surface charge, using Zetasizer, revealed all particles were negatively charged, with exomeres being the weakest negatively charged (−2.7 mV to −9.7 mV); Exo-L, the strongest (−12.3 mV to −16.0 mV); and Exo-S, intermediate (−9.0 mV to −12.3 mV) (FIG. 5A).
For particle stiffness, atomic force microscopy (AFM) was performed in solution (see Methods). Exomeres demonstrated the highest stiffness (145˜816 mPa) and Exo-L the lowest (26˜73 mPa), with Exo-S stiffness being intermediate (70˜420 mPa).
AFM analysis of exomeres derived from B16-F10. MDA-MB-4175, and AsPC-1 cell lines demonstrated exomere structural heterogeneity and average exomere heights of 5.9 nm, 7.0 nm and 5.8 nm, respectively (FIG. 5C, SD).
Collectively, these findings demonstrate the diverse biophysical properties exhibited by exomeres versus distinct exosome subpopulations. How size, charge, and mechanical properties influence the differential stability, trafficking and uptake of the nanoparticles in vivo requires further investigation (Beningo et al., “Fe-Receptor-Mediated Phagocytosis is Regulated by Mechanical Properties of the Target,” Journal of Cell Science 115:849-856 (2002); Key et al., “Soft Discoidal Polymeric Nanoconstructs Resist Macrophage Uptake and Enhance Vascular Targeting in Tumors,” ACS Nano 9:11628-11641 (2015), which are hereby incorporated by reference in their entirety).

Example 3—Distinct Proteomic Content and Cellular Functions Among Exomeres and Exosome Subpopulations

To characterize the molecular composition of exomeres and distinct exosome subpopulations, proteomic profiling of nanoparticles derived from B16-F10, Pan02, 4T1, AsPC-1, MDA-MB-4175 cells was conducted using label-free mass spectrometry. A range of 165-483 proteins were identified in exomeres, 433-1004 proteins in Exo-S, and 247-1127 proteins in Exo-L. Moreover, unique proteins were detected in each nanoparticle subtype (FIG. 6A), suggesting exomeres are unique entities released by cells rather than debris or fragments of exosomes.
Examination of the subcellular localization annotation of proteins revealed the specific enrichment of Exo-S/L in membrane-associated proteins, which were relatively depleted in exomeres (Table 10), consistent with the structural studies identifying Exo-SL as membrane-encapsulated particles and exomeres as non-encapsulated particles.

TABLE 10

EXOMERE	EXO-S	EXO-L

Pathways	FDR q-val	Pathways	FDR q-val	Pathways	FDR q-val

Proteasame accessory	<0.001	Intrinsic component of plasma membrane	<0.001	Escrt complex	<0.001
complex
Endoplasmic reticulum	<0.001	Late endosome membrane	<0.001	Cytoplasmic side of membrane	0.001
lumen
Cytosolic part	0.001	Endosomal part	<0.001	Intercalated disc	0.001
Vesicle lumen	0.001	Phagocytic vesicle	<0.001	Basolateral plasma membrane	0.002
Proteasome complex	0.001	Secretory granule membrane	<0.001	Extrinsic component of	0.002
				cytoplasmic side of plasma
				membrane
Secretory granule	0.002	Vacuolar membrane	<0.001	midbody	0.002
lumen
microtubule	0.004	Phagocytic vesicle membrane	0.001	Heterotrimeric g protein	0.002
				complex
sarcoplasm	0.029	Late endosome	0.001	Cell cell contact zone	0.002
Inclusion body	0.030	Lytic vacuole membrane	0.001	Apical junction complex	0.004
Myelin sheath	0.031	endosome	0.001	Cell division site	0.005
Platelet alpha granule	0.032	Vacuolar part	0.001	Extrinsic component	0.005
lumen				of plasma membrane
Blood microparticle	0.032	vacuole	0.007	Late endosome membrane	0.006
Extracellular matrix	0.033	Multivesicular body	0.008	Extrinsic component of	0.010
				membrane
Chromosome	0.070	Lytic vacuole	0.010	filopodium	0.011
centromeric region
mitochondrion	0.071	Endocytic vesicle	0.010	Side of membrane	0.012
Supramolecular fiber	0.074	Recycling endosome	0.011	Plasma membrane	0.012
				protein complex
Proteinaceous	0.083	Extracellular matrix_component	0.011	synapse	0.013
extracellular matrix
Extracellular space	0.087	escrt_complex	0.013	postsynapse	0.015
Dna packaging	0.114	early_endosome	0.013	Synapse part	0.016
complex
Microtubule	0.146	snare_complex	0.013	Snare complex	0.016
cytoskeleton
Methyltransferase	0.151	plasma_membrane_raft	0.013	Cell junction	0.017
complex
Cytosketetal part	0.166	membrane_protein_complex	0.014	Anchoring junction	0.017
microbody	0.186	cell_cell_adherens_junction	0.016	ruffle	0.017
Motile cilium	0.236	basement_membrane	0.022	Membrane region	0.018
polysome	0.238	proton_transporting_two_sector_atpase_complex	0.024	Trans Golgi	0.018
				network transport
				vesicle
Ciliary part	0.238	dna_packaging_complex	0.025	Neuron spine	0.018
Nuclear pore	0.246	endacytic_vesicle_membrane	0.025	Plasma membrane receptor	0.018
				complex
podosome	0.257	secretory_vesicle	0.025	Cell leading edge	0.019
Sperm part	0.258	Plasma membrane	0.032	Intrinsic component	0.023
		protein complex		of plasma membrane

ESCRT- and Snare-related proteins, involved in vesicle budding, membrane fusion and exosome biogenesis (Colombo et al., “Biogenesis, Secretion, and Intercellular Interactions of Exosomes and Other Extracellular Vesicles,” Annu Rev Cell Dev Biol 30:255-289 (2014); Hessvik et al., “Current Knowledge on Exosome Biogenesis and Release,” Cell Mol Life Sci (2017), which are hereby incorporated by reference in their entirety), were identified within Exo-S/L. In particular, proteins associated with endosomes, multivesicular bodies, vacuoles, and phagocytic vesicles were enriched in Exo-S. Plasma membrane, cell-cell contact/junction, late-endosome, and trans-Golgi network proteins were enriched in Exo-L. Notably, proteins associated with extracellular matrix and space, proteasome accessory complex, endoplasmic reticulum, mitochondrion, and microtubule/cytoskeleton were packaged in exomeres. These findings imply possible fundamental differences in exomeres, Exo-S, and Exo-L biogenesis.

Principal component analysis (PCA) demonstrated closer correlation of protein expression for Exo-S and Exo-L compared to exomeres from the same cell-type (FIG. 7A). According to PCA and consensus clustering analysis, exomeres from different cell types exhibited a higher degree of similarity to each other than to Exo-S and Exo-L from the same cell type (FIG. 6B, 6C).
To identify the signature proteins in each particle subset, statistical analysis was performed on the expression levels of proteins identified in these datasets. 64 proteins were pinpointed for exomeres and 99 proteins for Exo-S/L (Tables 11-14), with a false discovery rate (FDR)<0.05, positive enrichment in each particle subset of interest, and detection frequency of >80% (i.e., a particular protein was positively enriched in at least 4/5 samples for each subtype of nanoparticles derived from 5 different cell lines).

TABLE 11

	Average expression	Frequency	Fold change	Fold change
Symbol	in exomere	(%, n = 5)	exomere vs Exo-S	exomere vs Exo-L

PPID	1.83E+08	80%	Inf	21.5
GANAB	3.03E+08	80%	16.4	Inf
MAT1A	9.11E+08	100%	10.4	Inf
DPYD	1.61E+08	80%	10.1	Inf
FAT4	3.48E+08	100%	9.3	91.9
GMPPB	1.15E+08	80%	8.3	Inf
ERP44	2.64E+08	100%	7.5	Inf
CALR	5.71E+08	100%	8.8	48.4
GPD1	2.93E+08	100%	7.1	Inf
BZW1	1.86E+08	100%	9.5	24.3
PFKL	5.34E+08	100%	6.4	134.6
OLFML3	1.98E+08	80%	8.1	Inf
HGD	4.03E+08	100%	5.9	Inf
LGALS3BP	3.40E+09	100%	7.9	21.2
GCLC	3.87E+08	100%	5.6	Inf
PEPD	8.00E+08	100%	5.8	87.6
MTHFD1	6.17E+08	100%	8.1	15.4
PGD	1.01E+09	80%	7.0	16.2
ACTR3	3.54E+08	100%	12.7	7.6
XPNPEP1	3.68E+08	100%	5.3	43.6
UGP2	8.77E+08	100%	5.8	24.6
SNX2	1.89E+08	80%	4.7	214.1
ALDOC	4.03E+08	80%	6.2	17.2
SEPT11	2.14E+08	80%	29.0	5.3
HSPA13	8.68E+08	100%	5.6	22.4
AARS	1.67E+08	80%	15.4	6.3
SERPINH1	6.42E+08	100%	4.7	48.8
CNDP2	4.63E+08	100%	4.4	76.7
PDE5A	2.22E+08	80%	4.4	79.7
AGL	3.14E+08	100%	4.4	72.8
EXT1	8.34E+08	100%	4.2	146.4
IDH1	4.82E+08	100%	5.1	20.6
SERPINC1	3.95E+09	80%	4.0	1601.2
RRM1	5.00E+08	100%	4.0	Inf
CKB	3.61E+08	80%	3.8	97.1
HMGCS1	4.25E+08	100%	4.4	17.9
HPD	1.10E+09	100%	3.9	38.6
PSMC4	3.13E+08	100%	3.9	35.6
NPEPPS	2.09E+08	80%	4.0	24.4
CAT	4.57E+08	100%	3.9	32.2
EXT2	6.05E+08	100%	3.8	38.6
CORO1C	6.60E+08	100%	3.9	26.1
B4GAT1	6.53E+08	100%	3.5	63.7
RACK1	3.10E+08	100%	4.4	13.3
MAPRE1	2.46E+08	80%	4.6	11.1
PGM1	1.12E+09	100%	3.5	37.8
PDSA3	6.64E+08	80%	4.4	11.2
ADK	1.23E+09	100%	3.6	25.9
SHMT1	2.30E+08	80%	3.8	24.5
ACO1	1.72E+09	100%	3.3	65.1
GSN	1.29E+10	100%	3.2	96.9
ESD	4.15E+08	80%	5.0	6.3
PPP2R1A	6.38E+08	100%	3.7	10.1
ALDH1L1	1.73E+09	100%	2.9	36.4
OLA1	2.81E+08	80%	5.0	5.8
ACLY	8.92E+08	100%	3.1	20.8
EEF1G	7.95E+08	100%	3.3	13.6
FLNB	3.08E+08	80%	4.0	7.9
PSMD11	2.26E+08	80%	3.1	17.8
ANGPTL3	3.01E+08	80%	2.8	31.7
FERMT3	7.54E+08	80%	2.8	27.6
PYGL	1.60E+09	100%	2.8	28.6
MDH1	3.34E+08	80%	8.0	3.7
EIF4A2	5.83E+08	80%	2.6	86.4

“inf” stands for “infinity”, indicating proteins that are absent in Exo-S or Exo-L.

TABLE 12

	Average expression	Frequency	Fold change
Symbol	in Exosome	(%, n = 20)	Exosome vs exomere

GNA13	1.16E+09	80%	Inf
DNAJA1	9.79E+08	100%	Inf
SLC38A2	9.48E+08	90%	Inf
TFRC	9.06E+08	100%	Inf
BSG	8.57E+08	80%	Inf
LAMP1	6.77E+08	90%	Inf
EHD2	6.74E+08	80%	Inf
ANXA5	6.56E+08	80%	Inf
SLC1A5	5.89E+08	90%	Inf
NRAS	5.84E+08	100%	Inf
CHMP5	5.69E+08	90%	Inf
DNAJA2	5.51E+08	90%	Inf
ANXA1	5.46E+08	90%	Inf
ANXA11	5.28E+08	80%	Inf
ATP1B3	5.28E+08	90%	Inf
SH3GL1	5.01E+08	90%	Inf
FLOT2	4.92E+08	100%	Inf
RAP2B	4.83E+08	90%	Inf
FLOT1	4.31E+08	100%	Inf
RALA	4.26E+08	80%	Inf
RAP2C	4.23E+08	80%	Inf
CEP55	4.18E+08	90%	Inf
STOM	4.13E+08	100%	Inf
MMP14	3.60E+08	90%	Inf
CHMP2A	3.59E+08	90%	Inf
TM9SF2	3.04E+08	80%	Inf
MYO1C	3.02E+08	80%	Inf
DIP2B	3.01E+08	90%	Inf
GNA11	3.00E+08	90%	Inf
MET	2.91E+08	80%	Inf
CTNNB1	2.78E+08	90%	Inf
ANXA4	2.78E+08	80%	Inf
LYN	2.58E+08	90%	Inf
ATP2B1	2.54E+08	80%	Inf
GNG12	2.54E+08	80%	Inf
GNAQ	2.24E+08	90%	Inf
YES1	2.22E+08	100%	Inf
RRAS	2.16E+08	80%	Inf
ITCH	1.86E+08	90%	Inf
ANTXR2	1.86E+08	90%	Inf
RRAS2	1.84E+08	80%	Inf
TGFBR2	1.81E+08	80%	Inf
ARF4	1.64E+08	90%	Inf
TOLLIP	1.60E+08	90%	Inf
ANXA7	1.59E+08	90%	Inf
SNAP23	1.58E+08	80%	Inf
VPS25	1.49E+08	80%	Inf
SLC12A2	1.46E+08	80%	Inf
CD2AP	1.44E+08	90%	Inf
STXBP3	1.40E+08	90%	Inf
EPS8	1.37E+08	80%	Inf
CHMP1A	1.36E+08	80%	Inf
JAK1	1.30E+08	90%	Inf
GRB2	1.20E+08	80%	Inf
MAP4K4	1.19E+08	80%	Inf
STX4	1.18E+08	80%	Inf
NEDD4L	1.15E+08	80%	Inf
RAB22A	1.04E+08	80%	Inf
ANXA2	1.08E+09	80%	54.6
MYOF	6.20E+08	100%	48.6
VPS4B	6.85E+08	90%	47.3
PDCD6	7.87E+08	90%	39.5
VPS37C	1.09E+09	90%	34.7
VPS4A	4.66E+08	90%	31.9
ITGAV	5.61E+08	100%	30.6
TSPAN14	4.39E+08	80%	26.6
TSPAN4	1.24E+09	80%	26.5
CHMP4B	8.65E+08	100%	26.1
ITGB5	2.93E+08	80%	19.5
IST1	1.03E+09	80%	19.0
EPHA2	9.45E+08	80%	16.5
GNAI3	2.15E+09	90%	12.5
RAB5B	2.99E+08	90%	11.6
GNAS	1.75E+09	100%	10.8
VPS37B	6.71E+08	100%	10.8
ITGA3	5.46E+09	80%	9.7
TSG101	1.11E+09	100%	9.5
CTNNA1	8.46E+08	90%	9.4
MVB12A	9.08E+08	90%	9.1
RDX	8.18E+08	80%	9.0
ATP1A1	1.96E+09	100%	8.9
PACSIN2	1.37E+08	80%	8.8
ITGB1	8.77E+09	100%	8.7
SLC3A2	2.39E+09	100%	8.6
RAB8B	5.71E+08	80%	8.6
ITGA6	6.84E+08	80%	8.6
RAB14	6.00E+08	100%	8.6
VPS28	1.37E+09	100%	8.0
CD9	8.89E+09	100%	7.9
LAMP2	3.95E+08	90%	7.8
RAB3S	7.70E+08	100%	7.5
BROX	4.16E+08	90%	7.2
CD44	9.51E+08	90%	7.0
MFGE8	5.72E+09	90%	6.9
CTNND1	3.39E+08	80%	6.8
ITM2B	5.79E+08	80%	6.7
GNAI2	2.49E+09	100%	6.3
ARRDC1	1.11E+09	80%	5.9
PDCD6IP	8.05E+09	100%	5.8

“inf” stands for “infinity”, indicating proteins that are absent in exomere.

TABLE 13

	Average expression	Frequency	Fold change	Fold change
Symbol	in Exo-S	(%, n = 5)	Exo-S vs exomere	Exo-S vs Exo-L

TTYH3	2.66E+08	100%	Inf	7.8
FLOT1	7.46E+08	100%	Inf	6.4
FLOT2	8.39E+08	100%	Inf	5.8
TSPAN14	7.39E+08	100%	44.8	5.3
LAMC1	1.44E+08	80%	6.8	11.4
CD63	5.95E+09	100%	Inf	3.3
MVB12A	1.44E+09	80%	14.5	3.8
ZDHHC20	1.56E+08	80%	Inf	3.0
VAMP3	1.08E+08	80%	Inf	2.8
VPS37B	1.03E+09	100%	16.6	3.3
ARRDC1	1.75E+09	80%	9.3	3.7
TGFBR2	2.60E+08	80%	Inf	2.6

“inf” stands for “infinity”, indicating proteins that are absent in Exomere or Exo-L.

TABLE 14

	Average expression	Frequency	Fold change	Fold change
Symbol	in Exo-L	(%, n = 5)	Exo-L vs exomere	Exo-L vs Exo-S

SQSTM1	2.34E+08	80%	Inf	Inf
STIP1	1.99E+08	100%	Inf	Inf
HINT1	1.68E+08	80%	Inf	Inf
WASF2	1.58E+08	80%	Inf	Inf
RASA3	1.48E+08	80%	Inf	Inf
EPB41L2	1.45E+08	80%	Inf	Inf
GIPC1	1.29E+08	80%	Inf	Inf
S100A10	3.76E+08	80%	Inf	89.7
MPP6	1.70E+08	100%	Inf	42.1
KIF23	3.38E+08	80%	Inf	35.8
RACGAP1	2.43E+08	80%	Inf	24.8
ANXA5	1.23E+09	100%	Inf	14.9
CASK	1.24E+08	80%	Inf	14.8
DLG1	2.18E+08	100%	Inf	14.4
TJP1	1.02E+08	80%	Inf	13.4
BAG5	1.36E+08	80%	Inf	12.3
TXN	4.79E+08	80%	Inf	12.3
ABI1	1.91E+08	100%	Inf	11.5
ANXA1	1.00E+09	100%	Inf	11.0
CAPG	1.17E+08	80%	Inf	9.9
DBI	2.68E+08	80%	Inf	9.0
S100A6	3.22E+09	80%	33.2	12.1
CHMP2B	1.78E+08	80%	Inf	8.6
CHMP3	1.76E+08	86%	Inf	7.9
ANXA2	1.91E+09	80%	96.7	7.7
MYO1C	5.24E+08	100%	Inf	6.6
ANXA4	4.79E+08	100%	Inf	6.2
SNX12	1.23E+08	80%	Inf	5.8
LIN7C	1.97E+08	80%	Inf	5.3
STXBP3	2.32E+08	100%	Inf	4.9
CEP55	6.92E+08	80%	Inf	4.8
ALCAM	2.93E+08	80%	Inf	4.7
VCL	2.95E+08	80%	20.0	6.0
CHMP1A	2.21E+08	100%	Inf	4.3
FARP1	3.59E+08	80%	14.1	6.2
ACSL4	1.64E+08	80%	Inf	4.3
BAIAP2	2.28E+08	80%	Inf	4.3
SH3GL1	8.10E+08	100%	Inf	4.2
DSTN	2.41E+08	100%	4.8	36.4
LGALS1	1.74E+09	80%	13.0	5.9
CYFIP1	1.66E+08	100%	9.4	7.1
CTNNA1	1.43E+09	100%	15.9	5.4
RAB31	2.28E+08	80%	Inf	4.0
ARF6	2.05E+08	80%	Inf	3.9
SLC1A5	9.38E+08	100%	Inf	3.9
EPS8	2.18E+08	80%	Inf	3.9
FMNL2	2.11E+08	100%	Inf	3.9
PGAM1	5.42E+08	100%	4.3	26.8
CNP	1.37E+08	80%	Inf	3.7
CHMP4B	1.39E+09	100%	41.9	4.0
ANXA3	4.14E+08	80%	Inf	3.7
VPS4B	1.09E+09	100%	75.0	3.9
GNG12	3.99E+08	100%	Inf	3.6
PACSIN3	1.20E+08	100%	Inf	3.4
GLG1	1.25E+08	80%	Inf	3.2
VTA1	3.19E+08	80%	Inf	3.2
LYN	3.91E+08	100%	Inf	3.1
VPS37C	1.68E+09	100%	53.2	3.3
CHMP5	8.59E+08	100%	Inf	3.1
F3	8.48E+08	80%	28.6	3.4
DNAJA1	1.47E+09	100%	Inf	3.0
RHOC	1.09E+09	80%	26.0	3.4
GNA13	1.72E+09	100%	Inf	2.9
CHMP2A	5.33E+08	100%	Inf	2.9
ATP2B1	3.74E+08	100%	Inf	2.8
RDX	1.26E+09	100%	13.9	3.4
ATP1B1	3.67E+08	80%	Inf	2.7
CAPZB	1.29E+08	80%	3.2	15.3
EHD1	4.39E+09	100%	6.2	4.5
DNAJA2	7.91E+08	80%	Inf	2.5
CTNND1	5.21E+08	100%	10.4	3.3

“inf” stands for “infinity”, indicating proteins that are absent in Exomere or Exo-S.

Remarkably, exomeres were significantly enriched in proteins involved in metabolism (see gene set enrichment analysis [GSEA] analysis below), including MAT1A, IDH1, GMPPB, UGP2, EXT1, and PFKL. The sialoglycoprotein galectin-3-binding protein (LGALS3BP) and key proteins controlling glycan-mediated protein folding control (CALR) (Molinari et al., “Chaperone Selection During Glycoprotein Translocation into the Endoplasmic Reticulum,” Science 288:331-333 (2000), which is hereby incorporated by reference in its entirety) and glycan processing (MAN2A1, HEXB, GANAB) (Fukuda et al., “Incomplete Synthesis of N-Glycans in Congenital Dyserythropoietic Anemia Type II Caused by a Defect in the Gene Encoding Alpha-Mannosidase II,” Proc Natl Acad Sci USA 87:7443-7447 (1990); Yang et al., “An intrinsic Mechanism of Secreted Protein Aging and Turnover,” Proc Natl Acad Sci USA 112:13657-13662 (2015); Martiniuk et al., “Identity of Neutral Alpha-Glucosidase AB and the Glycoprotein Processing Enzyme Glucosidase II. Biochemical and Genetic Studies,” The Journal of Biological Chemistry 260:1238-1242 (1985), which are hereby incorporated by reference in their entirety) are also enriched in exomeres, suggesting exomere cargo may mediate the targeting of recipient cells through specific glycan recognition and modulate glycosylation in recipient cells. Among proteins uniquely represented in Exo-S/L were annexins, ESCRT components (charged multivesicular body proteins/CHMPs, vacuolar protein-sorting proteins, HGS, Alix1/PDCD61P, and Tsg101), Hsp40 (DnaJ) family proteins, signaling transducer G protein subunits, integrins, Rab proteins, and solute carrier family members. Members of key signaling pathways, such as JAK1, TGFBR2, and MET, were also enriched in Exo-S/L. To evaluate the unique markers of Exo-S and Exo-L subpopulations, protein expression was compared between these two sample sets and exomeres separately using t-test. A second set of filters (protein intensity/area >10⁸and fold change ≥5.0) was applied to the identified signature for exomeres and Exo-L, but not for Exo-S (FIG. 6D). Fewer signature proteins were identified for Exo-S compared to exomeres and Exo-L, most likely due to similarity of Exo-S to the other particles. Representative signature proteins identified by proteomics in each subset were validated by western blot analysis (FIG. 6E).

These proteomic datasets were further mined for conventional exosome markers, including flotillins, CD9, CD63, CD81, Alix1, Tsg101, HSC70 (HSPA8) and Hsp90 (FIG. 6F). Among the five cell lines, flotillins (FLOT1 and FLOT2) represented bona fide markers of Exo-S, while HSP90AB1 was preferentially associated with exomeres. Although CD9, CD63 and CD81 all demonstrated specific association with Exo-S/L subsets, they all showed a cell type- and particle-dependent preferential expression. Consistent with Kowal et al., “Proteomic Comparison Defines Novel Markers to Characterize Heterogeneous Populations of Extracellular Vesicle Subtypes,” Proc Natl Acad Sci USA 113:E 968-977 (2016), which is hereby incorporated by reference in its entirety, combining CD63, CD9 or CD81 will be necessary to isolate/label exosomes.
Numerous Rab proteins were found in Exo-S/L subsets, but few of them in exomeres (FIG. 7B), suggesting critical roles of Rab proteins for Exo-S/L formation and trafficking, but not for exomere biogenesis.
Next, the most abundant proteins in each subset of nanoparticles were examined. Hemoglobin, histones, cytoskeleton proteins (actins and tubulins), peptidylprolyl isomerase A (PPIA) and HSP ranked as the most abundant top 50 proteins in all three nanoparticle subpopulations (Table 1). Hsp40/DnaJ family (HSP70 co-chaperones) members were also found in the top 50 proteins for Exo-L. Interestingly, HSP90AB1 was preferentially packaged in exomeres, while HSP70 members (HSPA8, HSPA2 and HSPA5) were more abundant in Exo-S/L. Other proteins relatively enriched in exomeres included inter-alpha-trypsin inhibitor heavy chain family members (IT1H), gelsolin (GSN), talin 1 (TLN1), WD repeat domain 1 (WDR1), and proteins involved in metabolism, such as phosphoglycerate kinase 1 (PGK1), pyruvate kinase muscle (PKM), and enolase 1 (ENO1). Consistent with the analysis above, SDCBP, PDCD61P/Alix, tetraspanins (CD9, CD63, CD81 and others), G protein family proteins and integrins were highly represented in both Exo-S and Exo-L. Tetraspanins were preferentially enriched in Exo-S while G proteins and integrins were more prominent in Exo-L. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was most often present in exomeres and Exo-L. Other proteins preferentially associated with Exo-S included immunoglobulin superfamily member 8 (IGSF8) and its paralog prostaglandin F2 receptor inhibitor (PTGFRN), milk fat globule-EGF factor 8 protein (MFGE8), and components of the ESCRT-1 complex. Notably, annexins and S100 proteins were only represented in the top 50 proteins of Exo-L.
Furthermore, to exclude the possibility of lipoprotein contamination in exomeres, proteins that are typically associated with purified lipoprotein particles (high-, low-, and very low-density lipoproteins, i.e., HDL, LDL, and VLDL) were examined by proteomic MS analysis and then evaluated their presence in exomeres and Exo-S/L. Much fewer proteins were found in lipoproteins (Table 2) and only some of these proteins were detected in exomeres and Exo-S/L, suggesting most nanoparticle proteins are distinct from lipoproteins. A rough estimation showed that the lipoprotein-associated proteins account for 0-8% of total nanoparticle proteins (FIG. 7C). Moreover, EM analysis revealed that lipoprotein morphology/structure was clearly distinct from exomeres and Exo-S/L (FIG. 7D). Taken together, these analyses ruled out the possibility that exomeres were mere lipoprotein contaminants.
The possible contamination of exomeres with other types of protein complexes with high molecular weights was also ruled out when exomere proteins were surveyed for subunits of known complexes. The co-existence of multiple subunits of protein complexes of similar size to exomeres were not detected (Table 2) except for 10 out of 59 subunits of Parvulin-associated pre-rRNP complex in 4T1 exomeres, 17 subunits of ribosomes in AsPC-1 exomeres, and 7 out of 16 subunits of Kinase maturation complex 1 in MDA-MB-4175 exomeres. However, these proteins account for only 1.8%, 2.1% and 1.8% of total exomere proteins in each case, respectively, suggesting their contribution diminishes the purity of exomeres by ˜2%.
To gain insight into the function of these particle subsets, GSEA was conducted utilizing gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and hallmark databases (Tables 3-5). Strikingly. GSEA demonstrated that exomere-specific proteins were selectively enriched in metabolic processes, including carbohydrate metabolism, protein synthesis, and small-molecules. At least 36 of the top 50 “GO-biological processes” pathways identified metabolic processes associated with exomeres in contrast to no metabolic processes associated with Exo-S/L (Tables 3-5). Genes encoding proteins involved in hypoxia, microtubule and coagulation were identified in exomeres (FIG. 7E). Exo-S were enriched in membrane vesicle biogenesis and transport, protein secretion and receptor signaling gene sets. For Exo-L, enriched gene sets included mitotic spindle, IL-2/Stat5 signaling, multi-organism organelle organization, and G-protein signaling. Profiles of top rank gene sets enriched in exomeres (glycolysis and mTORC1 signaling), Exo-S (endosome and protein secretion) and Exo-L (mitotic spindle and IL-2/Stat5 signaling) are displayed in FIG. 6G.

Example 4—Distinct N-Glycan Profiles of Exomeres and Exosome Subpopulations

Aberrant glycosylation is involved in pathological processes, including cancer (Pinho et al., “Glycosylation in Cancer: Mechanisms and Clinical Implications,” Nature Reviews Cancer 15:540-555 (2015), which is hereby incorporated by reference in its entirety). Here, the aim was to determine the N-glycan profiles of each particle subset in three cell lines by conducting lectin blotting analysis (FIG. 8A) and glycomic mass spectrometry.
E-PHA recognizing bisected N-glycans detected a major band at approximately 75 kDa in both Exo-S and Exo-L of B16-F10 and AsPC-1, with faint detection in exomeres across the three cell lines and Exo-S of MDA-MB-4175. E-PHA detected a high molecular-weight glycoprotein (240 kDa) in MDA-MB-4175 exomeres and a high molecular weight glycoprotein (150 kDa) in AsPC-1 and MDA-MB-4175 exomeres. L-PHA recognizing branched N-glycans detected a predominant band at 75 kDa in both Exo-S and Exo-L of B16F-10 and AsPC-1. Multiple bands ranging from 50 to 70 kDa were also detected in all exomeres (especially MDA-MB-4175). Using AAL, analysis of structures related to fucosylation (fucose linked α-1,6) to GlcNAc or fucose linked (α-1,3) to GlcNAc related structures revealed two abundant glycoproteins between 70 and 100 kDa in both Exo-S and Exo-L of B16-F10 and AsPC-1. Exomeres across all three cell lines and Exo-S of MDA-MB-4175 displayed strong fucosylation on high molecular-weight glycoproteins (200-280 kDa). SNA, recognizing α-2,6-linked sialic acid, detected the presence of high molecular-weight α-2,6-sialylated glycoproteins (200-250 kDa) in all exomeres. Moreover, a low molecular-weight protein (˜60 kDa) displaying α-2,6-linked sialic acid modification was uniquely detected in Exo-L (but not Exo-S) from B16-F10. For AsPC-1, exomeres were the major carriers of sialylated glycoproteins, while these sialylated structures were almost absent in Exo-L. Lectin-binding profiles did not overlap with the most abundant proteins in the SDS-PAGE gel, indicating the specificity of lectin recognition independently of protein abundance (FIG. 9A). Therefore, Exo-S and Exo-L versus exomeres display distinct N-glycosylation patterns. Notably, exomere and Exo-S/L-associated N-glycan profiles vary by cell type. Future studies will address the identity of these glycoproteins via glycoproteomic approaches.
The next aim was to identify profiles of the glycan structures enriched in each particle subset by MALDI-TOF mass spectrometry (MS). Two independent, semi-quantitative MS analyses were conducted on B16-F10-derived exomeres and Exo-S/L (FIG. 8B). FIG. 8C depicts the quantification of the top six most abundant N-glycan structures detected in one of the representative experiments. The ubiquitous expression of certain complex N-glycans was observed in all subsets, corresponding to peaks at m/z 2209.8, 2223.7, 2237.7 and 2365.5. Specifically, a complex N-glycan at m/z 2015.7 and a hybrid N-glycan at m/z 2404.8 were enriched in exomeres. Moreover, four of these six N-glycans contained sialic acid, and three of six were fucosylated. Similarly, the ions m/z 2015.7 and 2404.8 were enriched in exomeres from MDA-MB-4175 (FIG. 9B, 9D). The ion m/z 2404.8 was slightly enriched in AsPC1 exomeres, but the ion at m/z 2015.7 was not detected in AsPC-1 samples (FIG. 9B, 9C). Instead, the ion at m/z 2012.7 was strongly detected in AsPC1 exomeres and Exo-S. Two other ions, at m/z 2117.7 and 2389.9, demonstrating Exo-S enrichment, were detected in AsPC-1 only (FIG. 9B, 9D).
High-resolution MS analysis allowed further structural characterization of certain N-glycans (FIG. 9E-9J). This was the case of extracted ion chromatogram m/z 1111.39 (2-) and 1007.38 (2-) (corresponding to m/z 2223.7 and 2015.7 in FIG. 4c , respectively). In addition, the combination of CID-MS/MS de novo sequencing and PGC-LC relative retention times for extracted ion chromatogram m/z 1111.39 (2-) revealed that this N-glycan from exomeres contained both α2,3-linked and α2,6-linked sialic acids, whereas the glycan from Exo-S contained exclusively α2,3-linked sialic acids. The unique presence of m/z 1007.38 (2-) in exomeres was also further confirmed.
Taken together, the glycomics study demonstrated the prevalence of complex N-glycans in all particle subsets with relatively high levels of sialylation, consistent with previous findings of complex N-glycans and sialoglycoproteins in tumor microvesicles/exosomes (Escrevente et al., “Sialoglycoproteins and N-glycans from Secreted Exosomes of Ovarian Carcinoma Cells,” PloS One 8:e78631 (2013); Batista et al., “Identification of a Conserved Glycan Signature for Microvesicles,” Journal of Proteome Research 10:4624-4633 (2011); Saraswat et al., “N-linked (N-) Glycoproteomics of Urinary Exosomes,” Molecular & Cellular Proteomics 14:263-276 (2015), which are hereby incorporated by reference in their entirety). Furthermore, the study revealed differences in N-glycan composition and structures among exomeres, Exo-S, and Exo-L.

Example 5—Distinct Lipid Composition Among Exomeres and Exosome Subpopulations

To investigate the lipid composition of each subset of particles, quantitative lipidomics was performed on these nanoparticles derived from B16-F10, MDA-MB-4175 and AsPC-1. By lipid MS, it was found that Exo-S and Exo-L contained more lipids than exomeres for all cell lines (FIG. 10A, >5× fold in all subpopulations, except for Exo-S of MDA-MB-4175 (>3× fold)).
Eighteen lipid classes were commonly identified in all samples (Tables 6-8, FIG. 10B), and their relative frequency in each sample was compared. Phosphatidylcholine (PC) was the predominant lipid component in all subpopulations (46%˜89%) except for AsPC-1 exomeres (13%) (FIG. 10), which contained higher levels of diglyceride (DG, 38%) and triglyceride (TG, 26%) instead. Other phospholipids, including phosphatidylethanolamine (PE) and phosphatidylserine (PS), accounted for 2-6% of total lipids in Exo-S/L across all cell lines (FIG. 10B). However, PE and PS levels were lower in exomeres from MDA-MB-4175 and AsPC-1, but similar to Exo-S/L in B16-F10 (FIG. 10B, 10C). Phosphatidylinositol (P1) levels were lower than other phospholipids but had a pattern of distribution across nanoparticle subsets similar to that of PE and PS (FIG. 10B, 10C). Sphingomyelin (SM) accounts for 2-10% of the total lipid in all samples except for AsPC-1 Exo-S/L, which contained a higher level of SM (28%, FIG. 10B, 10C). Cholesterol data were not collected in this study.
The relative levels of ceramide (Cer), TG and lysophosphatidylglycerol (LPG) varied significantly between exomeres and Exo-S/L across cell lines (ANOVA test, q<0.05). Additionally, simple glycosphingolipid CerG2 and mitochondrion-specific cardiolipin (CL) were more abundant in exomeres of B16-F10 and MDA-MB-4175 compared to exosome subsets. In contrast, CerG2 and CL were more abundant in Exo-S/L compared to exomeres isolated from AsPC-1 cells. Monoglyceride (MG), phosphatidylglycerol (PG) and lysophosphatidylcholine (LPC) were more abundant in exomeres than in Exo-S/L from MDA-MB-4175 and AsPC-1, but present at equal levels in all three B16-F10 nanoparticle subsets. Lastly, lysophosphatidylethanolamine (LPE) was detected at higher levels in Exo-S/L from B16-F10 and MDA-MB-4175, but not from AsPC-1. Thus, the study revealed cell type-dependent differences in the total lipid content and composition among distinct nanoparticle subsets.
Collectively, these bioinformatic analyses of the proteomic content of each particle subset revealed the predominant link between exomere-associated proteins and metabolism and the link between Exo-S/L-associated proteins and multiple signaling transduction pathways, including biogenesis-related ESCRT complexes.

Example 6—Distinct Nucleic Acid Content Among Exomeres and Exosome Subpopulations

Since dsDNA was previously detected in tumor-derived exosomes (Thakur et al., “Double-Stranded DNA in Exosomes: a Novel Biomarker in Cancer Detection,” Cell Research 24:766-769 (2014), which is hereby incorporated by reference in its entirety), the relative abundance of DNA in exomeres and Exo-S/L was determined. DNA was detected in all three types of nanoparticles; however, relative abundance varied by cell-type (FIG. 11A). The relative amount of DNA was highest in exomeres derived from MDA-MB-4175 and in Exo-S from B16-F10 cells and AsPC-1. Bioanalyzer (Agilent) analysis revealed distinct size distribution of DNA associated with each subset of nanoparticles (FIG. 11B and FIG. 12). Exomere DNA was relatively evenly distributed in a broad range of sizes between 100 bp and 10 kb with a slight enrichment around 2 kb in several cases. In contrast, a strong enrichment between 2 kb to 4 kb was detected for Exo-S/L DNA, and the peak size of Exo-L DNA was slightly larger than that of Exo-S DNA. This phenomenon may be due to the structural capacity and different biogenesis mechanisms of each particle subset.
RNA was preferentially associated with Exo-S/L in both B16-F10 and AsPC-1 (FIG. 11C). RNA associated with exomeres and Exo-S showed a monomodal distribution (peak at 400 nt and 500 nt, respectively), whereas Exo-L RNA displayed a bimodal distribution (FIG. 11D) (additional peak >4000 nt). Specifically, 18S and 28S rRNAs were detected at very low levels in Exo-L, barely detected in Exo-S and absent in exomeres compared to cellular RNA. A strong small RNA peak (corresponding to tRNAs, microRNAs and other small RNAs) was detected in Exo-S and Exo-L, but not in exomeres. Remarkably, a unique RNA peak of unknown identity, of ˜315 nt in size, was detected only in Exo-L.

Example 7—Distinct Organ Biodistribution of Exomeres and Exosome Subpopulations

Next, the organ biodistribution of B16-F10-derived nanoparticle subsets in naïve mice was investigated. Twenty-four hours post intravenous injection of near infrared dye (NIR)-labeled exomeres, Exo-S and Exo-L into mice, organs were collected and analyzed using the Odyssey imaging system (LI-COR Biosciences; FIG. 13). Interestingly, all nanoparticles were uptaken by hematopoietic organs, such as the liver (˜84% of total signals), spleen (˜14%) and bone marrow (˜1.6%). The lungs (˜0.23%), lymph nodes (˜0.07%), and kidneys (˜0.08%) showed less uptake of all nanoparticle subtypes. Particle uptake was not detected in the brain. Subsequently, the dynamic range of signal intensity in each organ was adjusted to compare the uptake of each subset of nanoparticles in the same organ (FIG. 13A). Punctuated distribution patterns of nanoparticles were detected specifically in the lung and lymph nodes. This is in contrast to the homogenous distribution pattern found for all nanoparticle subsets in the liver, spleen, and bone marrow. Importantly, although exomeres and Exo-S/L were predominantly uptaken in the liver, Exo-L displayed lymph node tropism. In addition, though not statistically significant, a trend of higher uptake of exomeres in the liver was observed. Quantification is shown in FIG. 13B. Distinct organ distributions indicate that nanoparticle subsets may be involved in different aspects of tumor progression and metastasis.

Discussion of Examples 1-7

Dissecting the heterogeneity of EV populations by differential ultracentrifugation, immuno-affinity capture, ultrafiltration and size-exclusion chromatography, polymer-based precipitation, and microfluidics (Thery et al., “Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids,” Current Protocols in Cell Biology Chapter 3, Unit 322 (2006); Merchant et al., “Microfiltration Isolation of Human Urinary Exosomes for Characterization by MS,” Proteomics Clinical Applications 4:84-96 (2010); Lasser et al., “Isolation and Characterization of RNA-Containing Exosomes,” Journal of Visualized Experiments 59:e3037 (2012); Chen et al., “Microfluidic Isolation and Transcriptome Analysis of Serum Microvesicles,” Lab on a Chip 10:505-511 (2010); Jorgensen et al., “Extracellular Vesicle (EV) Array: Microarray Capturing of Exosomes and Other Extracellular Vesicles for Multiplexed Phenotyping,” Journal of Extracellular Vesicles 2 (2013); Tauro et al., “Comparison of Ultracentrifugation, Density Gradient Separation, and Immunoaffinity Capture Methods for Isolating Human Colon Cancer Cell Line LIM1863-Derived Exosomes,” Methods 56:293-304 (2012), which are hereby incorporated by reference in their entirety) in an attempt to separate nanoparticle populations has proven daunting. By employing state-of-the-art AF4 technology, two discernible exosome subpopulations, Exo-S and Exo-L, were separated and a distinct nanoparticle, named exomere, which differs in size and content from other reported particles, was identified. Unlike labor-intensive and time-consuming gradient methods, AF4 is highly reproducible, fast, simple, label-free and gentle. Moreover, the exosome subpopulations and exomeres were able to be efficiently resolved in a single AF4 run with real-time measurements of various physical parameters of individual particles.
The analyses revealed that exomeres were selectively enriched in proteins involved in metabolism, especially “glycolysis” and “mTORC1” metabolic pathways, suggesting their potential roles in influencing the metabolic program in target organ cells, as well as in proteins associated with coagulation (e.g., Factors VIII and X) and hypoxia. The proteomic analysis also showed that exomeres were enriched in key proteins controlling glycan-mediated protein folding control (CALR) (Molinari et al., “Chaperone Selection During Glycoprotein Translocation into the Endoplasmic Reticulum,” Science 288:331-333 (2000), which is hereby incorporated by reference in its entirety) and glycan processing (MAN2A1, HEXB, GANAB) (Fukuda et al., “Incomplete Synthesis of N-Glycans in Congenital Dyserythropoietic Anemia Type II Caused by a Defect in the Gene Encoding Alpha-Mannosidase II,” Proc Natl Acad Sci USA 87:7443-7447 (1990); Yang et al., “An Intrinsic Mechanism of Secreted Protein Aging and Turnover,” Proc Natl Acad Sci USA 112:13657-13662 (2015); Martiniuk et al., “Identity of Neutral Alpha-Glucosidase AB and the Glycoprotein Processing Enzyme Glucosidase 11. Biochemical and Genetic Studies,” The Journal of Biological Chemistry 260:1238-1242 (1985), which are hereby incorporated by reference in their entirety), suggesting exomere cargo may modulate glycosylation in distant recipient cells. Subcellular localization analysis of exomere-enriched proteins revealed their specific association with ER, mitochondria and microtubules, demonstrating the potential roles of these proteins in exomere biogenesis and secretion.
Proteins unique to exosomes (Exo-L and Exo-S) versus exomeres were also identified. Multiple components of ESCRT complexes were specifically associated with Exo-S and Exo-L, but not observed within exomeres, suggesting a major role for ESCRT complexes in Exo-S/L but not exomere production. Other exosome-enriched proteins included Rob proteins, annexins, Hsp40 members, and proteins involved in multiple signaling transduction pathways, such as integrins, G-proteins, JAK1 and TGFBRs.
Further differences were found between Exo-S and Exo-L protein cargo. Flotillin 1, flotillin 2, tweety family member 3, tetraspanin 14, and ESCRT-1 subunit VPS37B were specifically enriched in Exo-S. In contrast, levels of such proteins as annexin A1/A4/A5, charged multivesicular body protein 1A/2A/4B/5, vacuolar protein sorting 4 homolog B, DnaJ heat shock protein family (Hsp40) member A1, and myosin 1C were relatively higher in Exo-L. Interestingly, tissue factor, a well-studied exosome protein (Gardiner et al., “Extracellular Vesicles, Tissue Factor, Cancer and Thrombosis—Discussion Themes of the ISEV 2014 Educational Day” Journal of Extracellular Vesicles 4:26901 (2015), which is hereby incorporated by reference in its entirety), was enriched in Exo-L. It is thus plausible that exomeres and Exo-L cooperate to optimize the coagulation cascade in vivo.
Exo-S were predominantly enriched in proteins associated with endosomes, multivesicular bodies, vacuoles, and phagocytic vesicles, while Exo-L were specifically enriched in plasma membrane, cell-cell contact/junction, late-endosome, and trans Golgi network proteins. These data indicate that Exo-S are most likely bona fide/canonical exosomes (i.e., derived from intraluminal vesicles of endosomal compartments), whereas Exo-L may represent non-canonical exosomes or probably sEVs of different sub-cellular origin (i.e., plasma membrane budding).
Identifying specific exosome and exomere markers to better isolate and characterize these nanoparticles is critical to advancing knowledge of EV biology. Since Flotillin 1 and 2 were specifically associated with Exo-S, these proteins may represent reliable markers of conventionally defined exosomes. Other previously reported exosome markers, including CD9, CD63, CD81, Tsg101 and Alix1, were present in Exo-S and/or Exo-L in a cell type-dependent manner, and therefore would have to be combined with size exclusion to distinguish exosome subpopulations. Notably, Hsp90-0, highly represented in exomeres, could be a potential exomere marker, whereas several Hsp70 family members, such as HSC70/HSPA8 could serve as possible markers for Exo-S/L subpopulations.
The glycomic, lipidomic, and genomic studies also revealed additional distinct molecular signatures in exomeres and exosomes. Similar to the expression in metastatic tumor cells, exosome subsets were enriched with sialylated glycoproteins, supporting the role of these structures in exosome-mediated cellular recognition. One predominant sialoglycoprotein previously identified in exosomes (Escrevente et al., “Sialoglycoproteins and N-glycans from Secreted Exosomes of Ovarian Carcinoma Cells,” PloS One 8:e78631 (2013); Liang et al., “Complex N-linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment,” The Journal of Biological Chemistry 289:32526-32537 (2014), which are hereby incorporated by reference in their entirety), the galectin-3-binding protein (LGALS3BP), a modulator of cell communication and immune responses (White et al., “Galectin-3 Binding Protein Secreted by Breast Cancer Cells Inhibits Monocyte-Derived Fibrocyte Differentiation” Journal of Immunology 195:1858-1867 (2015); Laubli et al., “Lectin Galactoside-Binding Soluble 3 Binding Protein (LGALS3BP) is a Tumor-Associated Immunomodulatory Ligand for CD33-Related Siglecs,” The Journal of Biological Chemistry 289:33481-33491 (2014), which are hereby incorporated by reference in their entirety), was highly enriched in exomeres. This ligand could mediate the specific interaction of exomeres with target cells through proteins, such as collagens, fibronectin, nidogen, galectin-3 and integrin beta-1 (Hellstern et al., “Functional Studies on Recombinant Domains of Mac-2-Binding Protein,” The Journal of Biological Chemistry 277:15690-15696 (2002); Sasaki et al., “Mac-2 Binding Protein is a Cell-Adhesive Protein of the Extracellular Matrix which Self-Assembles into Ring-Like Structures and Binds Beta1 integrins, Collagens and Fibronectin,” The EMBO Journal 17:1606-1613 (1998), which are hereby incorporated by reference in their entirety).
Interestingly, the lipidomics analyses revealed that exomeres contained fewer lipids compared to Exo-S and Exo-L. Phospholipids and SM, the major structural components of plasma lipid bilayer membrane (Van Meer et al., “Membrane Lipids: Where They are and How They Behave,” Nat Rev Mol Cell Biol 9:112-124 (2008), which is hereby incorporated by reference in its entirety) ranked top in all nanoparticles examined. Such an observation is expected for Exo-S/L subsets due to their vesicular membrane structure, however, exomeres seem to lack external membrane structures. Yet, differences in several lipid classes distinguished exomeres from Exo-S and Exo-L. For instance, exomeres were found to contain higher levels of triglycerides and ceramides compared to exosome subpopulations and thus may serve to transport these metabolites to recipient cells. The study further revealed that DNA packaging in exomeres and exosomes varied by tumor-type, while RNA was packaged in Exo-S and Exo-L independent of tumor classification.
Collectively, the findings demonstrate that proteins, glycans, lipids, and nucleic acids are selectively packaged in exomeres, Exo-S, and Exo-L, further supporting the idea that these are distinct nanoparticle subsets.
The observation that nanoparticle subtypes have different organ biodistribution patterns suggests they mediate the pleiotropic effects of cancer. The punctate pattern of Exo-L uptake and its apparent tropism for lymph nodes implicate this nanoparticle in facilitating metastasis of disseminated tumor cells. Exomeres, along with exosomes, were uptaken by hematopoietic organs, including the liver, spleen, and bone marrow. Interestingly, the predominant exomere uptake by the liver and the exomere enrichment in protein cargo involved in metabolism lead us to speculate that exomeres may specifically target the liver for metabolic reprogramming during tumor progression. The data indicate that the size of nanoparticles, in addition to their specific cargo, may influence metastatic patterning and systemic effects of cancer.
The identification of exomeres highlights the diversity of EVs and particles secreted by cells. Elucidating their biogenesis will be essential to unravel their roles in cellular and organ function. Target cells and the functional outcomes exerted by each nanoparticle subset in organs need to be further delineated to advance the understanding of the collective, systemic effects of nanoparticles in the metastasis process. Undoubtedly, these discoveries will open avenues for translational studies of EVs and particles in diagnostic, prognostic, and therapeutic applications.

Materials and Methods for Examples 8-12

Preparation of small extracellular vesicles (sEVs) from cell culture. With the aim to separate distinct cellular nanoparticles, such as exomeres and exosome subsets, sEVs isolated using dUC as the input samples for AF4 were studied. Alternative methods, such as DGF, UF and SEC, can also be used for sEV input sampling. EVs captured by IAC can be applied, as well, if the antibody can be removed from the EVs.
This protocol has been developed and optimized using sEVs derived from cell culture model systems. Conditioned media was sequentially spun to remove cells, cell debris, and large EVs and finally pellet down the sEVs. It has been reported herein that fresh versus frozen sEV samples do not markedly differ in AF4 profiles, indicating that the structural integrity of EVs is well preserved during the freeze-thaw process. Of note, the culture conditions, such as growing cells in hypoxic conditions, and the passage of cells can influence EV production and composition (i.e. the percent of each particle type in a sample). Thus, these changes in ENP composition may require modifications of the AF4 methods for further optimization to achieve desired separation quality.
As described infra, this protocol could also be applied to sEVs prepared from other resources, such as bodily fluids, including plasma. AF4 parameters, such as the cross-flow gradient, can be further adjusted to meet the specific requirements of particular samples (for example, existence of additional types of ENPs). However, for certain sample types, the EV composition is more complicated than that derived from conditioned media of cell cultures. For example, the presence of lipoprotein particles in blood plasma may interfere with the separation of exosomes due to their partial overlap in size. In this case, other means of prior removal of lipoproteins from the plasma sample is desired before loading them onto the AF4 channel.
AF4 fractionation of sEVs and online data collection and analysis. The AF4 operative method for separation of exomeres and exosome subsets (i.e., Exo-S and Exo-L) from cell culture-derived sEVs is illustrated in FIG. 21. Based on the complexity of the EV samples and the goal of each specific study, this running method can be further adjusted, as described infra.
For real-time monitoring and analysis of the fractionation of particles, several online detectors are usually installed immediately downstream of the AF4 channel. The laboratory has the DAWN HELEOS-II (MALS detector) with QELS (DLS detector) installed at the detector 12 (100°) position (Wyatt Technology) and the Agilent 1260 Infinity Multiple Wavelength Detector (set at 280 nm for UV absorbance detection) in place. The DLS measurement is mainly used to determine the hydrodynamic size of the fractionated particles in real time. The primary data from a DLS measurement is the autocorrelation function, which plots the average overall changes in the scattered light intensity of molecules with time (see example in FIG. 22). The exponential decay rate of the autocorrelation function determines the translational diffusion coefficient (D_I) of molecules in solution based on the following equation:
G(τ)=1+β exp(−2D ₁ q ²τ) Auto correlation function

- τ—delay time; q=(4πη₀/λ₀/sin(0/2).
- β—intercept of the correlation function; η₀is the refractive index of the solution;
- q—scattering factor; D is the scattering angle

Based on the Stokes-Einstein relation,
R _h =kT/6πηD _t

- k—Boltzmann's constant
- T—temperature;
- ƒ—viscosity of solvent
  an effective hydrodynamic radius (R_h) can be further deduced from D₁. The assumption for this calculation is that the solute (EV, in the present case) is a sphere undergoing Brownian motion. R_his the radius of a sphere with the same translational diffusion coefficient as the analyzed solute. R_hdepends only on the physical size of the solute and its size-related behavior, such as diffusion and viscosity, but is not affected by its density or molecular weight. The measurement range of 0.5 nm to 1000 nm radius makes DLS an effective tool to measure the size of sEVs.

Combining AF4 with online DLS measurements is critical for accurate size determination. In a polydisperse sample, DLS measurement yields an average R_hand the specific information on each compositional species in a given sample is missing. However, fractionation results in the separation of solutes with different sizes and each fraction contains only a very small admixture of different R_hparticles. Thus, fractionation allows the size of each species to be more accurately measured. For such monodisperse samples, the resulting autocorrelation functions are single exponentials, which are simple to interpret. Fitting the data to a single exponential function is performed in the Astra software using the Cumulants model (Wyatt Technology Corporation. DYNAMICS User's Guide. Version 7.0 (M1400 Rev. 1) Appendix A-2 (2010), which is hereby incorporated by reference in its entirety). By examining the ideal fitting to a single exponential, one can further evaluate the separation quality.
One requirement for accurate DLS measurement of R_his that the sample concentration must be sufficient so that the sample scatters at least three times more light than the solvent to obtain an acceptable signal/noise ratio. In particular, small molecules, such as exomeres, scatter less light and require even higher concentrations of analyte to optimize results.
Besides DLS, static light scattering (SLS) detected by MALS measures the radius of gyration (R_g). R_gis defined as the mass averaged distance of each point in a molecule from its gravity center and is generally different from R_h. Comparing R_gto R_hcan further reveal the compactness of a solute (i.e., empty versus filled particles). In general, the MALS detector is more sensitive than DLS monitoring and thus it will be of specific use when only little amount of material available for analysis.
The UV detector is used as part of the instrumentation for online concentration measurements. The intensity of UV absorbance can provide us an approximation of the relative abundance of different species in the sample, despite not having defined extinction coefficients for different species in the EV sample mixture. The peaks of UV absorbance are useful in guiding the choice of combining fractions of similar particles. However, the bicinchoninic acid assay (BCA assay) and NTA is often conducted after fraction collection for quantification purposes. Once pure EVs are obtained and further characterization can be performed, the extinction coefficients of each species for improved interpretation of the UV absorbance data to concentration can then be determined.
One key consideration for online detectors is that it must have exceptional sensitivity due to the limited amount of material passing through the detector at each single time point. Besides the detectors mentioned above, other sensors, such as differential refractive index (dRI) and fluorescence detectors (FLDs), are often included as standard parts of the instrumentation for a variety of macromolecular characterization techniques. dRI, considered a universal concentration detector, is accurate and versatile in all types of solvents and independent of chromophores or fluorophores. FLDs are useful if autofluorescent molecules or artificial fluorescent labeling are present in specific subsets of EVs. It should be noted that, with additional detectors assembled online, the fractionated particles take a longer path and more time to reach the fraction collector. As a result, diffusion of molecules will lead to broadening of peaks, dilution of fractionated samples and reduction in separation resolution. Therefore, only detectors considered essential for the real-time monitoring should be installed.
Fraction collection, concentrations and characterization. AF4 fractions can be collected automatically or manually for downstream offline characterization. The Agilent Fraction Collector (1260 series) has been installed to automatically collect fractions into 96-well plates, but similar fraction collectors can be utilized for accurate and reproducible fraction collections. Fractions can be collected either by volume or over time, and fractions of particles with the same identity based on online and offline characterization can be further pooled together for downstream analysis. For example, as described supra, to identify exomeres and distinct subsets of exosomes, representative fractions were first examined across the whole time course of fractionation by online DLS and offline transmission electron microscopy analysis and then the fractions of particles with similar size and morphology were pooled together for further characterization. This step was also guided by the peaks of UV absorbance (indicating the most abundant fraction of each type of particles). To validate that the pooled fractions are relatively pure and not contaminated significantly by other types of adjacent particles, only fractions centered around the peaks were pooled together. Depending on the resolution of the fractionation, this fraction combination step can be empirically determined. Due to the different composition of EV subpopulations in a given sample, occasionally the UV peaks are not identifiable and thus the fraction combination will rely more on other properties, such as size and morphology.
The individual or combined fractions can be directly utilized for downstream analysis or subjected to a concentration step before further characterization. The collected fractions are usually concentrated using the Amicon Ultra-series of centrifugal filter units with Ultracel-30 (30 KDa cutoff) membrane (Millipore). Other alternative means of concentration, such as tangential filtration centrifugation, direct UF, UC, or IAC, can be applied depending on the need for the downstream analysis. A variety of analyses can be performed on fractionated EVs, including but not limited to: BCA assay, NTA, atomic force microscopy, electron microscopy, mass spectrometry of molecular contents (e.g., proteins, lipids, glycan, and metabolites), western blotting or the enzyme-linked immunosorbent assay, sequencing of genetic material (DNA and RNAs), DLS measurement in batch mode, and zeta potential measurement. The functional roles of the fractionated EV subpopulations can be further investigated in vitro or in vivo.
Reagents.

- B16-F10 cell line (ATCC). Cell lines should be regularly checked to ensure that they are authentic and free of Mycoplasma contamination.
- DMEM (VWR, Catalog No. 45000-304)
- Premium Grade Fetal Bovine Serum (FBS) (VWR, Catalog No. 97068-085)
- L-Glutamine, 100× (Corning, Catalog No. 25-005-C1)
- Penicillin-Streptomycin Solution, 50× (Corning, Catalog No. 30-001-C1)
- Sterile PBS (VWR, Catalog No. 45000-446)
- TrypLE (Thermo Fisher Scientific, Catalog No. 12604-039P)
- Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Catalog No. 23225)
- ATCC Universal Mycoplasma Detection kit (ATCC, Catalog No. 30-1012K)
- Bovine serum albumin (BSA)(Sigma, Catalog No. A 1900)
- Water filtered using the Milli Q system
- Ethanol (Sigma, Catalog No. 459828)
- Contrad 70 (Decon Labs, Inc Catalog No. 1003)
- Sodium Dodecyl Sulfate (SDS)(Omnipur, Catalog No. 7910)
- Nitric Acid (Fisher Scientific, Catalog No. 7697-37-2)

Consumable Equipment.

- 150×25 mm tissue culture dish with Grid (VWR, Catalog No. 25383-103)
- 5/10/25 mL Serological pipettes (VWR, Catalog No. 82050-478/82050-482/82051-182)
- Disposable Tips (Denville, Catalog No. P1096-FR/P1121/P1122/P1126)
- 500 ml Supor machV PES Filter Units (VWR, Catalog No. 73520-984)
- mL 50 mL conical tubes (VWR, Catalog No. 82050-276/82050-346)
- 1.7 ml Microcentrifuge Tubes (VWR. Catalog No. 53550-698)
- 96-well plate (VWR, Catalog No. 62406-081)
- Blue screw caps (Agilent, Catalog No. 5182-0717)
- Screw cap vials (Agilent, Catalog No. 5182-0714)
- vial insert, 250 ul pulled point glass (Agilent, Catalog No. 5183-2085)
- 96-well plate, 1.0 ml, polypropylene (Agilent, Catalog No. 8010-0534)
- Sealing tape, clear polyolefin (Thermo Fisher Scientific, Catalog No. 232701)
- Ultracentrifuge tube (Beckman Coulter, Catalog No. 355628)
- Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane (Millipore, Catalog No. UFC903024)
- Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-30 membrane (Millipore, Catalog No. UFC803024)
- Millipore Reg. Cellulose membrane 10 KD SC (Wyatt technology, Catalog No. 4057)
- Nadir Polyethersulfone membrane 10 KD SC (Wyatt technology, Catalog No. 1903)
- Inline filter membrane 0.1 μm (Wyatt Technology, Catalog No. 1871)
- Dry wipes (Kimtech, Catalog No. 7552)
- 2 L glass bottles (VWR, Catalog No. 10754-822)
- Hemocytometer (Weber Scientific, Catalog 3048-12)

Equipment.

- Labconco Purifier Class II biosafety cabinet
- Tissue culture incubator
- EVOS FL Microscope (Thermo Fisher Scientific)
- Optima XPN-100 Ultracentrifuge (Beckman Coulter, Catalog No. A94469)
- Type 45 Ti Rotor, Fixed-Angle, Titanium (Beckman Coulter, Catalog No. 41103909)
- Table-top Heraeus Multifuge ×3R Centrifuge Series (Thermo Fisher Scientific, Catalog No. 75004501)
- Microcentrifuge (Eppendorf, 5424R)
- AccuScan GO UV/Vis Microplate Spectrophotometer (Fisher Scientific, Catalog No. 14-377-579)
- Milli Q system (Millipore)
- 4° C. Refrigerator (Thermo Fisher Scientific)
- −20° C. freezer (Thermo Fisher Scientific)
- −80° C. freezer (Thermo Fisher Scientific)
- 37° C. Incubator (Thermo Fisher Scientific)
- Autoclave (Tuttnauer)
- Dishwasher (Steelco)

AF4 Instrument Parts.

- Agilent 1260 Infinity Analytical- and Preparative-scale Fraction Collectors (G1364C)
- Agilent 1290 Thermostat (G1330B)
- Agilent 1260 Infinity Standard Autosampler (G1329B)
- Agilent 1260 Infinity Multiple Wavelength Detector (G365D)
- Agilent 1260 Infinity Isocratic Pump (G1310B)
- GASTORR TG-14 HPLC vacuum degasser
- Wyatt Technology DAWN HELEOS-II with QELS installed at detector 12 (100°)
- Wyatt Technology Eclipse AF4
- Wyatt Technology short channel
- Computer installed with Chemstation and Astra 6 softwares

Software.

- Chemstation (Agilent Technologies) integrated with the Eclipse module (Wyatt Technology) to operate the AF4 flow
- Astra 6 (Wyatt Technology) for MALS, DLS and UV data acquisition and analysis

Reagent Setup.

- B16-F10 cell culture medium 500 mL of DMEM is supplemented with 10% (vol/vol) FBS (exosome-depleted), 50 units/mL Penicillin, 50 μg/mL Streptomycin, and 2 mM L-Glutamine, and stored at 4° C. for up to a month.
- Exosome-depleted FBS FBS is depleted of exosomes by ultracentrifugation at 100,000 g for 90 min and sterilized with a 0.22 μm filter unit. Aliquots of exosome-depleted FBS can be stored at −20° C. for long term. To avoid contamination, the rotor needs to be first sterilized by wiping with 70% Ethanol and all tubes for ultracentrifugation should be autoclaved. The entire FBS handling process should be carried out in a Biological Safety Cabinet for tissue culture.
- 20% (vol/vol) Ethanol. Milli Q filtered water is used to make the dilution of ethanol. The final solution is made freshly and filtered with a 0.22 μm filter unit.
- 0.5 mg/mL BSA solution. Dissolve BSA powder in PBS at a concentration of 0.5 mg/mL and store aliquot at −20° C. for long term.
- 1% (vol/vol) Contrad 70. Dilute Contrad 70 with Milli Q water to a final concentration of 1% (vol/vol), and store at room temperature (RT, ˜22° C.) for long term.
- 10% (wt/vol) SDS. Dissolve SDS powder in Milli Q water at a concentration of 10% (wt/vol) (i.e. 10 g per 100 mL) and store at RT for long term. This chemical is corrosive and toxic, and can cause severe skin and eye irritation. Wear protective gloves, mask, eyeshield, faceshield, and protective clothing to handle.
- 10% (vol/vol) Nitric Acid. Dilute nitric acid with Milli Q water to a final concentration of 10% (vol/vol), and store at RT for long term. This chemical is highly corrosive and can cause severe eye and skin burns, severe respiratory and digestive tract burns. Wear proper protective equipment (gloves, eyeshield, faceshield, clothing, respirators) and handle it in a chemical hood. The waste should be treated as a hazardous waste following state and local hazardous waste regulations.

Instrument setup. All the parts of the AF4 instrument should be installed, configured, calibrated and certified by the manufactures (Agilent and Wyatt Technology) before utilization.
Preparation of sEVs from the conditioned media of cell culture. B16-F10 murine melanoma is used as a model system in this protocol. A schematic flow diagram summarizing the key steps of the entire procedure and the flow route of AF4 is shown in FIG. 21.

- 1. Seed 2.25×10⁶B16-F10 cells per P150 tissue culture plate in 25 mL of the DMEM complete medium supplemented with exosome-depleted FBS, and seed a total of 12 plates. Cell lines should be regularly checked to ensure that they are authentic and free of Mycoplasma contamination. The passage number of the B16-F10 cells influences sEV composition, reflected by the changes in the relative abundance of different subsets of sEVs (7). So, avoid comparing experimental data using B16-F10 cells with a big difference in their passage numbers.
- 2. Keep cells in a humidified tissue culture incubator for 72 hours under standard conditions (5% CO₂, 37° C.). The cell culture should just reach confluence without cell death and any abnormal phenotypical changes. Cells should be allowed to reach confluence to get highest sEV yield, but no cell death and stressed phenotype should be apparent by the harvesting time to ensure the purity of the sEVs.
- 3. Collect the conditioned media into 50 mL conical tubes and centrifuge at 500×g at 10° C. for 10 minutes in the table-top centrifuge. The supernatant can be spun at 3000×g at 10° C. for 20 minutes in the table-top centrifuge, transferred to new tubes and placed at −80° C. for long term storage.
- 4. Transfer the supernatant to ultracentrifuge tubes (6×50 mL/tube) and centrifuge at 12,000×g at 10° C. for 20 minutes in Type 45-Ti ultracentrifuge rotor (pre-chilled at 4° C.). For ultracentrifugation, the opposing pair of tubes across the center of rotation need to be balanced with each other. Do not load more than 50 mL per tube to avoid sample spilling. The rotors should be kept at 4° C. when not in use.
- 5. Transfer the supernatant to new ultracentrifuge tubes and centrifuge at 100,000×g at 10° C. for 70 minutes in the same rotor. At the end of each ultracentrifugation step, make sure the supernatant is transferred immediately to avoid the loosening of the pellet and either contaminating the supernatant or losing the pelleted samples.
- 6. Discard the supernatant and resuspend the pellets in one milliliters of ice-cold PBS gently. Avoid introducing air bubbles. Combine all the samples into one ultracentrifuge tube and bring the final volume to 50 mL with PBS.
- 7. Centrifuge at 100,000×g at 10° C. for another 70 minutes. Resuspend the final pellet in ˜0.5 mL of PBS and transfer to 1.7 mL microcentrifuge tubes on ice (the sEV sample for AF4 fraction in the next section). The pellet sometimes may be hard to break and resuspended into a homogeneous suspension. If so, the samples can be kept on ice for another 15 to 30 minutes or an extra volume of PBS can be added to the sample. Then gently pipette up and down to resuspend the samples and transfer to Eppendorf tubes for quantification. Avoid introducing air bubbles.
- 8. Quantify the sEV yield by measuring the protein concentration using the Pierce BCA Protein Assay Kit. Follow the manufacture's instruction to mix the samples or BSA standards provided by the kit with the reagents in a 96-well plate and incubate at 37° C. for 30 minutes. Read the absorbance at 562 nm using the AccuScan GO UV/Vis Microplate Spectrophotometer and calculate the concentration of the samples based on the BSA standards included.
- 9. The samples can be immediately applied for AF4 fractionation, or kept on ice overnight to be processed further the next day. The samples can be frozen at −80° C. for long term storage.

AF4 Fractionation of sEVs.

Assemble the AF4 Channel

- 10. Before assembling the AF4 channel, one should select the membrane type and cutoff size (e.g., RC membrane with 10 KDa cutoff size) and the spacer with desired thickness (i.e. the channel height, preferably 490 μm) first.
- 11. Rinse all parts with Milli Q water and assemble in the order of the top plate, spacer, membrane and bottom plates with the frit/O-ring in place). Bolt the parts together using a torque wrench with 5 Nm and 7.5 Nm torques applied sequentially. Since the sample specimen is positioned in the channel laminae very close to the membrane, it is critical that the membrane should be smooth and unruffled. Wear gloves and do not bend the membrane during the assembly procedure. Since ethanol can cause the “membrane swelling” phenomena and reduce the effective channel height for fractionation, avoid exposing the membrane to ethanol. It is critical that the channel be tightly sealed and its height be precise and even across the whole channel. To assemble the AF4 channel, a metered wrench such as a torque wrench should be used to apply force precisely. A good practice is to tighten the two bolts in the center first and then the ones at the corners in diagonal order,
- 12. Connect the tubing to the inlet, crossflow and injection ports but with the outlet port unconnected. Start to run water at a channel flow rate of 1 mL/min (program the flow rate settings and operate using ChemStation) through the system for at least 30 minutes and let air in the channel run out from the outlet port first. Then connect the tubing from the outlet to the detectors. No air bubbles should be observed in the channel. The instrument can be operated in the Night Rinse mode with a constant channel flow of 0.2 mL/min overnight, up to a few days. For short term storage, keep the system running in the Night Rinse mode. Do not leave the system in still aqueous solvents for a long period. For long term storage, dissemble the membrane from the channel and maintain the system in 20% ethanol. Keep the tubing from the channel to the detectors and the fraction collector as short as possible to reduce the peak broadening and sample dilution effects and to avoid decreases in separation resolution.

Equilibrate and Coat the Membrane with BSA

- Change the aqueous solvent to PBS, and keep running at a channel flow of 1 mL/min for at least 30 minutes to 1 hour. The instrument can be operated in the Night Rinse mode overnight, with a constant channel flow of 0.2 mL/min. If the system has been maintained in ethanol or isopropanol, it should be flushed completely with water first before switching to PBS. Mixing PBS with alcohol will cause salt precipitation.
- 13. Load 30 to 40 μg of BSA (0.5 mg/mL) onto AF4 and run the sample using the same AF4 method as for sEV fractionation except for the elution step, using a constant crossflow of 3 mL/min for 15 minutes for BSA instead. Repeat, by running BSA 1-2 more times. The instrument can be operated in the Night Rinse mode overnight, up to a few days after membrane coating. The purpose of this step is to block non-specific binding of the samples to the membrane. The sample to be analyzed, if extra sample is available, can be used for this blocking step, too. This step is only needed when a new membrane is installed. At the end of the day, after all samples have been processed, turn on the COMET of the DAWN HELEOS II detector for about 30 min to clean the flow cell.

AF4 Fractionation of sEVs.

- 14. Instrument initialization:
  - (a) Open Chemstation and load the AF4 running method as described in Table 15 (The running method is programmed, edited and saved in the “Method” module of Chemstation).

TABLE 15

		Channel flow	Cross flow Start	Cross flow End
Time (min)	Mode	(mL/min)	(mL/min)	(mL/min)

2	Elution	1.0	0.5	0.5
1	Focus	1.0	—	—
2	Focus + Inject	1.0	—	—
45	Elution	1.0	0.5	0
5	Elution	1.0	0	0
5	Elution + Inject	1.0	0	0

Inject flow 0.2 mL/min: Focus fow 0.5 mL/min
Focus valve position- Focusing (%): 30

- - (b) Set both thermostats for the autosampler and the fraction collector at 4° C.; turn on the UV lamp for the MW D detector (280 nm) at least 30 minutes before sample analysis; turn on the laser for DAWN HELEOS II (664 nm).
  - (c) Turn on the fraction collector and choose the collection mode (based on volume or time interval); install 96-well plates for fraction collection (Ensure plates are installed according to the configuration of the fraction collector). Fractions are collected based on time intervals of 0.5 minutes, so two plates are needed to collect the fractions from one sample). All operations should be done using the ChemStation software except for switching on the laser of DAWN HELEOS II using the instrument's front control panel.
- 15. Open Astra 6 and start a new experiment file for data collection:
  - (a) For Configuration, select “PBS, aqueous' as the system solvent; specify UV wavelength at 280 nm” and enable “Band Broadening” option; for HELEOS, enable “Band Broadening” and “Temperature Control” options; and for QELS, select “Use QELS dithering”.
  - (b) For Procedure: specify the time interval for MALS data collection at 1 second and QELS interval at 2 seconds; set the duration for data collection at 60 minutes; select “Trigger on Auto-Inject”;
  - (c) Click the “Run” button and the data collection will automatically start once triggered by the signal from the autosampler.
- 16. Prepare the AF4 input samples by adjusting the concentration of the sEVs isolated in Step 9 to 1 μg/μl with PBS. Spin at 12,000×g at 4° C. for 5 minutes to remove insoluble aggregates right before loading onto AF4.
- 17. Transfer the supernatant into a pre-chilled screw cap glass vial (250 ul pulled point glass vial insert can be used if the total volume of the sample is small), and put it onto the autosampler platform at the designated position from which the autosampler is set to pick up the sample automatically. Pre-spinning of the sample before loading onto AF4 is critical to avoid analyzing artifacts of aggregates formed during the high-speed ultracentrifugation.
- 18. In the Autosampler module of Chemstation, specify the sample volume to analyze (40 to 100 μl; i.e. 40 to 100 μg at 1 μg/μl), and then click the “Single Sample” to start the fractionation, real time data collection (MALS, DLS, and UV absorption), and fraction collection (by time slice of 0.5 mL). The pilot study has determined a range from 40 to 100 sg of B16-F10 sEVs is suitable for the current AF4 running method that has been developed. This can be further adjusted for specific samples due to their composition complexity. It is critical to avoid getting air bubbles into the system. Make sure that no air bubbles trapped in the sample vial and have a larger volume of sample in the vial than the volume to be analyzed.
- 19. During the running of the sample, check the real flow rates in the panel of “Wyatt Eclipse Status” and make sure they are close enough to the set flow rates. If the real flow rates are quite different from the set ones, something is wrong with the flow control and repairing/maintenance by the manufacturer is needed to ensure the fractionation quality. It is critical to keep the channel flow rate (the detector flow) constant during the fractionation. Changes in the flow rate can cause artifact signal detection by the monitors.
- 20. Once the fractionation is finished, take the 96-well plates out of the fraction collector and seal them using adhesive tape. Keep the plates on ice or at 4° C. for the next procedure.
- 21. Click “Reset the fraction collector” so that the starting position for fraction collection is reset to its original position. Otherwise, the instrument will resume the fraction collection of the next sample from the last fraction position of the previous sample.)
- 22. Proceed to “Online data analysis” described infra to evaluate the fractionation quality.
- 23. Proceed to “Fraction offline characterization” as described infra or start the fractionation of the next sample (first install new 96-well fraction collection plates and then start fractioning the next sample by repeating steps 16-23). If multiple samples need to be analyzed but the collection of separated fractions is not required, a “Sequence” (a series of methods and samples programmed to be run sequentially) can be run instead of the method for a single sample. Users can refer to the manual from the manufacturer for details.
- 24. A good practice includes running a PBS blank control (or other types of running buffer used to analyze the samples) using the same AF4 running method for the samples on the same day. This blank control can help evaluate the instrument performance such as background noise level and identify systemic problems than may influence sample analysis.
- 25. After all samples have been analyzed for the day, turn on the COMET of the DAWN HELEOS II for ˜30 minutes and switch off the laser, UV lamp, thermostats and fraction collector. Change the aqueous solvent to water and run in the Night Rinse mode overnight or up to a few days. For long term storage, please refer to Step 12.

Online data analysis. Online data analysis is performed using Astra 6. First, select the experiment to be analyzed and

- (a) adjust the baselines: set up the baseline for the MALS signal collected from the LS 11 (90°) detector first, and then apply it to all the other detectors. Make sure to check individual detectors to ensure the baselines are set up correctly.
- (b) Select peak regions to analyze: a single or multiple regions can be selected to analyze simultaneously.
- (c) Examine the fractionation quality by checking upon the fitting of the autocorrelation function at representative fractions to a single exponential model. The closer of R²to 1, the better purity of the separation.
- 26. Open a new window of EAST Graph, and plot Hydrodynamic Radius (R_h), QELS (DLS) and UV signals versus time. The hydrodynamic radius (Rh) of particles is deduced solely from DLS signal using equations described above. The Hydrodynamic Radius (R_h) plot displays the size of particles eluted at each time point. The UV signal intensity can reveal the relative abundance of particles with different sizes. Based on these plots (and together with potential offline characterizations), one can judge the AF4 separation quality and the sample composition (i.e. the relative abundance of particles with different sizes). Other types of analysis can be plotted as well by choosing different axes to display in EAST Graph according to the need. Besides online UV detection, other means of quantification such as BCA assay and NTA analysis can be used to measure the concentration of the fractions. The sample concentration should be high enough to scatter enough light for accurate R_hdetermination, especially for small size particles as they scatter much less light.

Fraction collection and concentration for offline characterization.

- 27. Depending on the characterization to be conducted, the individual fractions can be examined directly or further concentrated before examination. Adjacent fractions with similar properties (especially from the same peak region) can be combined and concentrated for downstream analysis. If a high amount of material is required for the downstream characterization, multiple runs of the same sEV sample can be carried out following the same procedure and similar fractions from each run can be combined.
- 28. Individual fraction or pooled fractions are concentrated using Millipore centrifugal filter columns with Ultracel-30 membrane (30 kDa cutoff) in the following steps.
  - (a) The filter columns are first pre-rinsed by adding 5 mL (for Ultra-4 filter column) or 15 mL (for Ultra-15 filter column) of ice-cold PBS followed by spinning at 3,700×g at 4° C. for 5 minutes. The flow-through and liquid remaining in the top filter columns are discarded.
  - (b) Pooled fractionated samples are then transferred into the top filter column and spun at 3,700×g at 4° C. for 7-8 minutes. The concentrated samples are retained in the top filter columns and buffer is collected at the bottom of the collection tubes (the flow-through). Discard the flow-through.
  - (c) Repeat step 31 (b) until each sample is concentrated to the desired volume. For each sample, the same filter column can be repeatedly loaded and spun to concentrate the sample.
  - (d) Transfer the concentrated samples to 1.7 mL microcentrifuge tubes on ice. Record the volume and take an aliquot for BCA measurements to determine the protein concentration.
  - (e) The concentrated samples can be kept on ice for short-term storage (up to 2˜3 days) or frozen at −80° C. for long term storage. Downstream molecular characterizations and functional study can be followed up on these concentrated fractionated samples. For an unknown sample that is analyzed using AF4 for the first time, check the morphology of representative individual fractions by TEM first before pooling fractions together for further analysis. It is possible that particles with same hydrodynamic size but different morphology are eluted together from AF4. Other means to separate these particles based on their distinct biophysical/biochemical properties (such as density, surface molecule expression, and charge) should be explored in combination with AF4 for further fractionation, Pool fractions together based on the hydrodynamic size, morphology and purity of representative fractions. If baseline separation of two adjacent, distinct populations of particles is not achieved, avoid collecting those fractions in the “valley” between the peaks of two populations for further characterization.

Troubleshooting. Table 16. Troubleshooting table.

TABLE 16

Step	Problem	Possible reason

9	Low sEV yield	Low cell confluence by the CM harvest time
		Lost the sEV pellet of 100.000x g
		ultracentrifugation
	Abnormally high sEV yield	Contamination due to inefficient washing
		Too much carry over of media in Step 6
		Contamination from the pellet of 12.000x g
		ultracentrifugation

12	Leaking channel and/or tubing connection	The AF4 channel was not assembled properly
		The Q-ring is damaged or not placed properly
		The screw thread is damaged or worn out
13	high LS background noise	Particle contaminants in the system
14. 19	Nosignal or much lower	Membrane installed improperly
	signal than expected for the
	sample
		Lost sample due to leaking
		channel connection
		Lost sample due to damaged membrane
		Inefficient focus
14. 19	High noise	Contaminant present in the mobile phase
		solution
		Particle contaminants in the system
14. 19	Baseline drifting	Particle contaminants in the system
		Laser performance quality decreased
14. 19	Sharp jump in signal	Unstable voltage
	intensity
		Air bubble introduced into the system
14. 19	Sample elutes too early or	Aberrant flow rate
	too late than expected
18	Too large pellet	Insufficient resuspension of sEV pellet in Step 9.
19	A shift toward late elution	Membrane used for extended period and
		bound non-specifically with contaminant
19	The signal not reaching the	Not enough elution time
	baseline level by the end of
	AF4 fractionation
28	Curling-up tail of the R_hplot	Inaccurate R_hdetermination from the DLS
	at the small R_hend	measurement due to insuffient amount of the
		sample. especially for particles of small size
		Insufficient separation of the particles,
		especially at the beginning of AF4
		fractionation
	Scattered R_hplot	Inaccurate R_hdetermination from the DLS
		measurement due to insuffient amount of the
		sample, especially for particles of small size
		High background noise
28	Too big P5-corresponding	Not enough elution time
	peak

30	No sample recovered	Lost sample due to damaged membrane of the
		filter unit

Step

Solution



	9	Seed a higher number of cells per plate or a bigger
		number of plates. or use a longer cell culture time
		Remove the supernatant immediately from the sEV
		pellet of 100.000x g ultracentrifugation
		Resuspend the pellet from Step 6 completely and use
		a large volume of PBS to wash in Step 7
		Invert the tubes from Step 6 on paper towel to drain
		the leftover of media or suck it off using the vacuum
		system before washing with PBS
		Transfer the supernatant immediately to new tubes
		from the pellet of 12.000x g ultracentrifugation in Steps 4-5
	12	Reassemble the channel following Steps 11-12.
		Make sure the channel is assembled using the proper
		and precise force with a torque wrench
		Change to a new O-ring if it is damaged; install it
		evenly and smoothly in the groove along the frit on
		the bottom plate of the channel
		Replace with new screws. or use a piece of Teflon
		(polytetrafluoroethylene (PTFE) film) tape to help
		seal the thread
	13	Change the inline filter for mobile phase solution
		Thoroughly rinse the membrane with Milli Q water
		before assembling: equilibrate the membrane in the
		AF44 channel with PBS overnight: flush the channel
		thoroughly before connecting to detectors.
	14. 19	Reassemble the channel and make
		sure the smooth side of the membrane
		facing the inside of the channel
		See above troubleshooting for the Step 12 “Leaking
		channel and/or tubing connection”
		Replace with new membrane
		Use a colored sample such as blue Dextran to test the
		focus efficiency. A narrow band of the sample
		should be located close to the inject port. If not,
		increase the focus time post the injection step
	14. 19	Filter the buffer before use and use an inline filter
		(0.1 μm. and change it routinely. about once a
		month)
		If compatible with downstream analysis. include
		sodium azide in the mobile phase solution
		Use COMMET after running samples to clean the
		MALS flow channel
		Flush the channel and the system thoroughly with a
		large volume of filter water:
		If flushing with water does not solve the problem,
		clean the detectors by running and incubating in 10%
		SDS. 1% Contrad 70. or 10% Nitric acid for 30 min
		up to overnight. then thoroughly rinse with filtered
		water
	14. 19	See above troubleshooting for the Step 14. 19 “High
		noise”
		Collect AF4 profile of PBS blank control right
		before or after the AF4 fractionation of the samples
		of interest. and then use the PBS control profile to
		perform baseline subtraction from the profile of the samples.
		Replace with new laser
	14. 19	Use the power supply that provides stable voltage
		and current
		Degas the buffer before use and/or use an online
		degaser: make sure there is no air bubble in the
		sample vial and have an excess of sample to inject
	14. 19	Compare the real flow rate versus the set flow rate
		shown in the panel of “Wyatt Eclipse Status”
		If the difference is big, repair by the
		manufacture isrequired.
	18	Repeat pipetting up and down gently to resuspend
		the pellet. use a larger volume of PBS to resuspend
		the pelleted sEVs. To get more accurate loading of
		the sample, conduct the BCA assay upon the
		supernatant from the brief spin at 12.000 x g in Step
		18.
	19	Replace with a new membrane
	19	Use a longer cross flow gradient time and/or a longer
		time for the last two steps (Elution. Elution + Inject)
		of the AF4 running method
	28	Increase the amount of the sample to analyze
		Use other means such as EM and NTA
		analysis to validate the purity
		Increase the Focus time after the injection step
		Increase the initial cross flow rate and n longer time
		span of fractionation to allow better separation
		Increase the amount of the sample to analyze
		See above troubleshooting for the Step 14. 19
		“High noise”
	28	Use a longer cross flow gradient time
	30	Save the flow through and apply to
		new filter unit for concentration

Timing.

I. Step 1-9, cell culture and isolation of sEVs: ˜3 days.
II. Step 10-12, the AF4 channel assembling: ˜1 hour.
- Step 13-14, Equilibration and coating the membrane with BSA: 2˜3 hours.
- (Step 10-14 are only needed when a new membrane is installed.)
- Step 15-26, AF4 fractionation of sEVs: 1-2 hours per sample.
III. Step 27-28, online data analysis: ˜20 minutes per sample.
IV. Step 29-30, fraction collection and concentration for offline characterization: 1-2 hours.

Example 8—Cross-Flow

According to the AF4 theory, cross-flow is the driving force counteracting the Brownian motion of particles to resolve particles with different hydrodynamic sizes at different channel-flow laminae at steady state. Thus, cross-flow is a defining factor in AF4 fractionation quality. To determine the optimal cross-flow for exosome fractionation, various cross-flow settings were evaluated. Exosome fractionation profiles (fractograms of UV absorbance and DLS) were devised from representative cross-flow settings, as shown in FIG. 16. Specifically, linear gradients of cross-flow with different starting now rates (at 0.3, 0.5 and 1.0 mL/min) and slopes (i.e., how fast the cross-flow drops to 0 mL/min; tested conditions: a decrease in flow rate from 0.5 to 0 mL/min within 15, 30 and 45 min) were examined. Three major peaks (P2, P3 and P4) were observed when the cross-flow decreased from 0.5 to 0 mL/min within 45 min. These peaks represented the exomeres and two exosome subsets (i.e., small exosomes [Exo-S] and large exosomes [Exo-L]), respectively, as reported supra (FIG. 16A). Among the other peaks, P0 is the void peak, resulting from flow disturbance when switching from the focus/injection mode to the elution mode. P1 is a very minor peak, generated by the concomitant elution of the void peak and species that were smaller than exomeres. Depending on the ENP preparation, P1 was sometimes barely detected. PS was generated due to loss of control on flow rate when it decreased below ˜0.08 mL/min and all retained sample components (larger microparticles and/or aggregates of small particles) were eluted out. As shown in FIG. 16B, when the initial cross-flow rate was increased to 1.0 mL/min, no additional shoulder peaks were observed to separate further from peaks P2-P4, indicating the uniformity of these three populations of particles. A delay in the elution of all three peaks was observed. Moreover, a much higher P5 peak was observed and this is due to insufficient time for elution of large particles, including Exo-L, in the given time and based on the channel size. In contrast, when the initial cross-flow rate was set to 0.3 mL/min, the samples eluted much earlier, indicating that this flow rate was not fast enough to retain the sample constituents inside the channel and resolve them efficiently. Therefore, an initial cross-flow rate of 0.5 mL/min was used throughout the procedure.
Next, the impact of different slopes of the cross-flow gradient on separation quality was evaluated. A linear decrease of the cross-flow rate from 0.5 to 0 mL/min within a time span of 15, 30 and 45 minutes were compared. Clearly, the peaks became narrower and the separation quality was compromised when shorter time spans were used (FIG. 16C). In addition, a larger P5 peak was observed when a shorter time span was used, indicating insufficient time for elution of large particles.
On the contrary, when longer time spans were used, the peaks broadened but with improved separation quality. This setting is desired when high-purity particles in discrete fractions need to be recovered for further offline characterization. However, when longer time spans are used, other practical issues, such as the dilution of samples and the sensitivity limit of online detectors for accurate measurement, have to be taken into account. Therefore, it was aimed to separate distinct subsets of exosomes (<150 nm), which were clearly separated from each other when a time span of 45 minutes was applied (longer time spans were not tested). A linear gradient of the cross-flow decreasing from 0.5 to 0 mL/min for 45 minutes was chosen for the study.

Example 9—Channel Height

Based on the working principle of AF4, the channel's geometry, including its width, height and shape, is critical for fractionation quality. The short channel utilized in this study is a product of Wyatt Technology (Santa Barbara, USA), which has a trapezoidal geometry (Cölfen & Antonietti, “Field-Flow Fractionation Techniques for Polymer and Colloid Analysis,” Adv. Polym. Sci. 150:67-187 (2000); Litzen & Wahlund, “Zone Broadening and Dilution in Rectangular and Trapezoidal Asymmetrical Flow Field-Flow Fractionation Channels,” Analytical Chemistry 63:1001-1007 (1991), which are hereby incorporated by reference in their entirety) with a tip-to-tip length of 152 mm and a linear decrease of the channel width from 21.5 mm (close to the injection port and about 12 mm away from the inlet tip) to 3 mm. With the shape and width already optimized and fixed, the height (i.e., the thickness of the channel, determined by the spacer used between the upper wall and the bottom accumulation membrane) was the only parameter available for further optimization. A series of spacers with different thicknesses (190, 250, 350 and 490 μm) were provided by the manufacturer. The channel height affects the parabolic laminar flow rate profile and thus the separation resolution. It also affects the channel capacity, with a thicker channel allowing for analysis of larger sample amounts. Since enough sample needed to be recovered for downstream analysis, the loading capacity is an important factor for the fractionation and so employing only spacers with a thickness of 350 μm and 490 μm were considered. As shown in FIG. 17, the channel with the 350-μm spacer eluted samples earlier but with narrower peaks and a reduced separation resolution compared to the channel with the 490-μm spacer. Therefore, the 490-μm spacer was chosen for the work.

Example 10—Focusing

A 100-μL sample loop was used in the instrument for sample loading. It is a significant portion of the total channel capacity, which usually ranges from 200 μL to 1000 μL. Once injected into the channel, the sample would spread throughout the channel and lead to insufficient fractionation. To avoid this, a flow opposing the channel forward flow was introduced from the outlet and, together with the channel flow, focused the sample into a narrow band close to the injection port (i.e., focus mode). First, the focus flow was established and then the samples were injected in the focus mode and given enough time to reach steady-state equilibrium before elution. The focusing flow rate and focusing time determine focusing efficiency. Here, the focusing flow rate was fixed at 0.5 mL/min, the same as the initial cross-flow rate for elution, and then tested different time periods (2, 5, and 10 minutes) for focusing efficiency. As shown in FIG. 18, different focusing times did not significantly affect peak shape or resolution power. Moreover, before exomere elution occurred, the fractograms of both UV and DLS reached similar baselines. Notably, it was observed that the PS signal intensity increased as focusing time increased, suggesting potential particle aggregation caused by extensive focusing. Therefore, a focusing time of 2 minutes was chosen for the study.

Example 11—Membrane Choice

Since the sample fractionation is performed close to the membrane, in addition to the pore size of the membrane, the compatibility of the membrane material with the samples also needs to be considered. For example, the sample may bind to the membrane non-specifically. Two different types of membranes that are commonly used for biological material concentration or filtration, regenerated cellulose (RC) and polyethersulfone (PES), were tested for exosome fractionation. While keeping other AF4 parameters exactly the same, a delay of sample elution and broader peaks was observed in the channel with PES compared with RC, suggesting potential non-specific interactions between samples and the membrane (FIG. 19). Therefore, the RC membrane was selected for the studies.

Example 12—Amount of Input Sample

Once the key fractionation parameters were determined, the loading capacity of AF4 was then examined. The minimal amount of material required for AF4 is determined primarily by the sensitivity limit of the online detectors, such as DLS and UV monitors. The signal/noise ratio must be adequate for accurate data collection and interpretation. The maximal amount of material is determined by the required resolution of fractionation, which depends on the purpose of the experiment and the complexity of the sample to be analyzed. To efficiently separate exomeres and the two exosome subsets that are reported herein from small (s)EVs prepared using UC, different amounts of B16-F10-derived sEV input samples ranging from 15 μg to 165 μg were tested. As shown in FIG. 20, 15 μg was the lower limit of material for this analysis, as a high level of noise began to be detected, especially at the low end of hydrodynamic size. Inputs of 40 μg and 100 μg yielded almost identical fractionation profiles and hydrodynamic size determinations, indicating comparable fractionation resolution and robust signal detection. However, when the amount of input increased to 165 μg, the elution of all peaks was delayed significantly, resulting in incomplete elution of Exo-L. Bleed-through of each particle population to the adjacent populations increased (and thus poorer separation occurred), as indicated by the increased signal intensity at the valleys between peaks. Therefore, an input ranging from 40 μg to 100 μg was used for this study.

Discussion of Examples 8-12

As illustrated in the above assessments, AF4 technology provides unique capabilities to separate nanoparticles with high resolution within a large size range. Through the highly robust and straightforward means of AF4, distinct exosome subpopulations and exomeres were able to be efficiently separated. These findings exhibit AF4s potential usefulness in identifying other distinct EV subpopulations. Coupled with online monitoring (e.g., multi-angle light scattering [MALS]. DLS, UV absorbance and fluorescent detection) and offline analyses (e.g., microscopy, mass spectrometry of proteins, lipids, glycans and metabolites, and DNA and RNA sequencing), AF4 can yield valuable data on ENP analytes, including particle morphology and size, relative abundance, molecular composition, and other biophysical and biochemical properties. A powerful tool, AF4 can help researchers decipher the complexities and heterogeneity of ENPs that cannot be well addressed with other existing techniques.
The AF4 protocol describes the fractionation of exomeres and exosome subsets from sEVs isolated from the conditioned media of B16-F10 cells and a panel of more than 20 different cancer cell lines and 5 normal cell lines. This AF4 method can be used to fractionate and characterize sEVs isolated from an array of bodily fluids (including blood plasma or serum, lymphatic fluid, bone marrow plasma, cerebrospinal fluid, urine, saliva, bronchioalveolar lavage, milk and amniotic fluid), given their similar particle compositional complexity. Since all cells are capable of shedding EVs, this protocol can be employed to study the EV biology of any organism.
Not only for biological discovery, this protocol can also be modified for use in the field of quality control in exosome-based pharmaceutical production. Exosomes have become attractive therapeutic delivery vehicles for treating cancer and other types of diseases (Batrakova & Kim, “Using Exosomes, Naturally-Equipped Nanocarriers, for Drug Delivery,” J Control Release 219:396-405 (2015), which is hereby incorporated by reference in its entirety). AF4 coupled with sensitive molecular assays can serve as an improved analytic tool to evaluate purity, drug loading efficiency, and the integrity of the exosome product by detecting debris or aggregates.
Last but not least, this protocol can serve as a reference to further develop and optimize methods for fractionating and characterizing other types of ENPs. Some unique advantages of AF4 are its high resolution and large size range of fractionation and that different conditions, such as cross-flow setting and focus time, can be easily tested by simply programing the settings into the software, with minimal handling of the channel. Besides their use in analyzing exosomes and other sEVs, fractionation protocols for large EVs, such as larger microparticles and oncosomes, can be further developed. Specific caution should be taken when fractionating large particles since they may be too large to elute in the normal mode (when the particle is small and considered as point-mass compared to the channel height) but in the steric model instead (FIG. 15). Moreover, other fields, such as electric field, can also be applied to AF4 to stratify particles based on additional biophysical properties other than size, allowing even broader application of AF4 technology.
Taken together, the separation and characterization of distinct EV subpopulations by AF4 are critical to advancing knowledge of the biology of EVs and their functional roles in physiological and pathological conditions. By profiling the molecular cargo of EVs, signature proteins, lipids, glycans and genes as well as specific signaling pathways associated with disease progression can be identified, facilitating the identification of potential diagnostic/prognostic biomarkers, including those related to cancers. Such knowledge will also provide a rationale for developing ENP-based therapies in clinical trials.
Comparison with Other Methods
A multitude of technologies, in addition to AF4, have been developed to isolate pure exosomes and other EV subpopulations. The most commonly used technique makes use of dUC and separates the particles based on their hydrodynamic size and density. Successive centrifugation at different centrifugal forces eliminates dead cells and cellular debris (500×g, 10 min), large oncosomes and apoptotic bodies (2000˜3000×g, 20 minutes) and larger microparticles (10,000-12,000×g, 20 minutes), and subsequently pellets sEVs (100,000×g, 70 minutes)(Théry et al., “Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids,” Curr Protoc Cell Biol Chapter 3, Unit 3.22 (2006); Jeppesen et al., “Comparative Analysis of Discrete Exosome Fractions Obtained by Differential Centrifugation,” J Extracell Vesicles 3:25011 (2014); Cvjetkovic et al., “The Influence of Rotor Type and Centrifugation Time on the Yield and Purity of Extracellular Vesicles,” J Extracell Vesicles 3 (2014), which are hereby incorporated by reference in their entirety). Centrifuge rotor type, centrifugal force and centrifugation time are key factors influencing the product yield and purity of this method. Its performance also varies depending on the cell types studied (Willms et al., “Cells Release Subpopulations of Exosomes with Distinct Molecular and Biological Properties,” Sci Rep 6:22519 (2016), which is hereby incorporated by reference in its entirety), dUC can process large volumes and high amounts of sample, but the purity of the material recovered is poor. It can only roughly partition particles into groups, such as large vesicles, microparticles, and sEVs (enriched for exomeres and exosomes), with expected heterogeneity within each group and contamination for other groups. The high centrifugal force may also cause sample aggregation. With the present protocol, the advantage of dUC was used by first stratifying and concentrating the sEV population and then analyzing particles at much higher resolution to further fractionate exomeres and exosome subsets.
Density gradient floatation (DGF) is often used to further purify sEVs first isolated using dUC. In DGF, EVs are overlaid upon a gradient of increasing dilutions of a viscous solution (sucrose or iodixanol are commonly used) and, upon centrifugation, they migrate to the equilibrium density determined by the EV's size, shape and density. DGF is often used to remove non-membranous particles from EVs and has also been employed in several studies to address exosome heterogeneity (Aalberts et al., “Identification of Distinct Populations of Prostasomes that Differentially Express Prostate Stem Cell Antigen. Annexin A1, and GLIPR2 in Humans,” Biol Reprod 86:82 (2012); Bobrie et al., “Diverse Subpopulations of Vesicles Secreted by Different Intracellular Mechanisms are Present in Exosome Preparations Obtained by Differential Ultracentrifugation,” J Extracell Vesicles 1 (2012); Willms et al., “Cells Release Subpopulations of Exosomes with Distinct Molecular and Biological Properties” Sci Rep 6:22519 (2016), which are hereby incorporated by reference in their entirety). The major drawbacks of DGF, when compared to the performance of AF4, include its time-consuming preparations, lack of automation, operator-dependent reproducibility and low yield. Long periods of incubation with high sucrose concentrations can also damage EV integrity, necessitating additional washing steps for its removal. In contrast, AF4 is rapid, fully automated, highly reproducible, robust, and compatible with many buffer choices that mimic physiological conditions. Resolution and size range in EV fractionation is far superior with AF4 than with DGF (Tauro et al., “Comparison of Ultracentrifugation, Density Gradient Separation, and Immunoaffinity Capture Methods for Isolating Human Colon Cancer Cell Line LIM1863-Derived Exosomes,” Methods 56:293-304 (2012), which is hereby incorporated by reference in its entirety).
SEC, a gentle means of nanoparticle fractionation, has been extensively used for protein and protein complex analysis in biochemical and biophysical studies. Recently, it has been adopted to fractionate EVs (Mol et al., “Higher Functionality of Extracellular Vesicles Isolated Using Size-Exclusion Chromatography Compared to Ultracentrifugation,” Nanomedicine 13:2061-2065 (2017); Nordin et al., “Ultrafiltration with Size-Exclusion Liquid Chromatography for High Yield Isolation of Extracellular Vesicles Preserving Intact Biophysical and Functional Properties,” Nanomedicine 11:879-883 (2015); Böing et al., “Single-Step Isolation of Extracellular Vesicles by Size-Exclusion Chromatography,” J Extracell Vesicles 3 (2014); Willis et al., “Toward Exosome-Based Therapeutics: Isolation, Heterogeneity, and Fit-for-Purpose Potency,” Front Cardiovasc Med 4:63 (2017), which are hereby incorporated by reference in their entirety). In SEC, particles are separated in a column filled with porous polymer beads (stationary phase) based on their size and shape. Smaller-sized particles with a globular shape can penetrate the porous beads more readily, taking a longer route and more time to elute, whereas the larger particles are excluded from penetrating the pores and subsequently elute more rapidly. The elution of particles with abnormal shapes is more complicated due to its potential steric interference with particle traveling through the pores. Compared to other technologies, SEC has a resolution most similar to that of AF4. Still, AF4 demonstrates superior resolution over a much wider size range (Fraunhofer et al., “The Use of Asymmetrical Flow Field-Flow Fractionation in Pharmaceutics and Biopharmaceutics.” Eur J Pharm Biopharm 58:369-383 (2004), which is hereby incorporated by reference in its entirety). SEC resolution drops when particles are close to or larger than the upper limits of pore size. Furthermore, SEC is not as flexible as AF4 in changing separation parameters and its size range of separation is fixed for a given column with a specific solitary phase. Moreover, AF4 contains a hollow channel with only a membrane at the accumulation wall but, unlike SEC, requires no stationary phase. This stationary phase in SEC generates shear stress and renders a much larger surface area than AF4 for nonspecific binding of analytes. Similar to AF4 methods, the input sample loading volume for SEC must be restricted and there is an upper limit for the sample capacity to compromise a balance between sufficient yield and exemplary fractionation quality. Sample stratification by dUC and concentration methodologies prior to separation greatly facilitates the separation power of SEC.
UF allows for straightforward isolation of EV populations based on their size by filtering the sample through a series of semipermeable membranes with defined pore sizes (i.e., as reflected by molecular weight cutoffs) (Xu et al., -Highly-Purified Exosomes and Shed Microvesicles Isolated from the Human Colon Cancer Cell Line LIM1863 by Sequential Centrifugal Ultrafiltration are Biochemically and Functionally Distinct,” Methods 87:11-25 (2015); Xu et al., “A Protocol for Isolation and Proteomic Characterization of Distinct Extracellular Vesicle Subtypes by Sequential Centrifugal Ultrafiltration,” Methods Mol Biol 1545:91-116 (2017), which are hereby incorporated by reference in their entirety). Smaller particles below the cutoff size can penetrate through the pores while larger ones are retained. UF provides a crude separation of EVs due to limitations of membrane pore size availability. Most EVs are not rigid spheres but rather flexible particles and can transfigure to pass through the pores, especially when pressure is applied. Another concern is the uniformity of membrane pore size, which is critical for separation purity. Though the separation power of UF is inferior to AF4, UF can serve as a means to pre-stratify and concentrate input samples for further analysis by AF4.
Distinguishable from methods which separate EVs mainly by their size, IAC relies on the antigenic recognition of EV surface molecules (primarily proteins). IAC is highly selective, fast and flexible to scale for either preparation or analytic purpose. This separation principle has been adapted for different formats of analysis and preparation, including precipitation using immunomagnetic beads, flow analysis, detection by microarray, microscopy, western or ELISA assay, and microfluidic separation (Thery et al., “Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids,” Curr Protoc Cell Biol Chapter 3. Unit 3.22 (2006); Chen et al., “Microfluidic Isolation and Transcriptome Analysis of Serum Microvesicles,” Lab Chip 10:505-511 (2010); Jorgensen et al., “Extracellular Vesicle (EV) Array: Microarray Capturing of Exosomes and Other Extracellular Vesicles for Multiplexed Phenotyping” J Extracell Vesicles 2 (2013); Ko et al., “miRNA Profiling of Magnetic Nanopore-Isolated Extracellular Vesicles for the Diagnosis of Pancreatic Cancer,” Cancer Res. 78:3688-3697 (2018), which are hereby incorporated by reference in their entirety). The inherent limitation of IAC is that knowledge about the surface antigen is a prerequisite. The other concern is that the IAC antigen may be represented in multiple subpopulations of EVs with divergent sizes and/or origins. Thus, the application of AF4 may further necessitate EV separation based on size. EVs captured by IAC are ideal for molecular content characterization but not for further functional studies due to inefficient removal of the capturing antibody, which may interfere with the functional assay or targeting and uptake by recipient cells. In contrast, AF4 is label-free and enables these functional analyses feasible.

Level of Expertise Needed to Implement the Protocol

The AF4 instrument is commercially available (e.g., Wyatt Technology) and the manufacturer can perform the initial set-up. The method development for specific sample analysis, routine maintenance of instruments, and troubleshooting require a good understanding of the working principles of AF4 and installed detectors, training for handing the AF4 channel and detectors, and being familiar with software used for AF4 operation and data collection. Previous experience with chromatography and/or microfluidics is helpful in mastering the AF4 application. However, once the AF4 fractionation method training has been achieved, only minimal skills, such as familiarity with software interface and proper instructions, are necessary to complete the fractionation process since nearly all the steps are automatic and programmed.

Limitations

One inherent limitation of AF4 is that it fractionates samples based on their size. As a consequence, particles with the same hydrodynamic size but with different morphologies, surface molecules and other biophysical properties cannot be separated from each other via AF4 alone. However, other fields, such as electric field, can also be applied in conjunction with AF4 to provide further separation according to additional characteristics such as particle surface charge. Special consideration is also required when developing a protocol for large particles whose sizes are too large to be considered as point-mass compared to the channel height. These large particles will elute in the steric mode rather than the normal mode, as illustrated in FIG. 1.
A second inherent drawback is that AF4 can accommodate only small amounts of sample (e.g., 40 μg to 100 μg in the present case), which is often not efficient for large-scale preparations in more detailed assessments of nanoparticle properties. The sample instead can be divided into multiple fractionation analyses for improved characterization of specific nanoparticle subsets. A third limitation of AF4 is that due to the loading capacity limitation, the input sample requires UC preparation or other means to first stratify and concentrate the analytes (i.e. sEVs in the present study) prior to fractionation.
Furthermore, it has to be pointed out that no single formula can be universally applied for analysis of different types of samples. The fractionation method and key parameters discussed above in the protocol development section have to be developed and optimized based on the complexity (i.e. size and abundance of each component) of the sample of interest. In certain cases, different running methods and instrument settings may have to be combined sequentially to efficiently separate different components within a complex sample.
A detailed protocol has been described herein for optimal sEV preparation and fractionation via AF4. The key steps for successful of AF4 separation are: (i) the preparation of sEVs from conditioned media of cell culture; (ii) development and optimization of the AF4 running methods; (iii) online data analysis and fraction collection for offline characterization.
Pre-stratification of the sEVs using methods such as UC is critical to reduce the complexity of the samples to be analyzed in their particle composition. This allows enough material for each subpopulation of sEVs present in the samples to be analyzed by a single run of AF4. Otherwise, a series of AF4 methods for best separation of particles within different size ranges have to be adapted. Another key factor for successful AF4 analysis and fractionation is the amount of input samples loaded onto the AF4 system. Overloading the system will result in poor resolution and inefficient separation of nanoparticles; whereas loading too little a sample will lead to poor signal detection and inaccurate data deduction, as shown in FIG. 19.
Five major parameters for AF4 running method optimization have been discussed, including cross flow, channel height, focus time, loading amount, and membrane type (see FIGS. 16-19). A representative AF4 fractionation profile of B16-F10 derived sEVs is shown in FIG. 22. Based on the method described here, three major subpopulations of sEVs are identified (FIG. 22A, i.e. exomeres, Exo-S and Exo-L, corresponding to peaks P2, P3 and P4, respectively). The autocorrelation function is a key factor to determine the purity of each fraction (FIG. 22B). The separated particles can be further recovered and usually need further concentration for a variety of offline analyses, such as TEM, NTA, BCA assay, biophysical/biochemical property characterization, molecular composition determination, and functional studies. Shown in FIG. 22C is TEM imaging analysis of combined fractions for B16-F10 exomeres, Exo-S and Exo-L, revealing the distinct morphology of each sEV subset.

Example 13—Examination of Systemic Functions of Exomeres, Exo-S, and Exo-L

To investigate their systemic functions, especially in liver, B16-F10 murine melanoma-derived exomeres, Exo-S and Exo-L were intravenously injected into naïve, syngeneic C57BL/6 mice. An equal volume of PBS was injected as the control. 24 hours later, livers were harvested from each group of treated mice and subjected to total RNA extraction and RNA sequencing analysis. As shown in FIG. 23A, a total of 5700, 5320, and 6291 genes were identified to be significantly (p<0.05) changed in their expression levels in the liver of mice treated with exomeres, Exo-S and Exo-L when compared with the PBS control group, respectively. Specifically, a list of 140 and 810 genes are uniquely changed in exomeres when compared with Exo-S and Exo-L, respectively.
To further compare the changes in gene expression among each group, one-way ANOVA analysis was performed and the result was illustrated in FIG. 23B for the top 2000 genes that are significantly changed. A large similarity was identified in all three groups of exomeres, Exo-S, and Exo-L treated mice when compared to the PBS control group. Specifically, shown in FIG. 24 are the top 50 gene lists that are up-regulated (FIG. 24A) or down-regulated (FIG. 24B) in all three groups when compared to the PBS control group, respectively. These genes can therefore serve as potential biomarkers for detection of disease, monitoring liver dysfunction, and therapeutic targets to intervene with tumor progression in cancer patients. For example, the Serum Amyloid A family genes (Saa1, Saa2 and Saa3), S100 calcium-binding protein AF4 (S100A4), and a subset of ribosomal protein subunits (Rpl41, Rps24, Rpl36, Rpl35, Rpl21, Rps12, Rps18, Rps14, Rpl12, Rpl34, Rps100, and Rpl17) are specifically upregulated in the liver of mice treated with exomeres, Exo-S and Exo-L when compared to the PBS control. On the other hand, transcription factors such as Forkhead box protein N3 (Foxn3), TEA domain family member 1 (Tead1), Nuclear Factor Of Activated T Cells 5 (Nfat5), Forkhead box Q1 (Foxq1), Forkhead box K1 (Foxk1), Kruppel Like Factor 12 (Klf12), and ETS domain-containing protein (Elk4) are among the top 50 genes that are significantly down-regulated genes in all three groups.
To further investigate the functional pathways that are remarkably influenced by exomeres, Exo-S, and Exo-L, Ingenuity Pathway Analysis was conducted upon each dataset. Shown in FIG. 25 are the top five canonical pathways that were identified in each dataset: exomere versus PBS (FIG. 25A); Exo-S versus PBS (FIG. 25B); Exo-L versus PBS (FIG. 25C); exomere versus Exo-S (FIG. 25D), and exomere versus Exo-L (FIG. 25E). Importantly, pathways including E2F signaling, mTOR signaling, and regulation of eIF4 and p70S6K signaling are identified to be remarkably changed in all three groups of exomeres, Exo-S, and Exo-L when compared to the PBS control, indicating their fundamental influence on the proliferation and metabolism of the liver. Beyond these findings, pathways of Molecular Mechanisms of Cancer and Glucocorticoid Receptor Signaling are specifically recognized among the top five canonical pathways in the exomere versus PBS group; pathways of Mitochondrial Dysfunction and Oxidative Phosphorylation in the Exo-S versus PBS group; and pathways of Nerve Growth Factor Signaling and Insulin Receptor Signaling in the Exo-L versus PBS group. Furthermore, when the exomere-treated group was compared to the Exo-S or Exo-L-treated groups, the following pathways are specifically recognized: Acute Phase Response Signaling, FXR/RXR Activation, Toll-like Receptor Signaling, LPS/IL-1 Mediated inhibition of RXR Function, and Aryl Hydrocarbon Receptor Signaling in the comparison of exomeres versus Exo-S; Superpathway of Cholesterol Biosynthesis. Cholesterol Biosynthesis I, Cholesterol Biosynthesis II (via 24,25-dihydrolanosterol), Cholesterol Biosynthesis III (via Desmosterol), and IGF-1 Signaling in exomere versus Exo-L. Collectively, detection of the alteration in these signaling pathways may assist in detecting and monitoring tumor progression in cancer patient. They also represent potential therapeutic targets.
Proteomic analysis of exomeres has indicated its potential role in the metabolism of the target cells. To specifically follow up on this hypothesis, the livers harvested from mice 24 hours post injection of B16-F10 derived exomeres, Exo-S and Exo-L in comparison with the PBS control were subjected for metabolite extraction and mass spectrometry analysis. FIG. 26A listed the number of metabolites whose abundance are significantly affected in each comparison group using unpaired t test. One-way ANOVA analysis was utilized to further identify metabolites that are changed in all three experimental groups and those are uniquely affected in each group. FIG. 26B illustrated all the metabolites identified with significant changes and FIG. 26C showed the clustering analysis of the metabolites that are uniquely affected in each group. In particular, the abundance of metabolites including thymine, taurine, and adenylosuccinate are found increased in all three groups of exomeres. Exo-S and Exo-L-treated mouse livers (FIG. 27A); whereas the abundance of the following metabolites including glucose-6-phosphate, L-arginino-succinate, methylcysteine, sn-glycerol-3-phosphate, and tyrosine decreased in all three groups when compared to the PBS control (FIG. 27B). Furthermore, representative metabolites that are uniquely affected in each group (FIG. 26C) are illustrated in FIGS. 28A-28C. Besides the altered gene expression, the aberrant regulation of these metabolites can be utilized as biomarkers to detect and monitor tumor progression and serve as potential therapeutic targets as well for cancer patients.
To further dissect the functional roles of exomeres in liver, immunofluorescent colocalization analysis was conducted and Kupffer cells, the resident macrophages in liver, were identified as the primary cell type that uptakes B16-F10 melanoma derived exomeres. When intravenously administrated into the naïve and syngeneic C57BL/6 mice, more than 95% of exomeres previously labeled with the PKH67 fluorescent dye were observed colocalized with the F4/80 positive Kupffer cells in the liver (FIG. 29). This finding implicates that Kupffer cell function can potentially be manipulated by tumor-derived exomeres and initiate a cascade of systemic effect to favor the tumor growth in vivo, therefore representing a potential target to develop therapeutic strategy for blocking cancer development.
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefrom considered to be within the scope of the invention as defined in the claims which follow.

Claims

1. A method of diagnosing cancer in a subject, said method comprising:

selecting a subject having cancer;

obtaining, from the selected subject, a population of either exomeres having a diameter of less than 50 nm, small exosomes having a diameter of 60-80 nm, or large exosomes having a diameter of 90-120 nm;

recovering the exomeres, small exosomes, or large exosomes from the sample;

contacting the exomeres, small exosomes, or large exosomes or portions thereof with one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more proteins contained in said exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more N-glycans contained in said exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more lipids contained in said exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in said exomeres, small exosomes, or large exosomes, or (5) combinations thereof, and

diagnosing the cancer based on said contacting.

2. The method of claim 1, wherein said sample is blood, serum, plasma, ascites, cyst fluid, pleural fluid, peritoneal fluid, cerebrospinal fluid, tears, urine, saliva, sputum, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary trances, breast milk, intra-organ system fluid, peritoneal fluid, conditioned media from tissue explant culture, or combinations thereof.

3. The method of claim 1, wherein exomeres are recovered.

4. The method of claim 1, wherein small exosomes are recovered.

5. The method of claim 1, wherein large exosomes are recovered.

6. The method of claim 3, wherein said contacting is carried out by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more proteins contained in said exomeres.

7. The method of claim 6, wherein said method is carried out to determine if the subject has cancer by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of PPID, GANAB, MAT1A, CPYD, FAT4, GMPPB, ERP44, CALR, GPD1, BZW1, PFKL, OLFML3, HGD, LGALS3BP, GCLC, PEPD, MTHFD1, PGD, ACTR3, XPNPEP1, UGP2, SNX2, ALDOC, SEPT11, HSPA13, AARS, SERPINH1, CNDP2, PDE5A, AGL, EXT1, IDH1, SERPINC1, RRM1, CKB, HMGCS1, HPD, PSMC4, NPEPPS, CAT, EXT2, CORO1C, B4GAT1, RACK1, MAPRE1, PGM1, PD1A3, ADK, SHMT1, ACO1, GSN, ESD, PPP2R1A, ALDH1L1, OLA1, ACLY, EEF1G, FLNB, PSMD11, ANGPTL3, FERMT3, PYGL, MDH1, and EIFA2.

8. The method of claim 4, wherein said contacting is carried out by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more proteins contained in said small exosomes.

9. The method of claim 8, wherein said method is carried out to determine if the subject has cancer by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of TTYH3, FLOT1, FLOT2, TSPAN14, LAMC1, CD63, MVB12A, ZDHHC20, VAMP3, VPS37B, ARRDC1, and TGFBR2.

10. The method of claim 5, wherein said contacting is carried out by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more proteins contained in said large exosomes.

11. The method of claim 10, wherein said method is carried out to determine if the subject has cancer by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of SQSTM1, STIP1, HINT1, WASF2, RASA3, EPB41L2, GIPC1, S100A10, MPP6, KIF23, RACGAP1, ANXA5, CASK, DLG1, TJP1, BAG5, TXN, ABI1, ANXA1, CAPG, DB1, S100A6, CHMP2B, CHMP3, ANXA2, MYO1C, ANXA4, SNX12, LIN7C, STXBP3, CEP55, ALCAM, VCL, CHMP1A, FARP1, ACSL4, BA1AP2, SH3GL1, DSTN, LGALS1, CYFIP1, CTNNA1, RAB31, ARF6, SLC1A5, EPS8, FMNL2, PGAM1, CNP, CHMP4B, ANXA3, VPS4B, GNG12, PACSIN3, GLG1, VTA1, LYN, VPS37C, CHMP5, F3, DNAJA1, RHOC, GNA13, CHMP2A, ATP2B1, RDX, ATP1B1, CAPZB, EHD1, DNAJA2, and CTNND1.

12. The method of claim 6, wherein said method is carried out to determine if the subject has melanoma by detecting, higher or lower levels, relative to a standard for subjects not having melanoma, or the presence or absence, of one or more of the proteins selected from the group consisting of IT1H2, IT1H3, H2AFX, PMEL, MAT1A, HPD, ALB, B4GAT1, ARF1, GCLC, HGD, PPP2CB, PAH, AGL, RNPEP, PPID, BZW1, ME1, DPYD, CA6, OLFML3, NPEPPS, PREP, ERP44, RELN, GPD1, GFPT1, CNDP2, PFKL, ALDH8A1, ATP6V1A, ENO2, THBS3, CORO1C, EXT1, CAT, XPNPEP1, PYGL, CALR, and LGALS3BP.

13. The method of claim 8, wherein said method is carried out to determine if the subject has melanoma by detecting, higher or lower levels, relative to a standard for subjects not having melanoma, or the presence or absence, of one or more of the proteins selected from the group consisting of TYRP1, SDCBP, SDCBP, CD63, IGSF8, HSPA8, MLANA, HBA1/HBA2, GPNMB, DCT, HSPA2, HSPA1L, HSPA5, Fv4, PDCD61P, RAB7A, ENV1, CD81, GNB1, SYT4, GNB2, HIST1H2AH, GNAI2, GAPDH, APOE, BC035947, Hist1h2a1, ACTG1, ACTB, GNB4, GNA13, SLC3A2, ACTC1, GNAS, SLC38A2, HIST2H2BF, ATP1A1, TFRC, TMEM176B, VAMP8, TSPAN10, ADGRG1, Hist1h4a, PMEL, UBL3, PPIA, ACTBL2, CD9, BACE2, and TSPAN4.

14. The method of claim 10, wherein said method is carried out to determine if the subject has melanoma by detecting, higher or lower levels, relative to a standard for subjects not having melanoma, or the presence or absence, of one or more of the proteins selected from the group consisting of HSPA8, TYRP1, SDCBP, HSPA2, RPS27A, HSPA1L, MLANA, HSPA5, CD63, IGSF8, GPNMB, Fv4, ENV1, PDCD61P, HSPA1A/HSPA1B, DCT, ACTG1, ACTB, PPIA, SLC3A2, ACTC1, CD81, ITM2C, RAB7A, GNB1, TSPAN4, DNAJA1, GNB2, TFRC, HBA1/HBA2, GNAI2, SYT4, GAPDH, APOE, PMEL, MFGE8, GNB4, GNA13, GNAO1, DNAJA2, ATP1A1, ITGB1, TMEM59, SLC38A2, GNA12, ITM2B, GNAS, HIST1H2AH, LAMP1, and EEF1A1.

15. The method of claim 6, wherein said method is carried out to determine if the subject has breast cancer by detecting, higher or lower levels, relative to a standard for subjects not having breast cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of FGB, HIST2H2AB, COMP, HIST1H2BJ, C7, GSTA5, ENO3, ARF3, SULT1C4, E1F4A2, MAT1A, GNB2, UGDH, AKR1B10, MTHFD1, CTSC, DPP3, RPSA, OTUB1, ALDH8A1, F11, CTPS2, MGAT1, HYH1, LGALSL, GSTM3, GSTM5, PSMC1, F8, PRKAR2B, RPL10A, HNRNPK, SEMA6D, SNX5, IARS, LCP2, ARPC4, PPP6C, PSMD6, PTPRS, TIE1, PSMD8, PABPC4, RPS18, CHAD, IPO5, FABP3, GALNT2, QPCT, STAT5A, SEMA3A, NT5C2, IDE, STAT3, DPYSL3, PDXK, ARF5, PSMD5, GNE, NBEAL2, FHL1, TIMP3, POSTN, MAPRE2, IT1H3, C3, ENO2, PPID, O1T3, CAND1, SEPT2, UBE2N, DPYSL2, CKB, PTGES3, DSTN, PKLR, THBS3, RAP1B, HIST2H2AB, ACTBL2, TUBB2A, F10, CNTN1, HPD, ACE, EML2, HSPA13, TNXB, HEXB, CALR, ADH5, GPX1, CFL2, KRT76, TCP1, COTL1, DYNLL1, HGD, ALDOC, EPRS, GLO1, MAN2A1, FLT4, NAP1L4, RARS, HMGCS1, GANAB, SEPT7, FKBP4, COL12A1, ADSL, AKR1C20, VASN, DDX39B, ME1, COMT, ALDH1A1, EIF4A3, CDH11, PRPS1I3, PNPEP, NPEPPS, SEPT11, CMBL, PSMD1, ACTR1B, PSMD3, GCLC, FAT4, LPL, GPD1L, GCLM, VARS, PHPT1, CACNA2D1, SEPT9, GLRX3, AARS, GMPPB, SNX2, GLOD4, PTPRF, CSAD, PXDN, AGL, DPYD, PRKACB, LARS, PPID, LTA4H, PSMD7, CAPNS1, ETF1, IARS, VPS35, TKFC, HYOU1, PGM2, TKT, HMCN1, CYB5R3, GPS1, UMPS, SND1, RTCB, RPL26, CARM1, PLCG2, P4HA2, CORO6, GMPS, IGSF8, PPP1R7, TIMP3, UXS1, DNM2, MEMO1, RPS3, ARHGD1A, PTGES3, NRP2, RAB1A, HBG2, and YWHAQ.

16. The method of claim 8, wherein said method is carried out to determine if the subject has breast cancer by detecting, higher or lower levels, relative to a standard for subjects not having breast cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of SDCBP, HIST1H2BN, HIST1H2AH, HBA1/HBA2, ITGB1, Hist1h4a, PDCD61P, HIST3H2BB, H2AFX, CD9, CD63, ITGA3, IT1H2, MFGE8, H2AFZ, PTGFRN, Hist1h3b, HSPA8, ACTG1, ACTB, ARRDC1, ACTC1, IT1H3, IGSF8, GSN, TUBA4A, HIST1H1D, TUBA1A, HIST1H1C, THBS1, HSPA2, ENO1, MVB12A, HTRA1, GAPDH, Hist1h1e, VPS28, TSG101, TUBB, TUBB4A, RAP1B, PFN1, CD81, VPS37B, TUBB6, RAP1A, EPCAM, Hist1h1b, PPIA, ADAM10, HBA1, HIST1H2BK, A2M, ED1L3, SDCBP, MFGE8, GSN, HIST2H2AC, HIST1H2AC, H2AFX, ACTB, THBS1, IT1H4, TUBB, TUBB2A, TUBB4B, F10, H2AFZ, TUBB4A, TUBB6, TUBB1, HSPA8, CD9, CD81, GAPDH, PFN1, HIST1H4A, HSP90AA1, HSP90AB1, HSPA2, HIST2H3A, PGK1, THBS2, EEF1A1, GPX3, ITGB1, PPIA, PDCD61P, EEF1A2, FBLN1, ATIC, CPNE8, TLN1, HSPA5, PKM, HIST1H1C, WDR1, RAN, PYGL, and ITGA3.

17. The method of claim 10, wherein said method is carried out to determine if the subject has breast cancer by detecting, higher or lower levels, relative to a standard for subjects not having breast cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of PDCD61P, SDCBP, EHD1, ITGB1, S100A6, ITGA3, CD9, VPS37C, Hist1h4a, RAP1B, CTNNA1, MSN, HIST1H2AH, ITGA2, PTGFRN, ACTG1, HIST1H2BN, Calm1, EPCAM, ITGA6, YWHAE, HSPA1A/HSPA1B, GNB1, SLC3A2, GNB2, EHD2, H2AFX, PPIA, NT5E, VPS4B, GNB4, Cdc42, SLC1A5, GNAI2, CFL1, YWHAH, EEF1A1, YWHAB, Hist1h3b, TSG101, YWHAG, ANXA5, GNA13, F5, H3F3A/H3F3B, CHMP4B, HSPA5, EZR, GAPDH, CD81, ED1L3, HBA1/HBA2, UBC, SDCBP, HSPA8, ITGB1, CD9, HSPA2, ACTC1, ACTB, ACTG1, PDCD61P, AFP, HBG2, ANXA2, ITGA3, HIST1H2BK, GAPDH, CD81, SLC3A2, GNAI2, GNA13, GNAI1, ATP1A1, HIST2H2AC, CPNE8, IST1, PFN1, TUBA4A, H2AFX, TUBA1C, HSPA5, YWHAZ, ENO1, ANXA5, GNAS, DNAJA1, CHMP5, EEF1A1, RHOA, KRT1, CEP55, GNB1, ACTBL2, ITGA2, EPHA2, GNA13, PPIA, RAP1A, and CD59.

18. The method of claim 6, wherein said method is carried out to determine if the subject has pancreatic cancer by detecting, higher or lower levels, relative to a standard for subjects not having pancreatic cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of SULT1E1, PKLR, ENO2, AKR1B1, FH, MGAT2, GPX1, DPP3, SEMA4B, GPD1, CSAD, NCAM1, PCMT1, NARS, THOP1, UMPS, PDE5A, CACNA2D1, TIE1, CDH11, AOX1, F8, GLB1, RPL10A, ACAP2, UXS1, ADSL, BMP1, PSMD3, LANCL1, GLO1, PPP2CA, ESD, PSMD5, FARSB, PAFAH1B1, SNX5, XPO1, MAPRE1, APRT, NEO1, GBA, THBS1, PYGL, THBS2, FAT4, CNTN1, AKR1C20, E1F4A2, ESD, BPGM, VASN, MAT1A, MAT2A, PFKL, CLIC5, HGD, GLOD4, AGL, PLEKHB2, CLSTN1, ST13, CMBL, AKR1E2, PRKAR2A, GPD1, LGALSL, GLA, IL1RAP, GMPPB, PCSK6, SEPT9, PSMC6, FYN, PAFAH1B1, VPS37C, CTNND1, NRBP1, ERP44, SHMT1, DARS, ADSL, GCLM, ALDOC, EPHA4, PEPD, CKB, PCMT1, UGDH, PRKAR1A, and GNAS.

19. The method of claim 8, wherein said method is carried out to determine if the subject has pancreatic cancer by detecting, higher or lower levels, relative to a standard for subjects not having pancreatic cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of SDCBP, PDCD61P, HSPA8, IGSF8, CD9, PTGFRN, ACTC1, LY6E, ACTB, MFGE8, HSPA2, CD81, ITGA3, ITGB1, IT1H2, VPS28, CD63, HTRA1, ENV1, Fv4, GSN, ENO1, ED1L3, MVB12A, IF1TM3, SERPINC1, ACTBL2, TUBA4A, PPIA, HSPA1A/HSPA1B, HSPA5, GAPDH, TSG101, TUBB, PLEKHB2, TUBA1C, TUBB4B, PFN1, GPC1, GJA1, EHD1, GNB2, TSPAN4, GNAI2, SLC3A2, VPS37B, GNA13, RAB7A, EEF1A1, GNAS, ALB, HBA1/HBA2, CD9, UBC, SDCBP, F2, ACTG1, ACTB, CD59, ACTC1, ACTA2, A2M, HIST1H2BK, HIST1H2BJ, HSPA8, TSPAN3, HIST2H2AC, CD55, H3F3A/H3F3B, HIST2H3PS2, PDCD61P, ITGB1, POTEJ, SERINC5, H2AFZ, ARRDC1, CLDN3, NT5E, EPCAM, CDH17, ATP1A1, ALPPL2, HIST2H2AB, ALPP, HSPA2, TSPAN8, MVP, ADAM10, THBS1, VNN1, ITGAV, IGSF8, MYOF, ATP1A2, AHCY, GSN, TSPAN1, PPIA, SDCBP2, and HSPA5.

20. The method of claim 10, wherein said method is carried out to determine if the subject has pancreatic cancer by detecting, higher or lower levels, relative to a standard for subjects not having pancreatic cancer, or the presence or absence, of one or more of the proteins selected from the group consisting of ACTC1, ACTG1, ACTB, MFGE8, ITGB1, HSPA8, ITGA3, SDCBP, GAPDH, LGALS1, ENV1, Fv4, YWHAZ, PPIA, GNB1, GNAI2, GNB2, ACTBL2, GNA13, CFL1, Marcks, GNAS, EEF1A1, ENO1, BSG, Calm1, S100A4, MSN, EZR, RDX, PTGFRN, PKM, SLC3A2, HBA1/HBA2, ED1L3, GNA13, RHOA, RHOC, S100A6, YWHAE, ALDOA, PDCD61P, PFN1, HSP90AB1, YWHAQ, ANXA1, ANXA2, ATP1A1, ITGA6, UBC, HBA1/HBA2, CD9, ACTG1, ACTB, CD59, MVP, SDCBP, ACTC1, ACTA2, HIST1H2BK, HIST2H2AC, ALB, HSPA8, HIST1H2BJ, CD55, H3F3A, TSPAN3, HIST2H3PS2, POTEJ, DPP4, NT5E, EPCAM, VNN1, H2AFZ, ITGB1, ALPPL2, HIST2H2AB, ATP1A1, ALPP, IST1, PDCD61P, MUC13, ANXA11, HSPA2, CDH17, GPA33, ANXA2, S100A6, ATP1A2, PPIA, EGFR, TSPAN8, MYOF, GNAI1, GNAI2, GNA13, S100A4, CLDN3, and A2M.

21. The method of claim 1, wherein said one or more reagents suitable for detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more proteins contained in said exomeres, small exosomes, or large exosomes measure protein expression level.

22. The method of claim 3, wherein said contacting is carried out by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more N-glycans contained in said exomeres.

23. The method of claim 22, wherein said one or more N-glycans are selected from the group consisting of N-glycan (Fucose)+GlcNAcβ1-6(GlcNAcβ1-2)Manα1-6(GlcNA β1-4(GlcNAcβ1-2)Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAcβ1-Asn and N-glycan (Fucose)+Neu5Acα2-8Neu5Acα2-3Galβ1-3/4GlcNAcβ1-2Manα1-3(Manα1-3(Manα1-6))Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn.

24. The method of claim 4, wherein said contacting is carried out by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more N-glycans contained in said small exosomes.

25. The method of claim 5, wherein said contacting is carried out by detecting, higher or lower levels relative to a standard for subjects not having cancer, or the presence or absence, of one or more N-glycans contained in said large exosomes.

26. The method of claim 3, wherein said contacting is carried out by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more lipids contained in said exomeres.

27.-28. (canceled)

29. The method of claim 4, wherein said contacting is carried out by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more lipids contained in said small exosomes.

30.-31. (canceled)

32. The method of claim 5, wherein said contacting is carried out by detecting, higher or lower levels, relative to a standard for subjects not having cancer, or the presence or absence, of one or more lipids contained in said large exosomes.

33.-34. (canceled)

35. The method of claim 1, wherein said contacting is carried out by detecting the presence or absence, relative to a standard for subjects not having cancer, of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in said exomeres, said small exosomes, or said large exosomes.

36.-37. (canceled)

38. The method of claim 1 further comprising:

selecting a suitable cancer therapeutic based on said diagnosing and administering the selected cancer therapeutic to said selected subject.

39. A method of prognosing cancer in a subject, said method comprising:

selecting a subject having cancer;

recovering the exomeres, small exosomes, or large exosomes from the sample;

contacting the exomeres, small exosomes, or large exosomes or portions thereof with one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more proteins contained in said exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more N-glycans contained in said exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more lipids contained in said exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in said exomeres, small exosomes, or large exosomes, or (5) combinations thereof, and

prognosing the cancer based on said contacting.

40. A method of managing treatment of a subject having cancer, said method comprising:

selecting a subject undergoing treatment for cancer;

recovering the exomeres, small exosomes, or large exosomes from the sample;

contacting the exomeres, small exosomes, or large exosomes or portions thereof with one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more proteins contained in said exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more N-glycans contained in said exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard or to the level in a prior sample obtained from the subject, or the presence or absence, of one or more lipids contained in said exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in exomeres, small exosomes, or large exosomes, or (5) combinations thereof, and

modifying said treatment, as necessary, based on said contacting.

41.-78. (canceled)

79. A kit suitable for diagnosing cancer, said kit comprising:

one or more reagents suitable to detect: (1) higher or lower levels, relative to a standard or to a sample from a subject, or the presence or absence, of one or more proteins contained in exomeres, small exosomes, or large exosomes, (2) higher or lower levels, relative to a standard or to a sample from a subject, or the presence or absence, of one or more N-glycans contained in exomeres, small exosomes, or large exosomes, (3) higher or lower levels, relative to a standard or to a sample from a subject, or the presence or absence, of one or more lipids contained in exomeres, small exosomes, or large exosomes, (4) the presence or absence of one or more genetic mutations in nucleic acid molecules associated with cancer and contained in exomeres, small exosomes, or large exosomes, or (5) combinations thereof, wherein said exomeres have a diameter of less than 50 nm, small exosomes have a diameter of 60 to 80 nm and said large exosomes have a diameter of 90 to 120 nm.

80.-111. (canceled)