US20160024503A1 - miRNA BIOGENESIS IN EXOSOMES FOR DIAGNOSIS AND THERAPY - Google Patents

miRNA BIOGENESIS IN EXOSOMES FOR DIAGNOSIS AND THERAPY Download PDF

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US20160024503A1
US20160024503A1 US14/775,716 US201414775716A US2016024503A1 US 20160024503 A1 US20160024503 A1 US 20160024503A1 US 201414775716 A US201414775716 A US 201414775716A US 2016024503 A1 US2016024503 A1 US 2016024503A1
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mir
mirna
hsa
cancer
exosomes
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Raghu Kalluri
Sónia MELO
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Beth Israel Deaconess Medical Center Inc
University of Texas System
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University of Texas System
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Definitions

  • the invention was made with government support under Grant Nos. EB003472, EB006462, CA135444, CA125550, CA155370, CA151925, DK081576, and DK055001 awarded by the National Institutes of Health and Grant Nos. EFRI-1240410, CBET-0922876, and CBET-1144025 awarded by the National Science Foundation. The government has certain rights in the invention.
  • the present invention relates generally to the field of molecular biology, oncology and medicine. More particularly, it concerns methods for detecting cancer by their unique exosome content and methods for enhanced inhibitory RNA-based therapies.
  • Exosomes are capable of mediating such communications and achieve this across long distances (Mathivanan et al., 2010; Kahlert and Kalluri, 2013). Communication via exosomes can likely overcome the limitations associated with stability and diffusion of growth factors/cytokines/chemokines/hormones (Mathivanan et al., 2010).
  • Exosomes are nano-vesicles of 30-140 nm in size, which contain proteins, mRNA, and microRNAs (miRNAs) protected by a lipid bilayer (Cocucci et al., 2009; Simons and Raposo, 2009; Simpson et al., 2008; Thery et al., 2002).
  • mRNAs microRNAs
  • exosomes are increased in the circulation of cancer patients when compared to normal subjects (Logozzi et al., 2009; Taylor and Gercel-Taylor, 2008); however, a functional role remains unknown. Recent evidence suggests that exosomes may play an important role in cancer progression and metastasis (Luga et al., 2012; Peinado et al., 2012; Yang et al., 2011).
  • RNAi is a natural biological process within living cells that participates in the control of gene expression and activity. Extracellular miRNAs were initially only thought to be contained inside exosomes (Valadi et al., 2007).
  • miRNAs in exosomes offer the possibility of regulating gene expression of cells at distant sites (Guescini et al., 2010; Valadi et al., 2007; Mittelbrunn et al., 2011; van Balkom et al., 2013). Via their regulation of mRNA translation, miRNAs coordinate the expression of entire sets of genes and shape the organism's transcriptome (Bartel, 2009).
  • miRNAs are enriched in exosomes derived from many different cell types (Valadi et al., 2007). They are small non-coding RNAs of 18-24 nucleotides (nt) in length that control gene expression post-transcriptionally. They are synthesized via sequential actions of Drosha and Dicer endonucleases and loaded into the RISC (RNA induced silencing complex) to target mRNAs (Bartel, 2009; Maniataki and Mourelatos, 2005). In the Dicer knockout mice, failure of miRNA biosynthesis results in lethality due to defective embryonic stem cell proliferation and differentiation (Bernstein et al., 2003; Fukagawa et al., 2004).
  • RISC RNA induced silencing complex
  • MicroRNAs operate via sequence-specific interaction and pairing of the miRNA-associated RISC (composed of Dicer, TRBP and AGO2 proteins) with the target mRNAs (Bartel, 2009). This action consequently inhibits translation and/or causes mRNA destabilization (Filipowicz, 2005).
  • the degree of complementarity of the miRNA and its mRNA target dictates the process of mRNA silencing, either via mRNA destabilization/degradation or by inhibition of translation (Ambros, 2004; Bartel, 2009). If complete complementation is encountered between the miRNA and target mRNA sequence, the RISC complex acts to cleave the bound mRNA for degradation (Ambros, 2004; Bartel, 2009). If absolute complementation is not encountered, as in most cases of miRNAs in animal cells, translation is prevented to achieve gene silencing (Ambros, 2004; Bartel, 2009).
  • a miRNA For a miRNA to be functional and achieve efficient miRNA-mediated gene silencing, it must be complexed with the RLC (RISCloading complex) proteins Dicer, TRBP and AGO2. Within the RLC, Dicer and TRBP are required to process precursor miRNAs (pre-miRNAs), after they emerge from the nucleus via exportin-5, to generate miRNAs and associate with AGO2.
  • RLC RISCloading complex
  • AGO2 bound to the mature miRNA constitutes the minimal RISC and may subsequently dissociate from Dicer and TRBP (Chendrimada et al., 2005; Gregory et al., 2005; Haase et al., 2005; MacRae et al., 2008; Maniataki and Mourelatos, 2005; Melo et al., 2009).
  • Single-stranded miRNAs by themselves incorporate into RISC very poorly and therefore cannot be efficiently directed to its target mRNA for post-transcriptional regulation (Tang, 2005; Thomson et al., 2013).
  • siRNAs double-stranded cause mRNA decay through perfect base pairing with their target mRNAs (Ambros, 2004; Bartel, 2009). Such siRNAs are loaded directly into the RISC proteins Dicer, TRBP and AGO2 due to its double stranded nature (Tang, 2005). A single-stranded miRNA cannot incorporate into RISC and therefore, cannot be directed to its target mRNA for translation inhibition or degradation (Tang, 2005).
  • miRNAs contained in exosomes can influence gene expression in target cells (Ismail et al., 2013; Kogure et al., 2011; Kosaka et al., 2013; Narayanan et al., 2013; Pegtel et aL, 2010; Valadi et al., 2007; Zhang et al., 2010), but a question remains as to how efficient are these miRNAs in silencing mRNA if they are not incorporated into the RISC as pre-miRNAs for appropriate mRNA recognition and efficient arrest of translation.
  • Exosomes secreted by cancer cells are unique relative to non-cancer exosomes, the cancer exosomes comprising a unique repertoire of miRNAs as well as active RNA processing RISC complexes.
  • Such encapsulated RNA-RISC complexes could also be used for cell-independent miRNA biogenesis and highly efficient mRNA silencing in target cells.
  • the present disclosure provides a method of detecting a cancer biomarker in a subject comprising (a) obtaining a biological sample from the subject; (b) measuring the level of either (i) one or more miRNA(s) selected from the miRNAs provided in Table 5 in an exosome fraction of the sample; (ii) a precursor miRNA; (iii) a RISC protein in an exosome fraction of the sample; or (iv) a miRNA processing activity (e.g., primary miRNA and/or precursor-miRNA processing activity) in an exosome fraction of the sample; and (c) identifying the subject having or not having a cancer biomarker based on the measured level of said miRNA(s), precursor miRNA, RISC protein or miRNA processing activity.
  • the method comprises measuring the level of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 of said miRNAs.
  • the method comprises measuring the level of AGO2, TRBP, or DICER protein.
  • the biological sample is essentially free of cells.
  • the sample may have less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cell(s).
  • the biological sample does not contain cells.
  • the biological sample may be a lymph, saliva, urine or blood (e.g., plasma) sample.
  • the method my further comprise purifying an exosome fraction of the sample and/or increasing the production of an exosome fraction of the sample.
  • the cancer is a breast cancer, lung cancer, head & neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.
  • the cancer is a breast cancer.
  • the subject has previously been treated for a cancer or has previously had a tumor surgically removed.
  • identifying the subject as having or not having a cancer biomarker further comprises correlating the measured miRNA level(s), precursor miRNA level, RISC level or miRNA processing activity with a risk for cancer.
  • identifying the subject as having or not having a cancer biomarker further comprises analysis of the measured miRNA level(s), precursor miRNA level, RISC level or miRNA processing activity using an algorithm. In some cases, an analysis may be performed by a computer.
  • the method of the embodiments further comprises measuring the level of either (i) one or more miRNA(s) selected from the miRNAs provided in Table 5 in an exosome fraction of the sample and a reference sample; (ii) precursor miRNA; (iii) a RISC protein in an exosome fraction of the sample and a reference sample; or (iv) a miRNA processing activity in an exosome fraction of the sample and a reference sample; and (c) identifying the subject as having or not having a cancer biomarker by comparing the level of miRNA(s), a precursor miRNA, RISC or miRNA processing activity in the sample from the subject to the level of miRNA(s), a precursor miRNA, RISC miRNA processing activity in the reference sample.
  • measuring RISC protein levels comprises performing a Western blot, an ELISA or binding to an antibody array.
  • measuring miRNA levels comprises measuring processed miRNA levels.
  • measuring miRNA levels comprises performing RT-PCR, Northern blot or an array hybridization.
  • the method further comprises reporting whether the subject has or does not have a cancer biomarker.
  • Reporting may comprise preparing a written, oral or electronic report.
  • the report may be provided to the patient, a doctor, a hospital or an insurance company.
  • the present disclosure provides a method of treating a subject comprising selecting a subject identified as having a cancer biomarker in accordance with the embodiments and administering an anti-cancer therapy the subject.
  • the method can comprise (a) obtaining the level of (i) one or more miRNA(s) selected from the miRNAs provided in Table 5; (ii) a precursor miRNA, (ii) a RISC protein; or (iii) a miRNA processing activity, in an exosome fraction of a sample from the subject; (b) selecting a subject having a cancer biomarker based on the level of said miRNA(s), precursor miRNA, RISC protein or miRNA processing activity; and (c) treating the selected subject with an anti-cancer therapy.
  • the anti-cancer therapy is a chemotherapy, a radiation therapy, a hormonal therapy, a targeted therapy, an immunotherapy or a surgical therapy.
  • the present disclosure provides a method of selecting a subject for a diagnostic procedure comprising (a) obtaining the level of either (i) one or more miRNA(s) selected from the miRNAs provided in Table 5; (ii) precursor miRNA level, (iii) a RISC protein; or (iv) a miRNA processing activity, in an exosome fraction of a sample from the subject; (b) selecting a subject having a cancer biomarker based on the level of said mRNA(s), RISC protein or miRNA processing activity; and (c) performing a diagnostic procedure on the subject.
  • the diagnostic procedure comprises diagnostic imaging.
  • the imaging may be a biopsy, X-ray, CT, MRI or PET imaging.
  • the present disclosure provides a tangible computer-readable medium comprising computer-readable code that, when executed by a computer, causes the computer to perform operations comprising (a) receiving information corresponding to a level of either (i) one or more miRNA(s) selected from the miRNAs provided in Table 5; (ii) a precursor miRNA, (iii) a RISC protein; or (iv) a miRNA processing activity, in an exosome fraction of a sample from the subject; and (b) determining a relative level of one ore more of said miRNAs, precursor miRNA, RISC proteins or a miRNA processing activity compared to a reference level, wherein altered level compared to a reference level indicates that the subject has a cancer biomarker.
  • the operation of the tangible computer-readable medium further comprises receiving information corresponding to a reference level of (i) one or more miRNA(s) selected from the miRNAs provided in Table 5; (ii) a a precursor miRNA; (iii) a RISC protein; or (iv) a miRNA processing activity, in an exosome fraction of a subject no having a cancer.
  • the tangible computer-readable medium further comprises computer-readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising: sending information corresponding to the relative level of miRNA; a precursor miRNA, RISC protein or miRNA processing activity, to a tangible data storage device.
  • receiving information comprises receiving from a tangible data storage device information corresponding to a level of miRNA; a precursor miRNA level, RISC protein or miRNA processing activity, in a sample from a subject. In some aspects, receiving information further comprises receiving information corresponding to a level of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of said miRNAs in a sample from a subject.
  • the computer-readable code when executed by a computer, causes the computer to perform operations further comprising (c) calculating a diagnostic score for the sample, wherein the diagnostic score is indicative of the probability that the sample is from a subject having a cancer.
  • the present disclosure provides a method of detecting cancer biomarker in a subject comprising (a) obtaining a biological sample from the subject; (b) measuring the level of one or more miRNA(s) in the sample selected from the miRNAs provided in Table 5 or a precursor miRNA thereof; and (c) identifying the subject having or not having a cancer biomarker based on the measured level of said miRNA(s).
  • the biological sample is essentially free of cells.
  • the biological sample may be a lymph, saliva, urine or plasma sample.
  • the method my further comprise purifying an exosome fraction of a body fluid.
  • the present disclosure provides a method for delivery of active inhibitory RNA comprising contacting a cell with an inhibitory RNA that is provided in association with a RISC protein complex.
  • the RISC protein complex comprises TRBP, DICER and AGO2.
  • the inhibitory RNA is a siRNA or shRNA.
  • the inhibitory RNA is a human miRNA.
  • the inhibitory RNA and RISC protein complex are comprises in a liposome, a nanoparticle or a microcapsule comprising a lipid bilayer.
  • the microcapsule is an exosome.
  • a method further comprises transfecting a cell with the inhibitory RNA and RISC protein complex. In another aspect, the method further comprises administering the inhibitory RNA and RISC protein complex to a subject.
  • the present disclosure provides a composition comprising a recombinant or synthetic inhibitory RNA in association with a RISC protein complex, said complex comprised in a liposome, a nanoparticle or a microcapsule.
  • the RISC protein complex comprises TRBP, DICER and AGO2.
  • the inhibitory RNA is a siRNA or shRNA.
  • the inhibitory RNA is a human miRNA.
  • the complex is comprised in a synthetic liposome, a nanoparticle or a microcapsule.
  • the microcapsule is an exosome.
  • a method can comprising measuring a level of one or more miRNA selected from the group consisting of mmu-miR-709, hsa-miR-1308, mmu-miR-615-3p, hsa-miR-1260b, mmu-miR-1937a, mmu-mir-321-A, hsa-miR-615-3p, hsa-miR-1979, mmu-miR-1937b, hsa-mir-373, mmu-miR-1937c, hsa-miR-1273d-P, mmu-miR-720, mmu-miR-1274a, hsa-mir-565-A, mmu-miR-1931, hsa-m
  • FIGS. 1A-F Characterization of Exosomes—Oncosomes are enriched in oncogenic miRNAs compared to normosomes.
  • A Transmission electron micrograph of oncosomes (upper left photo and lower left photo and inset zoom; doted lines depict the zoom area). Lower right images produced by immunogold labeling using anti-CD9 antibody and transmission electron microscopy. Gold particles are depicted as black dots.
  • Graph represents the average size of exosomes preparations analyzed from 112 TEM pictures.
  • C Immunoblot using anti-Dicer antibody in exosomes harvested from: non-tumorigenic mouse (NMuMG) and human (MCF10A) cell lines (left blot, first panel); mouse cancer cell lines, 67NR and 4T1 (middle blot, first panel); human cancer cell lines MCF7 and MDA-MB231 (right blot, first panel).
  • NMuMG non-tumorigenic mouse
  • MCF10A human
  • Controls used were: exosomes treated with TritonX followed by proteinase K (Triton+PK), to induce lysis of exosomes and subsequent degradation of exosomal proteins; exosomes treated with proteinase K to degrade extra-exosomal proteins (PK); supernatant after ultracentrifugation to harvest exosomes (Supernatant).
  • TSG101 second row
  • CD9 third row
  • E Sizing exosomes with Light Scattering Spectroscopy (LSS). Calibration of the system was done using signals from phosphate buffered saline (PBS) suspensions of glass microspheres with nominal diameters of 24 nm and 100 nm and polystyrene microspheres with nominal diameters of 119 nm, 175 nm, 356 nm and 457 nm. The experimental spectra and resulting fits are shown in the left graph for glass microspheres with nominal diameter of 100 nm and polystyrene microspheres with nominal diameter of 356 nm. Right graph represents the size measurement of a PBS suspension of cancer exosomes.
  • PBS phosphate buffered saline
  • FIGS. 2A-F Oncosomes become enriched in miRNAs.
  • A Correlation graph of expressed miRNAs in MDA-MB231 exosomes and MCF10A exosomes.
  • B Correlation graphs between miRNAs in cells and respective exosomes using 6 of the differentially expressed miRNAs between normosomes and oncosomes (miR-10a, miR-10b, miR-21, miR-27a, miR-155, and miR-373) after 72 h of cell-free culture.
  • C Normosomes and oncosomes were resuspended in DMEM media and maintained in cell-free culture for 24 and 72 h.
  • exosomes were recovered and 15 miRNAs (see Table 4) were quantified by qPCR.
  • the fold-change of each miRNA in exosomes after 72 h cell-free culture was quantified relative to the same miRNA in exosomes after 24 h cell-free culture.
  • the graphical plots represent an average of fold-change for the tumor suppressor (TS) and oncogenic (ONC) miRNAs in exosomes harvested after 72 h compared to those harvested after 24 h.
  • TS tumor suppressor
  • ONC oncogenic miRNAs in exosomes harvested after 72 h compared to those harvested after 24 h.
  • D Northern blots of miR-10b and miR-21 from normosomes after 24 and 72 h of cell-free culture and oncosomes without culture and with 24 h, 72 h and 96 h of cell-free culture.
  • the tRNAMet was used as a loading control. Quantification was done using Image J software.
  • E Correlation plots between the 15 quantified miRNAs in MCF10A, MDA-MB231 and 4T1 cells and their respective exosomes after 72 h of cell free culture. Oncosomes present low correlation values with their cell of origin (middle and right graphs) when compared to normosomes (left graph).
  • F Bioanalyzer graph representation depicted in fluorescence units (FU) per seconds (s) and gel images of exosomes RNA content of normosomes and oncosomes.
  • FIGS. 3A-E Exosomes contain pre-miRNAs.
  • A Fifteen pre-miRNAs corresponding to the mature miRNAs studied were quantified using qPCR of MCF10A and MDA-MB231 exosomes. The inverse of the ⁇ Ct value for each pre-miRNA was plotted to reflect their abundance and values are represented as ⁇ s.d.
  • B Oncosomes and normosomes were resuspended in DMEM media and maintained for 24 and 72 h in cell-free culture conditions. After 24 and 72 h exosomes were extracted once again and 15 pre-miRNAs were quantified by qPCR.
  • Graphs show fold-change of each pre-miRNA in MCF10A and MDA-MB231 exosomes after 72 h of cell-free culture relative to 24 h cell-free culture and are represented as ⁇ s.d.
  • C Northern blots of premiR-10b and pre-miR-21 using MCF10A normosomes after 24 h and 72 h of cell-free culture, and MDA-MB231 oncosomes with 0 h, 24 h, 72 h and 96 h of cell-free culture.
  • the tRNAMet was used as a loading control. Quantification was done using Image J software.
  • Oncosomes and normosomes were resuspended in DMEM media and maintained for 0 h, 24 h, 72 h and 96 h in cell-free culture conditions. Exosomes were extracted from the different time points and pre-miRNAs were quantified by qPCR. The inverse of the ⁇ Ct value for each pre-miRNA in the different time points was plotted to reflect their abundance.
  • FIGS. 4A-N Oncosomes contain RLC proteins.
  • A Immunoblot using anti-Dicer antibody in exosomes harvested from: nontumorigenic mouse (NMuMG) and human (MCF10A) cell lines; mouse cancer cell lines, 67NR and 4T1; and human cancer cell lines MCF7 and MDAMB231. Controls used were: exosomes treated with TritonX followed by proteinase K treatment (Triton+PK) to induce lysis of exosomes and subsequent degradation of exosomal proteins; and exosomes treated with proteinase K to degrade extra-exosomal proteins (PK). TSG101 (second row) and CD9 (third row) immunoblots were used to confirm presence of exosomes.
  • CD9 immunoblot was used to confirm the presence of exosomes and as a loading control (lower panel).
  • D Immunoblot for Dicer in exosomes harvested from MCF10A and MDA-MB231 cells treated with the calcium ionophore A23187 (upper panel). Exosomes extracted from untreated cells were used as control. CD9 immunoblot (lower panel) was used as control to show increased exosomes secretion.
  • E Immunoblot for Dicer in exosomes extracted from MCF10A and MDA-MB231 parental cells and cells transfected with shScramble and shDicer plasmids (upper blot).
  • CD9 immunoblot was used to show exosomes presence and as a loading control (lower blot). Immunoblot quantification was done using Image J software.
  • F Transmission electron micrographs of immunogold labeling using anti-Dicer antibody in oncosomes derived from MDAMB231shDicer cells. Gold particles are depicted as black dots. Right graph depicts quantification of gold particles in EM pictures.
  • G Immunoblot using anti-AGO2 antibody in exosomes harvested from oncosomes (MCF7 and MDA-MB231) and normosomes (MCF10A).
  • Controls used were: exosomes treated with Triton X followed by proteinase K (Triton X+PK) to induce lysis of exosomes and subsequent degradation of exosomal proteins; exosomes treated with proteinase K to degrade extra-exosomal proteins (PK); and supernatant after ultracentrifugation to harvest exosomes (Supernatant).
  • TSG101 second row
  • CD9 third row immunoblots were used to confirm the presence of exosomes.
  • H Immunoblot using anti-TRBP antibody in exosomes harvested from oncosomes (MCF7 and MDA-MB231) and normosomes (MCF10A).
  • TSG101 second row
  • CD9 third row
  • immunoblots were used as exosomes markers.
  • I Immunoblot using anti-GFP antibody in MCF10A and MDA-MB231 cells transfected with GFP-AGO2 plasmid (upper panel). Beta actin was used as loading control (lower panel).
  • J Immunoblot using anti-GFP antibody in exosomes extracted from MCF10A and MDA-MB231 cells transfected with GFP-AGO2 plasmid (upper panel). TSG101 (middle panel) and CD9 (lower panel) were used as exosomes markers and loading controls.
  • K AGO2 mRNA expression in MCF10A and MDA-MB231 cells transfected with siAGO2. MCF10A and MDA-MB231 parental cells were used as relative controls for fold change comparison. Data are the result of three biological replicates and are represented as SD.
  • FIGS. 5A-E Oncosomes process pre-miRNAs to generate mature miRNAs.
  • A Exosomes were harvested from MCF10A, MCF10A shScramble, MCF10A shDicer cells (upper graph), MDA-MB231, MDA-MB231 shScramble and MDA-MB231 shDicer cells (lower graph) and maintained under cell-free culture conditions for 24 and 72 h. After 24 and 72 h exosomes were recovered and 15 pre-miRNAs were quantified by qPCR. Graphs show the fold-change of each pre-miRNA in the different exosomes after 72 h of cell-free culture relative to 24 h cell-free culture and are represented as ⁇ s.d.
  • Exosomes were harvested from MCF10A, MCF10A shScramble, MCF10A shDicer cells (upper graph), MDA-MB231, MDA-MB231 shScramble and MDA-MB231 shDicer cells (lower graph) and maintained under cell-free culture conditions for 24 and 72 h. After 24 and 72 h exosomes were extracted once again and 15 miRNAs were quantified by qPCR. Graphs show the fold-change of each miRNA in the different exosomes after 72 h of cell-free culture relative to 24 h cell-free culture and are represented as ⁇ s.d.
  • C Immunoblot using antirabbit and anti-mouse secondary antibody to detect heavy chain (HC) and light chain (LC) primary Dicer antibody and primary Actin antibody electroporated in exosomes of MDA-MB231 cells. Electroporated exosomes without antibody derived from MDA-MB231 cells were used as negative control. Proteinase K treatments were performed after electroporation to ensure depletion of antibodies not included in exosomes.
  • D Oncosomes (MDA-MB231) were harvested in duplicate (bottom graph) or quadruplicate (top graph). Samples were electroporated with anti-Dicer antibody, anti-actin antibody, or anti-TRBP antibody. The samples plus control were left in cell-free culture conditions for 24 and 72 h.
  • FIGS. 6A-F Oncosomes process pre-miRNAs to generate mature miRNAs.
  • A Exosomes from MDA-MB231 cells were harvested and electroporated with Geldanamycin. The samples were left in cell-free culture conditions for 24 and 72 h, after which exosomes were extracted and the 6 miRNAs were quantified by qPCR. The fold-change of each miRNA in exosomes after 72 h cell-free culture was quantified relative to the same miRNA in exosomes after 24 h cell-free culture in each sample. The graphical plots represent the average fold-change for the miRNAs in 72 h exosomes relative to 24 h exosomes and are represented as ⁇ s.d.
  • C Synthetic pre-miRNAs-10b, -21 and -cel-1 were electroporated into exosomes harvested from MCF10A (MCF10A electrop.), MCF10AshDicer (MCF10AshDicer electrop.), MDA-MB231 (MDA-MB231 electrop.) and MDAMB231shDicer (MDA-MB231shDicer electrop.) cells. Exosomes were recovered after cell-free culture conditions for 72 h. MiR-10b, -21 and -cel-1 were quantified by qPCR before and after 72 h of electroporation and culture.
  • Each bar on the plots show the fold-change of miR-10b, -21 and -cel-1 72 h after electroporation relative to 0 h (top graphs) or 24 h (bottom graph) after electroporation and are represented as ⁇ s.d.
  • MCF10A and MDA-MB231 exosomes electroporated in the absence of pre-miRNAs were used as controls to determined basal levels.
  • D Northern blot without detection probe, using samples from dicing assay. Different exosomal protein extracts and synthetic pre-miR-10b internally labeled with biotin were used for the dicing assay.
  • FIGS. 7A-H Oncosomes induce transcriptome alterations in recipient cells and tumor formation in a Dicer-dependent manner.
  • A Immunoblot using anti-PTEN antibody and protein extracts of MCF10A cells treated for 0, 30 min, 1 h, 12 h and 24 h with MDA-MB231 oncosomes after cell-free culture. Beta actin was used as a loading control.
  • B Immunoblot using anti-HOXD10 antibody and protein extracts of MCF10A cells treated for 0, 30 min, 1 h, 12 h and 24 h with MDA-MB231 oncosomes after cell-free culture conditions. Beta actin was used as a loading control.
  • (C) Graph showing luciferase reporter activity in MCF10A cells transiently transfected with 3′UTR-PTEN-WT, 3′UTRPTEN-Mut, 3′UTR-HOXD10-WT and 3′UTR-HOXD10-Mut and treated with oncosomes derived from MDA-MB231 cells.
  • (F) Cell viability measured by MTT assay during 5 days of culture of MCF10A cells, MCF10A cells treated with MDA-MB231 exosomes with no cell-free culture time (MCF10A+MDA231 exos), MCF10A cells treated with MDA-MB231 exosomes with cell-free culture time (MCF10A cells+MDA231 exos culture) and MCF10A cells treated with MDA-MB231 exosomes electroporated with Dicer antibody with cell-free culture time (MCF10A cells+MDA231 exos Dicer AB) and are represented as ⁇ s.d. * p 0.0027.
  • the colony formation assay shows formation of colonies in culture plate and labeled with MTT reagent after 8 days MCF10A cells culture, MCF10A cells treated with MDA-MB231 exosomes with no cell-free culture time (MCF10A+MDA231 exos), MCF10A cells treated with MDA-MB231 exosomes with cell-free culture time (MCF10A cells+MDA231 exos culture) and MCF10A cells treated with MDA-MB231 exosomes electroporated with Dicer antibody with cell-free culture time (MCF10A cells+MDA231 exos Dicer AB).
  • Bottom graph MCF10A cells, MDA-MB231 cells and MCF10A cells exposed to oncosomes (MDA-MB231) were orthotopically injected in the mammary pads of athymic nude mice.
  • Graph depicts tumor volume with respect to time.
  • FIGS. 8A-I Serum from breast cancer patients contain Dicer and process pre-miRNAs.
  • A Immunoblot using anti-Dicer antibody, that recognizes human and mouse Dicer, and protein extracts from serum exosomes harvested from mice xenografted with human tumors (as shown in FIG. 18A ).
  • OVA1-5 represents human ovary xenografts;
  • END1-3 represents human endometrial xenografts;
  • BRST1 and 2 represent human breast xenografts.
  • 4T1 exosomes and cells were used as controls for murine Dicer.
  • hsa-Dicer represents human Dicer molecular weight and mmu-Dicer represents murine Dicer molecular weight.
  • FIG. 18D See FIG. 18D for Comassie staining of membranes as loading control.
  • B NanoSight particle tracking analysis showing size distribution of exosomes extracted from the serum of 8 healthy donors (left graph) and 11 breast cancer patients (right graph). Concentration of samples was standardized to better show size.
  • C Transmission electron micrograph of exosomes harvested from the serum of breast cancer patients.
  • E Exosomes were harvested from fresh serum from 8 healthy donors and 11 breast cancer patients. The extracted samples were left in cell-free culture conditions for 24 and 72 h.
  • exosomes were recovered and 6 pre-miRNAs were quantified by qPCR.
  • the fold-change of each pre-miRNA in exosomes after 72 h cell-free culture was quantified relative to the same pre-miRNA in exosomes after 24 h cell-free culture in each sample.
  • the graphical dot plots represent an average foldchange for the pre-miRNAs in 72 h exosomes relative to 24 h exosomes and are represented as ⁇ s.d.
  • Exosomes were harvested from fresh serum from 8 healthy donors and 11 breast cancer patients. The extracted samples were left in cell-free culture conditions for 24 and 72 h.
  • MCF10A cells MCF10A cells mixed with exosomes from healthy donors (H1-8) and MCF10A cells mixed with exosomes from breast cancer patients (BC1-11) were orthotopically injected into the mammary pads of athymic nude mice. The number of exosomes used was calculated per body weight reflecting the initial concentration collected from the serum. Samples that have not formed a tumor appear overlapped in the xaxis of the graph. This graph depicts tumor volume with respect to time and is represented as ⁇ s.d.
  • FIGS. 9A-B Dicer is present in multivesicular bodies and cytoplasmic CD43 mobilizes Dicer into exosomes.
  • A Immunoblot of CD43 in protein extracts of MDA-MB231 cells immunoprecipitated with Dicer antibody (IP Dicer) or with IgG (upper panel, right and middle lanes, respectively). Dicer alone immunoblot was used as control (lower panel).
  • B Immunoblot of Dicer in protein extracts of MDA-MB231 derived exosomes and MDA-MB231 siCD43 derived exosomes. CD9 immunoblot was used as a loading control. Quantification was done using Image J software.
  • FIGS. 10A-E Exosomes characterization.
  • A Photograph of PKH26 stained exosomes, at the bottom of the ultracentrifugation tube. Inset represents digital zoom image of the exosomes.
  • B Schematic representation of experimental system used to collect LSS spectra.
  • C Cell viability measured by MTT assay during 5 days of culture of MCF10A, NMuMG, MDA-MB231 and 4T1 cells.
  • D Flow cytometry analysis for propidium iodide (PI) and Anexin V of MDA-MB231 and 4T1 cells. MDA-MB231 cells treated with etoposide were used as a positive control for apoptosis.
  • PI propidium iodide
  • FIGS. 11A-E Oncosomes are enriched in miRNAs compared to normosomes.
  • A Bioanalyzer graphical representation depicted in fluorescence units (FU) per nucleotides (nt) (graphs) and gel images (right image) of the RNA content of human mammary MCF10A (non-tumorigenic) and MDA-MB231 (breast cancer) cell lines.
  • B Exosomes harvested from 4T1, MCF10A and MDA-MB231 cells were resuspended in DMEM media and maintained in cell-free culture conditions for 24 and 72 h. After 24 and 72 h exosomes were recovered and 15 miRNAs (see Table 4) were quantified by qPCR.
  • Graphs show fold change of each miRNA in oncosomes after cell-free culture for 24 h (upper graphs) and 72 h (lower graphs) relative to normosomes after 24 and 72 h of cell-free culture, respectively. Data represented are the result of three biological replicates and are represented as SD.
  • C Fifteen mature miRNAs (see Table 4) were quantified by qPCR in MCF10A (left graph), MDA-MB231 (middle graph) and 4T1 (right graph) cells and their respective exosomes. The fold change of each miRNA in exosomes was quantified relative to the same miRNA in cells.
  • TS tumor suppressor miRNAs
  • ONC oncogenic miRNAs.
  • Data are the result of three biological replicates and are represented as SD.
  • D Exosomes harvested from MCF10A, MDA-MB231 and 4T1 cells, were resuspended in DMEM media and maintained for 24 and 72 h in cell-free culture conditions. After 24 and 72 h exosomes were extracted once again and 15 miRNAs (see Table 4) were quantified by qPCR. The fold change of each miRNA in exosomes after 72 h cell-free culture was quantified relative to the same miRNA in exosomes after 24 h cell-free culture. Data corresponds to detailed graphs of the fold change average graphs in FIG. 2C .
  • FIGS. 12A-E Exosomes contain pre-miRNAs.
  • A Fifteen pre-miRNAs corresponding to the mature miRNAs previously quantified (see Table 4) were quantified by qPCR in NMuMG and 4T1 exosomes. The inverse of the ⁇ Ct value for each pre-miRNA was plotted to reflect their abundance. Data are the result of three biological replicates and are represented as ⁇ s.d.
  • Graphs show fold change of each pre-miRNA in NMuMG and 4T1 exosomes after 72 h of cell-free culture relative to 24 h cell-free culture. Data are the result of three biological replicates and are represented as SD.
  • C XPOS mRNA expression in MDAMB231 cells with two transiently transfected siRNAs targeting XPOS compared as a fold change to control cells.
  • D MDA-MB231 cells were transfected with XPOS siRNA constructs and miR-21 expression was assessed at several time points 12 h post-transfection (0 h, 6 h, 12 h, 24 h, 36 h, 48 h, 72 h and 96 h).
  • MDA-MB231 cells transfected with XPOS siRNA constructs were centrifuged at 4° C. for 3 h and put back in culture.
  • MiR-21 expression was assessed at several time points postcentrifugation (0 h, 6 h, 12 h, 24 h, 36 h, 48 h, 72 h and 96 h). Processing of premiR21 to miR-21 is delayed in centrifuged cells (green bar).
  • the presented data in this figure are the result of three independent experiments, each with three replicates and are represented as ⁇ s.d.
  • Exosomes harvested from NMuMG and 4T1 cells were resuspended in DMEM media and maintained for 0, 24, 72 and 96 h in cell-free culture conditions. Exosomes were extracted from the different time points and pre-miRNAs were quantified by qPCR. The inverse of the ⁇ Ct value for each pre-miRNA in the different time points was plotted to reflect their abundance. Data are the result of three biological replicates and are represented as SD.
  • FIGS. 13A-H Oncosomes contain Dicer.
  • A Transmission electron micrograph image produced by immunogold labeling using anti-Dicer antibody (right photos) and negative control (left photos) in MCF10A cells-derived exosomes. Compare with FIG. 4B for positive immunogold labeling of MDA-MB231 exosomes.
  • B Transmission electron micrograph image produced by immunogold labeling using anti-GFP antibody MDA-MB231-derived exosomes.
  • C Immunoblot using anti-flag antibody (upper panel) in MCF10A and MDAMB231 cells transfected with empty vector (pCMV-Tag4B; first and third lanes respectively) and Flag-Dicer vector (second and fourth lanes).
  • Beta actin immunoblot was used as a loading control (lower panel).
  • Beta actin immunoblot was used as a loading control (lower panel).
  • E Immunoblot using anti-Dicer antibody (upper panel) in MDA-MB231, MDA-MB231shScramble and MDA-MB231shDicer clones 1 and 2, respectively (MDA-MB231shDicer clone1 and MDA-MB231shDicer clone2) cells. Beta actin immunoblot was used as a loading control (lower panel). Immunoblots quantification was done using Image J software.
  • F Immunoblot using AGO2 antibody in exosomal proteins extracted from MCF10A and MDA-MB231 cells immunoprecipitated with Dicer antibody or IgG (upper panel).
  • FIGS. 14A-F Dicer detection in exosomes.
  • A Immunoblot using anti-Dicer antibody in 4T1, 4T1shScramble and 4T1shDicer cells and exosomes harvested from 4T1 (4T1 exos) and 4T1shDicer (4T1shDicer exos) cells (upper blot). GADPH immunoblot was used as loading control (lower blot). Quantification was done using Image J software.
  • Graphs show fold change of each miRNA in the different exosomes after 72 h of cell-free culture relative to 24 h cell-free culture. Data are the result of three biological replicates and are represented as SD.
  • D Exosomes were harvested from MDA-MB231 cells in duplicate. One of the samples was electroporated with anti-Dicer antibody. Both samples were left in cell-free culture conditions for 24 and 72 h. After 24 and 72 h exosomes were extracted once again and 15 pre-miRNAs (see Table 4) were quantified by qPCR. The fold change of each pre-miRNA in exosomes after 72 h cell-free culture was quantified relative to the same pre-miRNA in exosomes after 24 h cell-free culture in each sample.
  • the graphical plots represent fold change of pre-miRNAs in 72 h exosomes relative to 24 h exosomes and are a detailed analysis of graph represented in FIG. 5D . Data are the result of three biological replicates and are represented as SD.
  • E Exosomes were harvested from MDA-MB231 cells in duplicate. One of the samples was electroporated with anti-Dicer antibody. Both samples were left in cell-free culture conditions for 24 and 72 h. After 24 and 72 h exosomes were extracted once again and 15 miRNAs (see Table 4) were quantified by qPCR.
  • the fold change of each miRNA in exosomes after 72 h cell-free culture was quantified relative to the same miRNA in exosomes after 24 h cell-free culture in each sample.
  • the graphical plots represent fold change of miRNAs in 72 h exosomes relative to 24 h exosomes and are a detailed analysis of graph represented in FIG. 5E . Data are the result of three biological replicates and are represented as SD.
  • FIGS. 15A-C Dicer detection in exosomes.
  • A Exosomes were harvested from MCF10, MCF10AshDicer, MDA-MB231 and MDA-MB231shDicer cells and electroporated with synthetic pre-miRNA-10b, -21 and -cel-1. Each pre-miRNA was quantified by qPCR in the electroporated exosomes and represented as a fold change relative to exosomes that were electroporated with electroporation buffer only.
  • B Dot blot of biotin internally labeled pre-miR-21, -10b and -cel-1.
  • FIGS. 16A-I Dicer is present in multivesicular bodies and cytoplasmic CD43 mobilizes Dicer into exosomes.
  • A Graph represents the percentage of colocalization in the confocal images as quantified using image J software.
  • B Hrs, TSG101 and BiG2 mRNA expression after down regulation using two different siRNAs for Hrs and TSG101 and two different sh clones for BiG2. Non-transfected and shScramble transfected cells were used as control.
  • C Protein quantification by Bradford assay of exosomes extracted from MCF10A, MCF10AsiHrs, MDA-MB231 and MDA-MB231siHrs (left graph), MCF10shScramble, MCF10AshBiG2, MDA-MB231shScramble, MDA-MB231shBiG2 (middle graph) and MCF10AsiTSG101 and MDA-MB231siTSG101 (right graph). Parental non-transfected cells were used as relative controls for fold change analysis. Data was normalized by cell number and is the result of three biological replicates represented as SD.
  • MCF10A and MDA-MB231 parental cells were used as relative control for fold change comparison.
  • Data are the result of three biological replicates and are represented as SD.
  • MCF10A and MDA-MB231 parental cells were used as relative control for fold change comparison.
  • Data are the result of three biological replicates and are represented as SD
  • FIGS. 17A-G Oncosomes induce transcriptome alterations in receiving cells and tumor formation in a Dicer-dependent manner.
  • A NanoSight particle tracking analysis of exosomes derived from MDA-MB231 CD63-GFP cells. Black line represents a measure of total exosomes population and green line depicts the population of exosomes that is labeled with CD63-GFP using the NanoSight equipped with a 488 nm laser beam. Light gray and light green represent the error bars of each measure.
  • B Immunoblot using anti-PTEN antibody and protein extracts of MCF10A cells treated for 0, 30 min, 1 h, 12 h and 24 h with MDA-MB231 oncosomes freshly extracted.
  • Beta actin was used as a loading control.
  • C Immunoblot using anti-HOXD10 antibody and protein extracts of MCF10A cells treated for 0, 30 min, 1 h, 12 h and 24 h with MDA-MB231 oncosomes freshly extracted. Beta actin was used as a loading control.
  • D MCF10A cells were transfected with siRNA for XPOS to down regulate the flow of pre-miRNAs into the cytoplasm from the nucleus.
  • pre-miR15 The processing of pre-miR15 was assessed measuring the levels of miR-15 over time (6 h, 12 h, 24 h, 36 h and 48 h) in MCF10AsiXPO5 cells and MCF10AsiXPO5 cells treated with MDA-MB231 exosomes with and without Dicer antibody. No significant changes were denoted.
  • E miR182-5p expression was monitored in MDA-MB231 derived exosomes over time (0 h, 6 h, 12 h, 24 h, 36 h, 48 h, 72 h and 96 h). Each bar represents the fold change of each time point compared to 0 h. No significant differences were noted.
  • F Graph provides colony number quantification of FIG. 7G .
  • FIGS. 18A-D Breast cancer patient-exosomes contain Dicer, process pre-miRNAs and enter cells in different organs.
  • A Representative photos from orthotopic xenografts derived from fragments of fresh primary human ovary, endometrial and breast tumors in nude mice.
  • B Hematoxylin-eosin (HE) staining of ovary, endometrial and breast cancer orthotopic xenografts.
  • C Transmission electron micrograph of serum exosomes harvested from mice with orthotopic tumor xenografts.
  • D Comassie staining of membranes of immunoblots depicted in FIG. 8A .
  • Exosomes are nano-vesicles secreted by all cell types and contain proteins and nucleic acids. Exosomes secreted by cancer cells specifically contain microRNAs (miRNAs) associated with the RNA Induced Silencing Complex (RISC; Dicer/TRBP/AGO2) and possess cell autonomous capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs.
  • miRNAs microRNAs
  • RISC RNA Induced Silencing Complex
  • pre-miRNAs precursor microRNAs
  • RISC proteins in cancer cells are specifically directed into multivesicular bodies (MVBs) and subsequently into exosomes in a CD43-dependent manner.
  • RISC-incorporated miRNAs of exosomes stimulate non-tumorigenic epithelial cells to form tumors via specific induction of oncogenic pathways and activate stromal fibroblasts.
  • This study unravels the possible role of cancer exosomes in inducing oncogenic “field effect” that further subjugates normal cells to participate in cancer development and progression.
  • miRNA biogenesis can occur in a cell-independent manner in exosomes, which offers new opportunities to engineer efficient miRNA-mediated targeted therapy for a myriad of diseases.
  • Tumors contain cancer cells and stromal elements (Tse and Kalluri, 2011). Emerging evidence suggests that communication between cells of the tumors and their surroundings also determine the rate and intensity of systemic spread in cancer (Luga et al., 2012). Some studies suggest that primary tumors can educate and prepare secondary tumor sites for future metastasis via cancer cell secreted factors (Hood et al., 2011; Peinado et al., 2012).
  • mediators include soluble growth factors, glucose metabolites, chemokines, enzymes, microparticles, microvesicles, exosomes and free nucleic acids (Guermonprez et al., 2002; Luga et al., 2012; Peinado et al., 2012; Simons and Raposo, 2009; Thery and Casas, 2002).
  • single-stranded miRNAs are highly inefficient in silencing target mRNAs without RISC incorporation to facilitate mRNA recognition.
  • Proteins of the RLC recognize the pre-miRNA and process it into a 22-nucleotide RNA duplex. AGO2 selects one strand for subsequent gene silencing while the other strand is often degraded. The overall reaction is spontaneous and does not require any factors beyond the three proteins and the incorporated pre-miRNA (Maniataki and Mourelatos, 2005). Therefore, for a miRNA to be fully functional it needs RLC-incorporated processing of its pre-miRNA and AGO-mediated mRNA recognition and silencing.
  • Oncosomes specifically contain Dicer, TRBP and AGO2 as a functional complex with an ability to process pre-miRNAs to miRNAs.
  • the pre-miRNAs were present in all exosomes but only processed in the oncosomes due to the presence of RLC.
  • cancer cells overexpress miRNAs with oncogenic potential, such as miR-21 and miR-155, which provide them with a proliferative and survival advantage and are associated with advanced clinical stage, metastasis and poor prognosis (Yan et al., 2008). It has also been previously reported that these miRNAs are overexpressed in the circulation of cancer patients (Mao et al., 2013). The synthesis of miRNAs in cells is an enzymatic reaction and therefore depends on the amount of key enzymes, such as Dicer, present in their cytoplasm. Dicer has been described as down regulated in breast cancer cells and tumors (Grelier et aL, 2009; Martello et al., 2010).
  • miRNAs While certain miRNAs are up regulated in specific tumors (Volinia et al., 2006), a global reduction of miRNA is also reported to occur in human cancers (Kumar et al., 2007; Lu et al., 2005; Melo et al., 2011; Melo et al., 2010; Melo et al., 2009; Ozen et al., 2008). Dicer is described as suppressed in cancer cells but low levels are sufficient to sustain tumor growth (Kumar et al., 2009). Partial Dicer down regulation via miR-103/107 enhances cancer cell invasiveness without affecting cell proliferation (Martello et al., 2010).
  • Dicer protein Down regulation of Hrs, BiG2 and TSG101, components of the exosomal biogenesis pathway, led to dramatic changes in the cellular localization of Dicer protein.
  • One possible explanation for suppressed Dicer levels in cancer cells may be due to active export via exosomes. If exosomes secretion pathway is shut down, cancer cells sense the increase in Dicer protein and down regulate their mRNA expression. In addition, they shuttle the protein into the nuclear compartment, were it can no longer aid in the production of mature miRNAs. In this regard, Dicer up-regulation in aggressive cancer cells makes them more indolent (Park et al., 2011).
  • CD43 is transmembrane protein that is predominantly present in leukocytes. In some cancer cells, a truncated CD43 is observed in the cytoplasm and nucleus (Shelley at al. 2012). It has been previously shown that CD43 could target certain membrane proteins to exosomes (Shen et al., 2011a). Suppression of CD43 in a mouse model of orthotopic breast cancer reduces tumor burden by 76% (Shelley et al., 2012). Clinical studies suggest that CD43 expression correlates with poor survival of breast cancer patients (de Laurentiis et al., 2011). This report identifies that CD43 is functionally involved in directing Dicer into oncosomes.
  • Oncosomes mediate significant transcriptome alterations in target cells via RISC-associated miRNAs.
  • a myriad of biological process are affected in the target cells, inducing proliferation and converting non-tumorigenic cell into tumor-forming cells. Nonetheless, the potential in vivo effect of oncosomes on recipient cells likely depends on several other environmental parameters and accessibility barriers.
  • Oncosomes also activate stromal fibroblasts to acquire a myofibroblasts phenotype.
  • the capacity of oncosomes to silence tumor suppressors PTEN and HOXD10 via oncosomes derived miR-21 and miR-10b, respectively, were illustrated (Ma et al., 2007; Maehama, 2007). These results highlight the complex nature of communication adopted by cancer cells to achieve malignancy. These data illustrate that cancer cells can use exosomes to manipulate surrounding normal cells to accelerate cancer progression and recruit reactive stroma.
  • biomarkers or genes may be measured by a variety of techniques that are well known in the art. Quantifying the levels of the messenger RNA (mRNA) of a biomarker may be used to measure the expression of the biomarker. Alternatively, quantifying the levels of the protein product of a biomarker may be used to measure the expression of the biomarker. Additional information regarding the methods discussed below may be found in Ausubel et al. (2003) or Sambrook et al. (1989). One skilled in the art will know which parameters may be manipulated to optimize detection of the mRNA or protein of interest.
  • mRNA messenger RNA
  • said obtaining expression information may comprise RNA quantification, e.g., cDNA microarray, quantitative RT-PCR, in situ hybridization, Northern blotting or nuclease protection.
  • Said obtaining expression information may comprise protein quantification, e.g., protein quantification comprises immunohistochemistry, an ELISA, a radioimmunoassay (RIA), an immunoradiometric assay, a fluoroimmunoassay, a chemiluminescent assay, a bioluminescent assay, a gel electrophoresis, a Western blot analysis, a mass spectrometry analysis, or a protein microarray.
  • RNA quantification e.g., cDNA microarray, quantitative RT-PCR, in situ hybridization, Northern blotting or nuclease protection.
  • Said obtaining expression information may comprise protein quantification, e.g., protein quantification comprises immunohistochemistry, an ELISA, a radioimmunoa
  • a nucleic acid microarray may be used to quantify the differential expression of a plurality of biomarkers.
  • Microarray analysis may be performed using commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GeneChip® technology (Santa Clara, Calif.) or the Microarray System from Incyte (Fremont, Calif.).
  • single-stranded nucleic acids e.g., cDNAs or oligonucleotides
  • the arrayed sequences are then hybridized with specific nucleic acid probes from the cells of interest.
  • Fluorescently labeled cDNA probes may be generated through incorporation of fluorescently labeled deoxynucleotides by reverse transcription of RNA extracted from the cells of interest.
  • the RNA may be amplified by in vitro transcription and labeled with a marker, such as biotin.
  • the labeled probes are then hybridized to the immobilized nucleic acids on the microchip under highly stringent conditions. After stringent washing to remove the non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera.
  • the raw fluorescence intensity data in the hybridization files are generally preprocessed with the robust multichip average (RMA) algorithm to generate expression values.
  • RMA robust multichip average
  • Quantitative real-time PCR may also be used to measure the differential expression of a plurality of biomarkers.
  • the RNA template is generally reverse transcribed into cDNA, which is then amplified via a PCR reaction.
  • the amount of PCR product is followed cycle-by-cycle in real time, which allows for determination of the initial concentrations of mRNA.
  • the reaction may be performed in the presence of a fluorescent dye, such as SYBR Green, which binds to double-stranded DNA.
  • the reaction may also be performed with a fluorescent reporter probe that is specific for the DNA being amplified.
  • a non-limiting example of a fluorescent reporter probe is a TaqMan® probe (Applied Biosystems, Foster City, Calif.).
  • the fluorescent reporter probe fluoresces when the quencher is removed during the PCR extension cycle.
  • Multiplex qRT-PCR may be performed by using multiple gene-specific reporter probes, each of which contains a different fluorophore. Fluorescence values are recorded during each cycle and represent the amount of product amplified to that point in the amplification reaction. To minimize errors and reduce any sample-to-sample variation, qRT-PCR may be performed using a reference standard. The ideal reference standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment.
  • Suitable reference standards include, but are not limited to, mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and ⁇ -actin.
  • GPDH glyceraldehyde-3-phosphate-dehydrogenase
  • ⁇ -actin glyceraldehyde-3-phosphate-dehydrogenase
  • the level of mRNA in the original sample or the fold change in expression of each biomarker may be determined using calculations well known in the art.
  • Immunohistochemical staining may also be used to measure the differential expression of a plurality of biomarkers.
  • This method enables the localization of a protein in the cells of a tissue section by interaction of the protein with a specific antibody.
  • the tissue may be fixed in formaldehyde or another suitable fixative, embedded in wax or plastic, and cut into thin sections (from about 0.1 mm to several mm thick) using a microtome.
  • the tissue may be frozen and cut into thin sections using a cryostat.
  • the sections of tissue may be arrayed onto and affixed to a solid surface (i.e., a tissue microarray).
  • the sections of tissue are incubated with a primary antibody against the antigen of interest, followed by washes to remove the unbound antibodies.
  • the primary antibody may be coupled to a detection system, or the primary antibody may be detected with a secondary antibody that is coupled to a detection system.
  • the detection system may be a fluorophore or it may be an enzyme, such as horseradish peroxidase or alkaline phosphatase, which can convert a substrate into a colorimetric, fluorescent, or chemiluminescent product.
  • the stained tissue sections are generally scanned under a microscope. Because a sample of tissue from a subject with cancer may be heterogeneous, i.e., some cells may be normal and other cells may be cancerous, the percentage of positively stained cells in the tissue may be determined. This measurement, along with a quantification of the intensity of staining, may be used to generate an expression value for the biomarker.
  • An enzyme-linked immunosorbent assay may be used to measure the differential expression of a plurality of biomarkers.
  • an ELISA assay There are many variations of an ELISA assay. All are based on the immobilization of an antigen or antibody on a solid surface, generally a microtiter plate.
  • the original ELISA method comprises preparing a sample containing the biomarker proteins of interest, coating the wells of a microtiter plate with the sample, incubating each well with a primary antibody that recognizes a specific antigen, washing away the unbound antibody, and then detecting the antibody-antigen complexes.
  • the antibody-antibody complexes may be detected directly.
  • the primary antibodies are conjugated to a detection system, such as an enzyme that produces a detectable product.
  • the antibody-antibody complexes may be detected indirectly.
  • the primary antibody is detected by a secondary antibody that is conjugated to a detection system, as described above.
  • the microtiter plate is then scanned and the raw intensity data may be converted into expression values using means known in the art.
  • An antibody microarray may also be used to measure the differential expression of a plurality of biomarkers.
  • a plurality of antibodies is arrayed and covalently attached to the surface of the microarray or biochip.
  • a protein extract containing the biomarker proteins of interest is generally labeled with a fluorescent dye or biotin.
  • the labeled biomarker proteins are incubated with the antibody microarray. After washes to remove the unbound proteins, the microarray is scanned.
  • the raw fluorescent intensity data may be converted into expression values using means known in the art.
  • Luminex multiplexing microspheres may also be used to measure the differential expression of a plurality of biomarkers.
  • These microscopic polystyrene beads are internally color-coded with fluorescent dyes, such that each bead has a unique spectral signature (of which there are up to 100). Beads with the same signature are tagged with a specific oligonucleotide or specific antibody that will bind the target of interest (i.e., biomarker mRNA or protein, respectively).
  • the target is also tagged with a fluorescent reporter.
  • there are two sources of color one from the bead and the other from the reporter molecule on the target.
  • the beads are then incubated with the sample containing the targets, of which up to 100 may be detected in one well.
  • the small size/surface area of the beads and the three dimensional exposure of the beads to the targets allows for nearly solution-phase kinetics during the binding reaction.
  • the captured targets are detected by high-tech fluidics based upon flow cytometry in which lasers excite the internal dyes that identify each bead and also any reporter dye captured during the assay.
  • the data from the acquisition files may be converted into expression values using means known in the art.
  • In situ hybridization may also be used to measure the differential expression of a plurality of biomarkers.
  • This method permits the localization of mRNAs of interest in the cells of a tissue section.
  • the tissue may be frozen, or fixed and embedded, and then cut into thin sections, which are arrayed and affixed on a solid surface.
  • the tissue sections are incubated with a labeled antisense probe that will hybridize with an mRNA of interest.
  • the hybridization and washing steps are generally performed under highly stringent conditions.
  • the probe may be labeled with a fluorophore or a small tag (such as biotin or digoxigenin) that may be detected by another protein or antibody, such that the labeled hybrid may be detected and visualized under a microscope.
  • each antisense probe may be detected simultaneously, provided each antisense probe has a distinguishable label.
  • the hybridized tissue array is generally scanned under a microscope. Because a sample of tissue from a subject with cancer may be heterogeneous, i.e., some cells may be normal and other cells may be cancerous, the percentage of positively stained cells in the tissue may be determined. This measurement, along with a quantification of the intensity of staining, may be used to generate an expression value for each biomarker.
  • the marker level may be compared to the level of the marker from a control, wherein the control may comprise one or more tumor samples taken from one or more patients determined as having a certain metastatic tumor or not having a certain metastatic tumor, or both.
  • the control may comprise data obtained at the same time (e.g., in the same hybridization experiment) as the patient's individual data, or may be a stored value or set of values, e.g., stored on a computer, or on computer-readable media. If the latter is used, new patient data for the selected marker(s), obtained from initial or follow-up samples, can be compared to the stored data for the same marker(s) without the need for additional control experiments.
  • obtaining a biological sample or “obtaining a blood sample” refer to receiving a biological or blood sample, e.g., either directly or indirectly.
  • the biological sample such as a blood sample or a sample containing peripheral blood mononuclear cells (PBMC)
  • PBMC peripheral blood mononuclear cells
  • the biological sample may be drawn or taken by a third party and then transferred, e.g., to a separate entity or location for analysis.
  • the sample may be obtained and tested in the same location using a point-of care test.
  • said obtaining refers to receiving the sample, e.g., from the patient, from a laboratory, from a doctor's office, from the mail, courier, or post office, etc.
  • the method may further comprise reporting the determination to the subject, a health care payer, an attending clinician, a pharmacist, a pharmacy benefits manager, or any person that the determination may be of interest.
  • subject or “patient” is meant any single subject for which therapy or diagnostic test is desired. This case the subjects or patients generally refer to humans. Also intended to be included as a subject are any subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects used as controls.
  • “increased expression” refers to an elevated or increased level of expression in a cancer sample relative to a suitable control (e.g., a non-cancerous tissue or cell sample, a reference standard), wherein the elevation or increase in the level of gene expression is statistically significant (p ⁇ 0.05). Whether an increase in the expression of a gene in a cancer sample relative to a control is statistically significant can be determined using an appropriate t-test (e.g., one-sample t-test, two-sample t-test, Welch's t-test) or other statistical test known to those of skill in the art.
  • Genes that are overexpressed in a cancer can be, for example, genes that are known, or have been previously determined, to be overexpressed in a cancer.
  • “decreased expression” refers to a reduced or decreased level of expression in a cancer sample relative to a suitable control (e.g., a non-cancerous tissue or cell sample, a reference standard), wherein the reduction or decrease in the level of gene expression is statistically significant (p ⁇ 0.05).
  • the reduced or decreased level of gene expression can be a complete absence of gene expression, or an expression level of zero. Whether a decrease in the expression of a gene in a cancer sample relative to a control is statistically significant can be determined using an appropriate t-test (e.g., one-sample t-test, two-sample t-test, Welch's t-test) or other statistical test known to those of skill in the art.
  • Genes that are underexpressed in a cancer can be, for example, genes that are known, or have been previously determined, to be underexpressed in a cancer.
  • antigen binding fragment herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments.
  • primer is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • Primers may be oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form is preferred.
  • Exosomes were purified by differential centrifugation as described previously (Thery et al., 2006; Luga et al., 2012). In short, supernatant from cells cultured for 24 hr were subjected to sequential centrifugation steps of 800 g and 2000 g and supernatant was filtered using 0.2 ⁇ m filter in culture bottles. Exosomes were pelleted at 100,000 g in an SW40Ti swinging bucket rotor for 2 hr (Beckman). Supernatant was discarded and PBS was added for a 1 hr-washing step. The pellet was analyzed for exosome.
  • Exosomes for RNA extraction were resuspended in 500 ul of Trizol; exosomes for protein extraction were resuspended in 250 ul of lysis buffer (8M Urea/2.5% SDS, 5 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin and 1 mM phenylmethylsulphonyl fluoride); and exosomes for treatments were resuspended in PBS. Frozen serum samples were thawed on ice and 500 ⁇ l were added to 12 mL PBS and the same aforementioned procedure was followed.
  • Exosomes purified by centrifugation were treated (37° C., 60 minutes) with 500 g/mL proteinase K (Sigma-Aldrich) dissolved in RNase-free water, followed by heat inactivation of the protease (60° C., 10 minutes) and incubation (37° C., 15 minutes) with 2 g/mL protease-free RNaseA (Sigma-Aldrich) followed by addition of 10 ⁇ concentrated RNase inhibitor (Ambion).
  • exosomes treatment exosomes were purified in duplicate and one of the pellets was used for protein quantification.
  • Exosomes preparations (5-10 ⁇ g) were incubated with 5 ⁇ l of 4- ⁇ m-diameter aldehyde/sulfate latex beads (Interfacial Dynamics, Portland, Oreg.) and resuspended into 400 ⁇ l PBS containing 2% BSA.
  • Exosomes-coated beads (20 ⁇ l) were incubated with the following antibodies: anti-CD63 (Santa Cruz), anti-CD9 (abcam), anti-TSG101 (abcam), anti-flotillin-1 (Santa Cruz) for 30 minutes at 4° C. followed, when needed, by incubation with FITC-conjugated secondary antibody and analyzed on a FACS Calibur flow cytometer (BD Biosciences).
  • Exosomes at a total protein concentration of 100 ⁇ g (measured by Bradford Assay) and 5 ⁇ g of Dicer antibody (polyclonal SC-30226, Santa Cruz, Calif.), 5 ug of Actin antibody, or 10 ⁇ g of pre-miRNA-21, -10b and -cel1 were mixed in 400 ⁇ l of electroporation buffer (1.15 mM potassium phosphate pH 7.2, 25 mM potassium chloride, 21% Optiprep) and electroporated in a 4 mm cuvette using a Gene Pulser Xcell Electorporation System (Biorad) as described previously (Alvarez-Erviti et al., 2011). After electroporation, exosome were treated with proteinase K and/or RNAse when appropriate.
  • LSS Light Scattering Spectroscopy
  • LSS spectra were collected using the experimental system described in FIG. 10B .
  • the Fianium SC-450-2 broadband supercontinuum laser was used as a source of white light.
  • the light from the supercontinuum laser was focused into the sample with a long focus length lens.
  • the samples consisting of liquid suspensions of either exosomes or microspheres were placed in a custom cubic-shaped quartz sample holder.
  • the background signals were collected from the solvent samples with no exosomes or microspheres.
  • the light scattered by exosomes or microspheres at 90° to the incident beam was collected with the other long focus length lens and delivered to the Princiton Instrument Acton 2300i imaging spectrograph coupled with a high efficiency Andor Technology iXon DV885 EMCCD detector.
  • the detection was performed in the 470-870-nm wavelength range.
  • the detector was controlled by a computer, into which the data were transferred, stored, and processed.
  • the signals from from from phosphate buffered saline (PBS) suspensions of glass microspheres with nominal diameters of 24 nm and 100 nm and polystyrene microspheres with nominal diameters of 119 nm, 175 nm, 356 nm and 457 nm were measured.
  • the spectra predicted by Mie theory were fitted to the data using the previously developed least-squares minimization method (Fang et al., 2003).
  • the experimental spectra and resulting fits are shown in FIG. 1E for glass microspheres with nominal diameter of 100 nm and polystyrene microspheres with nominal diameter of 356 nm.
  • N—Rh-PE Advanti Polar Lipids, Alabaster, Ala.
  • N—Rh-PE Advanti Polar Lipids, Alabaster, Ala.
  • ice-cold 1 ⁇ Hanks buffer Invitrogen, Carlsbad, Calif.
  • N—Rh-PE cells were used for confocal imaging approximately 24 hr after labeling.
  • Grids were rinsed with PBS and then floated on drops of the appropriate secondary antibody attached with 10 nm gold particles (AURION, Hatfield, Pa.) for 2 hours at room temperature.
  • Grids were rinsed with PBS and were placed in 2.5% Glutaraldehyde in 0.1M Phosphate buffer for 15 minutes. After rinsing in PBS and distilled water the grids were allowed to dry and stained for contrast with uranyl acetate.
  • the samples were viewed with a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro, Oreg.) and images taken with an AMT CCD Camera (Advanced Microscopy Techniques, Danvers, Mass.).
  • blots were blocked for 1 hr at RT with 5% non-fat dry milk in PBS/0.05% Tween and incubated overnight at 4° C. with the following primary antibodies: 1:500 anti-Dicer (SC-30226) Santa Cruz; 1:1000 anti-Ubiquitinylated proteins, clone FK2 Millipore; 1:500 anti-Flag M2-Peroxidase Clone M2 Sigma; 1:500 anti-CD43 ab9088 Abcam; 1:500 anti-PTEN, ab32199, Abcam; 1:300 anti-CD9 ab92726, Abcam; 1:500 anti-GADPH ab9483, Abcam; 1:250 anti-TRBP ab72110, Abcam; 1:300 anti-TSG101 ab83, Abcam; 1:400 anti-AGO2 ab32381, Abcam; 1:4000 anti-3-actin Peroxidase Clone AC-15, Sigma; 1:500 anti-GFP ab6556, Abcam; 1:500 anti-HOXD10 ab
  • Real-time PCR for mRNAs was performed on an ABI PRISM 7300HT Sequence Detection System Instrument using SYBR Green Master Mix (Applied Biosystems) and 3-actin as the control. The primers are listed in Table 1.
  • Pre-miRNAs were quantified using 150 ng of DNase treated RNA and the SuperScript III Platinum One-Step RT-qPCR kit (Invitrogen) (Schmittgen et al., 2004). The primers are listed in Table 1.
  • RNA was mixed with TaqMan MicroRNA Reverse Transcription Kit reagent containing specific miRNA primers and reverse-transcribed according to the manufacturer's instructions (Applied Biosystems). Reaction mixes were incubated at 16° C. for 30 minutes, 42° C. for 30 minutes and 85° C. for 5 minutes. Real-time PCR was performed using ABI PRISM 7300HT Sequence Detection System Instrument (Applied Biosystems) using commercially available Assay-on-Demand for each miRNA studied (Applied Biosystems). Expression of miRNAs was normalized to the expression of 18S rRNA (TaqMan Pre-Developed Assay Reagent; Applied Biosystems) that served as internal control for the RNA amount and integrity.
  • Applied Biosystems ABI PRISM 7300HT Sequence Detection System Instrument
  • Threshold cycle the fractional cycle number at which the amount of amplified target reached a fixed threshold, was determined and expression was measured using the 2 ⁇ Ct formula, as previously reported (Livak and Schmittgen, 2001).
  • Northern blot was performed using 3′ Bio[TEG] DNA oligonucleotides of the reverse compliment to the mature miRNA as probes (see Table 2).
  • Urea/acrylamide 15% gels were used to load 40 ⁇ g of exosomal RNA (DNase treated) together with 1 ⁇ RNA loading dye after 2 minutes at 95° C. followed by a 2 minutes period on ice.
  • MicroRNA marker was used according to manufacturer's instructions (N2102, New England BioLabs).
  • Electrophoresis was done at 4° C. during 3 hr using TBE 1 ⁇ . Transfer was done using Whatman blotting papers and the BrightStar-Plus Positively Charged Nylon Membrane (Ambion) during 2 hr at 4° C. with TBE 0.5 ⁇ .
  • RNA was cross-linked to the membrane using a UV transilluminator for 20 minutes.
  • Membranes were pre-hybridized by rotating for 1 hr at 42° C. in Ambion's ULTRAhyb®-Oligo hybridization solution (Ambion). The probes were thawed on ice and 150 ⁇ g were added per mL of hybridization buffer after 5 minutes incubation at 95° C., after which membranes were left in rotation overnight at 42° C. The following washes were done: 2 ⁇ SSPE/0.5% SDS—twice for 15 minutes; 0.2 SSPE/0.5% SDS—twice for 30 minutes and 2 ⁇ SSPE—5 minutes.
  • MCF10A, MCF7, MDA-MB231, A549, SW480 and HeLa human cell lines as well as NMuMG, 67NR and 4T1 mouse mammary cell lines were cultured in DMEM 10% FBS (all cells are originated from the American Type Culture Collection—ATCC). Transfections were performed using Lipofectamine 2000 reagent (Invitrogen) for siRNA. For synthetic pre-miRNA transfections RNAiFect (Qiagen) was used in all cell lines. Sequences of siRNAs are listed in Table 3.
  • p-CMV-Tag4B-Dicer (Melo et al., 2009); p-CMV6-CD63-GFP from Origene (RG217238); GFP-hAGO2 from Addgene (plasmid 11590); pGFP-shBiG2 from Origene (TG314697); pGFP-shDicer from Origene (TG304991); synthetic pre-miR-10b, -21 and -cel-1 were purchased from Ambion; 3′UTR-WTPTEN, 3′UTR-Mutant-PTEN (Dr. Joshua Mendell laboratory), 3′UTR-WTHOXD10 and 3′UTR-Mutant-HOXD10 (Dr. Robert Weinberg laboratory) are from Addgene.
  • PBST PBS, 0.1% Triton
  • BSA 1:100 anti-Dicer (SC-30226) Santa Cruz; 1:500 anti-Flag Sigma; 1:50 anti-CD43 ab9088 (Abcam); 1:100 anti-TSG101 ab83 (Abcam); 1:500 anti-GFP ab6556 (Abcam); 1:100 anti-LAPM-1 ab25630 (Abcam); 1:100 anti-Hrs ab56468 (Abcam); 1:100 anti-BiG2 ab75001 (Abcam); 1:500 anti-biotin ab66233 (Abcam).
  • Exosomes were plated on top of coverslips in 12 well plates in 4% PFA for 45 min and washed with cold PBS. Images were obtained with a Zeiss LSM510 Upright Confocal System using the recycle tool to maintain identical settings. Aggregated exosomes lead to structures larger than 200 nm visible in confocal microscopy. For data analysis, images were selected from a pool drawn from at least two independent experiments. Figures show representative fields.
  • Exosomal protein extracts (10 ⁇ g) were incubated at 37° C. with 3 pmol of pre-miR-10b, -21 and -cel-1 biotin-internally labeled hairpins in the presence of 3 mM MgCl 2 , 30 mM NaCl and 100 mM Hepes, pH 7.5. The final volume of each reaction was 10 ⁇ l. Reactions were stopped by the addition of 10 ⁇ l of formamide gel loading buffer. RNA was resolved using denaturating polyacrylamide gel electrophoresis and developed with the BrightStar BioDetect Kit according to the manufacturer's instructions (Ambion).
  • Cells were plated in 96 well plates and harvested exosomes were added at day 1 at a concentration of 100 ⁇ g/mL. Cell viability was determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. For colony formation experiments, cells were plated in 12 well plates and exosomes were added at day 1 and day 5 of culture at a concentration of 100 ⁇ g/mL. After 8 days colonies were fixed and stained with MTT reagent.
  • MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
  • a custom miRNA array was used as described in9.
  • the array contains 1833 human microRNA probes, 1084 mouse microRNA probes and other 78 noncoding RNAs probes.
  • the probes are printed in duplicate.
  • GenBank accession ID associated with each probe is included.
  • Bioinformatic analysis was performed using R (version 2.14.2) (on the world wide web at r-project.org) and Bioconductor (on the world wide web at bioconductor.org/).
  • the raw intensity for each probe is the median feature pixel intensity with the median background subtracted. Setting an offset 1 ensures that there will be no negative values after log-transforming data. Data was quantile normalized followed by log 2 transform. Signals from probes measuring same miRNA were averaged.
  • the analysis was performed using the functions of LIMMA library.
  • the heatmaps were generated using the heatplot function of made4 library. When technical replicates were performed, the heatmap represented the average expression values obtained from replicate measurements.
  • mice Female athymic nu/nu mice (Harlan) between 4 to 6 weeks of age were housed in individually ventilated cages on a 12-hour light-dark cycle at 21 to 23° C. and 40% to 60% humidity. Mice were allowed free access to an irradiated diet and sterilized water. All animal protocols were reviewed and approved according to the Spanish Institutional Animal Care and Use Committees.
  • Non-necrotic tissue pieces (ca. 2-3 mm 3 ) from five representative resected human epithelial ovarian tumor (EOCs): serous, endometrioid, clear cell tumor and mucinous, were selected and placed in DMEM (BioWhittaker) supplemented with 10% FBS and penicillin/streptomycin at room temperature.
  • EOCs human epithelial ovarian tumor
  • Orthotopically engrafted tumors were allowed to grow and at the time of sacrifice 2 ml of blood were obtained from anesthesized mice by cardiac punction. Samples were centrifugated at 14,000 rpm and frozen at ⁇ 80° C.
  • a bead/slurry mix (100 n1) was added to 1 mL of cell lysate and 500 ⁇ l of exosomal lysate and incubated at 4° C. for 10 min. Beads were removed by centrifugation at 14,000 ⁇ g at 4° C. for 10 minutes and pellets discarded. Dicer antibody (5 ⁇ g for cells and 10 ⁇ g for exosomes) was added to 500 ⁇ l of cell lysate or 250 ⁇ l of exosomal lysate (1 ⁇ g/ ⁇ l cells, 10 ⁇ g/ ⁇ l exosomes) and incubated overnight at 4° C. on an orbital shaker.
  • Cells (8 ⁇ 10 7 cells) were seeded at 5 ⁇ 10 5 cells/ml in DMEM. To treat the cells, A23187 (200 nM final concentration, Calbiochem, La Jolla, Calif.) was added to the cultures four hours later. Media from treated and non-treated cells was harvested and exosomes collected.
  • Orthotopic tumor growth was measured by injecting MCF10A non-tumorigenic breast epithelial cells, MCF10A non-tumorigenic breast epithelial cells exposed to MDA-MB231-derived exosomes and MDA-MB-231 breast cancer cells (1 ⁇ 10 5 cells in 0.2 ml PBS) into the mammary fat pad of 3-week-old female athymic nude mice, as described previously (Welch, 1997). Tumor growth was monitored weekly by measuring the tumor length and width with a caliper and was reported as the mean tumor diameter as previously described (Welch, 1997). All animals were euthanized 21 days post tumor cell injection.
  • Exosomes from cancer cells (MDA-MB231 triple negative human metastatic breast carcinoma, MCF7 human breast adenocarcinoma, 67NR mouse non-metastatic breast carcinoma and 4T1 mouse metastatic breast carcinoma) and control cells (MCF10A non-tumorigenic human epithelial breast and NMuMG non-tumorigenic mouse epithelial breast) were isolated using established ultracentrifugation methods ( FIG. 10A ) (Luga et al., 2012; Thery et al., 2006). The harvested exosomes were analyzed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Particles between 40-140 nm in size were identified ( FIGS.
  • TEM transmission electron microscopy
  • AFM atomic force microscopy
  • LSS Light Scattering Spectroscopy
  • the NanoSight nanoparticle tracking analysis revealed particles with a size distribution peaking at 105 ⁇ 1.0 nm in diameter ( FIG. 1F ) further excluding the existence of potential contaminants of different size ranges that exist in solution when it is not filtered ( FIG. 1F , right graph).
  • Colorimetric cell viability assay MTT
  • terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay assay
  • flow cytometry analysis for Anexin V and propidium iodide cytochrome C immunoblots of exosomes ( FIGS.
  • Exosomes isolated from cancer cells are collectively termed as oncosomes, as defined previously (Lee et al., 2011).
  • Exosomes isolated from control cells are collectively termed as normosomes.
  • Oncosomes are Specifically Enriched in Oncogenic miRNAs when Compared to Normosomes.
  • the global miRNA content of oncosomes and normosomes were investigated. Microfluidics analysis of RNA isolated from exosomes revealed an increase in the small RNA content of oncosomes when compared to normosomes ( FIG. 2F ). Furthermore, a low correlation between the levels of miRNAs in normosomes (MCF10A-derived) and oncosomes (MDA-MB-231-derived) was observed, with an R 2 value of 0.35 ( FIG. 2A ). Global miRNA array analysis showed an enrichment of miRNAs content in oncosomes when compared with normosomes. This analysis also revealed a very distinct miRNA expression profile in oncosomes when compared to normosomes.
  • the miRNA array data showed 305 differentially expressed miRNAs between oncosomes and normosomes (Table 5), with an overall enrichment of miRNA content in oncosomes when compared with normosomes. Enrichment of miRNAs in oncosomes was not a mere reflection of an increase in miRNAs in the cancer cells because the cancer cells showed a decrease in the overall amount of total small RNAs when compared to non-tumorigenic cells ( FIG. 11A ). Therefore, accumulation of miRNAs in exosomes appeared to be specific and targeted.
  • the tumor-suppressive pre-miRNAs did not show any difference in oncosomes or normosomes ( FIG. 3B and FIG. 12B ). Moreover, decreasing amounts of oncogenic pre-miRNAs in oncosomes, but not in normosomes, was noted after 96 h of culture, at which point the oncogenic pre-miRNA levels reached the levels of tumor-suppressive pre-miRNAs ( FIG. 3E and FIG. 12E ). Down regulation of oncogenic pre-miRNAs in oncosomes was confirmed by Northern blotting for pre-miR10b and pre-miR21 ( FIG. 3C ). Next, a time-course analysis of pre-miRNAs and miRNAs in exosomes was performed.
  • XPOS is responsible for the transport of pre-miRNAs from the nucleus to the cytoplasm (Yi et al., 2003). Silencing XPOS prevents the flow of pre-miRNAs from the nucleus to the cytoplasm and allows for an evaluation of cytoplasmic premiRNA processing without the introduction of new cytoplasmic pre-miRNA from the nucleus.
  • MicroRNA-21 was monitored in MDA-MB-231siXPO5 cells before and after centrifugation ( FIGS.
  • MicroRNA biogenesis requires key protein components of the RLC, Dicer, TRBP and AGO2 (Chendrimada et al., 2005). It has been previously shown that Dicer and TRBP form a complex that provides stability to Dicer protein, while AGO2 is recruited later in the biogenesis pathway to help with strand selection and the RNA unwinding process (Chendrimada et al., 2005). Dicer protein was detected in oncosomes derived from MCF7, MDA-MB231, 67NR and 4T1 cancer cells ( FIG.
  • FIG. 1C and FIGS. 4A-B The possibility of detecting contaminating extra-exosomal Dicer protein was removed by treating all exosomes preparations with proteinase K before exosomal protein extraction as previously described (Montecalvo et al., 2012) ( FIG. 1C and FIGS. 4A-B ).
  • various cancer cell lines such as A549 (human lung cancer), SW480 (human colorectal cancer), HeLa (human cervical cancer) and 4T07 (mouse breast cancer) also produce Dicer-containing exosomes ( FIG. 13H ).
  • Dicer protein was further overexpressed with an N-terminal Flag tag in MCF10A and MDA-MB231 cells ( FIG. 13C ) Immunoblotting and confocal microscopy further confirmed the presence of the Flag-Dicer protein specifically in oncosomes and not normosomes ( FIG. 4C ). Increasing intracellular Ca2+ levels stimulates exosomes secretion (Savina et al., 2003). Ca 2+ ionophore A23187 was added to the culture media of MCF10A and MDA-MB231 cells and exosomes were collected. We observed a significant increase in exosomes production as judged by CD9 expression ( FIG. 4D ).
  • FIGS. 4G-H RLC proteins, AGO2 and TRBP, were also detected in oncosomes but not in normosomes.
  • Exosomes were extracted from MCF10A and MDA-MB231 cells transfected with a GFP tagged AGO2 ( FIG. 41 ).
  • an anti-GFP antibody the presence of GFP-AGO2 was detected in exosomes extracted from MDA-MB231-GFP-AGO2 cells ( FIG. 4J ).
  • siRNA silencing of AGO2 in MCF10A and MDA-MB231 cells Upon siRNA silencing of AGO2 in MCF10A and MDA-MB231 cells, a down regulation of AGO2 protein in MDA-MB231 derived oncosomes was observed ( FIGS. 4K-L ).
  • Oncosomes Use RLC to Process Pre-miRNAs to Generate Mature miRNAs.
  • RLC proteins the dicing and silencing properties
  • oncosomes were extracted from the MCF10AshDicer, MDA-MB231shDicer and 4T1shDicer cells ( FIG. 14A ).
  • Pre-miRNAs and miRNAs content did not reveal any significant changes in the Dicer down regulated exosomes with time, indicating that the pre-miRNAs were not processed to generate miRNA in absence of Dicer in oncosomes ( FIGS. 5A-B and FIGS. 14B-C ).
  • anti-Dicer and anti-TRBP antibodies was inserted into exosomes by electroporation and compared to oncosomes and normosomes electroporated with an anti-actin control antibody treated with proteinase K after electroporation to avoid the presence of antibodies outside exosomes ( FIG. 5C ).
  • Oncosomes electroporated with the control anti-actin antibody showed the same variations in pre-miRNA and mirNA levels as previously mentioned ( FIGS. 5D-E and FIGS. 14D-E ).
  • FIGS. 5D-e and FIGS. 14D-E In oncosomes with anti-Dicer and anti-TRBP antibodies, insignificant changes in levels of pre-miRNA and miRNA were observed with time, suggesting an inhibition of pre-miRNA processing ( FIGS. 5D-e and FIGS.
  • pre-miR-10b, -21 and -cel-1 were internally labeled with biotin-deoxythymidine (dT) and transfected them into MCF10A cells.
  • dT biotin-deoxythymidine
  • the dT-modified pre-miRNAs were processed and resulted in the generation of mature miRNAs, confirmed the labeling did not alter their processing potential ( FIGS. 15B-C ).
  • the modified pre-miRNAs were used in ‘dicing’ assays to show that Dicer containing exosomes were specifically capable of processing pre-miRNA and generate mature miRNAs ( FIGS. 6D-F ).
  • Multivesicular bodies are cellular organelles that contain endosomes that are released eventually as exosomes upon fusion with the plasma membrane (Pant et al., 2012).
  • a possible mechanism that allows the recruitment of RISC proteins into endosomes and their subsequent release into exosomes was explored.
  • Dicer associates with MVBs in cancer cells when compared to control cells was explored.
  • Hrs and BiG2 are early endosome markers and TSG101 is a marker for MVBs (Razi and Futter, 2006; Shin et al., 2004).
  • Dicer co-localized with Hrs, BiG2 and TSG101 in MDA-MB231 and 4T1 cells ( FIG. 16A ).
  • Exogenously delivered N-rhodamine-labelled phosphotidylethanolamine (NRhPE) is taken up by cells and retained within MVBs (Sherer et al., 2003).
  • Dicer staining in MDA-MB231 and 4T1 cells mostly co-localized with NRhPE in MVBs, which eventually generate exosomes.
  • FIG. 16A there was no co-localization of Dicer with Hrs, BiG2, TSG101 or NRhPE in control cells (NMuMG and MCF10A) ( FIG. 16A ).
  • Hrs and TSG101 genes were silenced using two different siRNAs, as well as BiG2 using two different shRNAS, in MDA-MB231 and MCF10A cells, and Dicer protein expression was evaluated ( FIG. 16B ). Silencing of Hrs, BiG2 and TSG101 impairs MVBs formation and led to down regulation of exosomes production ( FIGS. 16C-E ).
  • MVBs also sequester ubiquitinylated proteins for subsequent degradation by lysosomes (Luzio et al., 2009).
  • Dicer protein is not ubiquitinated and does not co-localize with LAMP-1, a widely used marker for lysosomes.
  • CD43 plasma membrane anchor proteins
  • FIG. 9A We show that Dicer immunoprecipitates with CD43 protein in MDA-MB231 cells ( FIG. 9A ).
  • CD43 is down regulated using siRNA in MCF10A and MDA-MB231 cells
  • Dicer levels significantly decrease in oncosomes ( FIGS. 9B and 16H ), with a nuclear and cytoplasmic accumulation of Dicer protein.
  • a down regulation of Dicer mRNA expression was observed in MDA-MB231siCD43 cancer cells but not in MCF10AsiCD43 non-tumorigenic cells, as also observed before with siHrs, shBiG2 and siTSG101 ( FIG. 16I ).
  • Oncosomes Alter the Transcriptome of Target Cells in a Dicer-Dependent Manner.
  • MDA-MB231 cells Cancer cells (MDA-MB231 cells) were transfected with CD63-GFP, a marker for exosomes (Escola et al., 1998).
  • the CD63-GFP MDA-MB231 cells were used to isolate GFP+ exosomes, which were subsequently incubated with MCF10A cells.
  • Exosomes from MDA-MB231-CD63-GFP were shown to be green by using NanoSight complemented with a laser beam that detects particles emitting green fluorescence ( FIG. 17A ).
  • the CD63-GFP+ oncosomes were shown to enter MCF10A cells, where they appeared in the cytoplasm.
  • miRNA expression arrays it was shown that MCF10A cells exposed to MDA-MB231 derived oncosomes acquire a new miRNA expression profile distinct from the parental MCF10A cells and resembling MDA-MB231 cells.
  • miRNA expression arrays it was shown that MCF10A cells exposed to MDA-MB-231-derived oncosomes acquire a new miRNA expression profile distinct from the parental MCF10A cells.
  • Global transcriptome profiling of MCF10A treated with oncosomes more closely resembles MDA-MB231 cells. Such significant alterations in the mRNA expression profile is reversed when MCF10A cells are exposed to MDA-MB231 oncosomes with Dicer antibody, and the expression pattern re-clusters with the parental MC10A cells.
  • miRNA-21 and -10b were up-regulated (4.6 and 2.3 fold respectively) in treated MCF10A cells, among with other oncogenic miRNAs.
  • MicroRNA-21 and -10b have been implicated in breast cancer progression, invasiveness and metastasis (Ma et al., 2007; Yan et al., 2011).
  • miR-21 and -10b were synthesized in oncosomes from their pre-miRNAs.
  • PTEN and HOXD10 are described as miR-21 and miR-10b targets and were suppressed in the expression array analysis of MCF10A cells treated with oncosomes when compared to control MCF10A cells.
  • Western blot analysis showed that PTEN and HOXD10 levels were suppressed in MCF10A cells exposed to oncosomes ( FIGS. 7A-B ).
  • MCF10A cells were transiently transfected with luciferase reporters containing the wild-type 3′UTR of PTEN or HOXD10 genes that are capable of binding miR-21 and miR-10b.
  • Mutant 3′UTR of PTEN or HOXD10 vectors were used as controls.
  • a decrease in luciferase reporter activity was seen in MCF10A cells incubated with oncosomes, confirming functional delivery of miRNAs from oncosomes to recipient cells ( FIG. 7C ).
  • PTEN and HOXD10 expression levels were evaluated at different time points. A significant decrease was detected in PTEN and HOXD10 expression immediately after treating the cells with 72 h cultured exosomes ( FIGS. 7A-B ).
  • FIGS. 17B-C MCF10A cells treated with 72 h cultured oncosomes with anti-Dicer antibody revealed an insignificant down regulation of PTEN and HOXD10 ( FIG. 7D and FIG. 17G ). Additionally, processing of miR-15 in cells, a miRNA not detected in MDA-MB231-derived oncosomes, was not altered due to treatment of MCF10A cells with MDA-MB-231 exosomes containing Dicer antibody, showing an insignificant effect of Dicer antibody in treated cells ( FIG. 17D ).
  • MiR-182-5p is one of the miRNAs up-regulated in MCF10A cells upon oncosomes treatment and Smad4, a miR-182-5p target (Hirata et al., 2012), is one of the genes down-regulated upon oncosomes treatment of these cells ( FIG. 7E ). Up-regulation of miR-182-5p in oncosomes during the culture period was not observed and pre-miR182-5p was not detected in oncosomes ( FIG. 17E ).
  • oncosomes also pack mature miRNAs without the need for processing pre-miRs. If such mature miRs are in relevant stoichiometric amounts, they may be able to regulate gene expression of recipient cells, as shown previously (Ismail et al., 2013; Kogure et al., 2011; Kosaka et al., 2013; Narayanan et al., 2013; Pegtel et al., 2010; Valadi et al., 2007; Zhang et al., 2010). However, if some mature miRNAs are not present in exosomes but their pre-miRNAs are, these can still have a biological effect on their targets since they will be processed into mature RLC associated miRNAs.
  • FIG. 7F Cell viability and proliferation of MCF10A cells treated with 72 h cultured oncosomes was increased, which was not observed when freshly isolated oncosomes were used ( FIG. 7F ). A difference was not observed when MCF10A cells were treated with MDA-MB231 derived oncosomes containing anti-Dicer antibodies ( FIG. 7F ). The same pattern holds true for the colony formation capacity of MCF10A cells treated with oncosomes ( FIGS. 7G and 17F ). MCF10A cells treated with 72 h-cultured oncosomes form colonies when compared to non-treated cells ( FIG. 7G ). Such colony formation was not observed when freshly isolated oncosomes or AB Dicer oncosomes were used ( FIG. 7G ).
  • Oncosomes Induce Tumor Formation of Non-Tumorigenic Epithelial Cells and Activate Fibroblasts.
  • MCF10A cells-oncosomes formed tumors after 21 days, as well as the control MDA-MB231 cells ( FIG. 7H ).
  • Exosomes of human tumors were examined for RISC proteins.
  • freshly isolated human primary ovarian, breast and endometrial tumor pieces were orthotopically grafted into appropriate organs of female athymic nu/nu mice ( FIGS. 18A-B ).
  • Serum exosomes from these mice were evaluated by electron microscope ( FIG. 18C ).
  • Size exclusion protein blotting of the content isolated from these exosomes demonstrated the existence of Dicer protein exclusively of human origin (hsa-Dicer) ( FIG. 8A and FIG. 18D ).
  • Protein extracts from 4T1-derived exosomes and 4T1 cells were used as controls to show Dicer of mouse origin, which exhibits a different molecular weight (mmu-Dicer) ( FIG. 8A ).
  • Oncosomes from MDA-MB231 cells were incubated with human dermal fibroblasts (HDF).
  • HDF human dermal fibroblasts
  • exosomes were isolated from 100 ⁇ l of fresh serum samples from 8 healthy individuals (H) and 11 patients with breast carcinoma (BC) ( FIG. 8B ). Lipid bilayer membranes were distinguished by electron microscopy on exosomes ( FIG. 8C ). Serum of breast cancer patients contained significantly more exosomes when compared to serum of healthy donors ( FIG. 8D ). When equal number of exosomes were placed in culture for 24 and 72 h, the 6 pre-miRNAs were found to be downregulated exclusively in breast cancer patients and their respective mature miRNAs were up-regulated after 72 h of culture, suggesting pre-miRNAs were processed into the mature form in the exosomes from fresh serum of breast cancer patients and not in the healthy controls ( FIGS. 8E-F ).
  • exosomes alone or combined with MCF10A cells were injected orthotopically in the mammary fat pad of female nu/nu mice.
  • Five out of 11 serum exosomes derived from breast cancer patients combined with MCF10A cells formed tumors while none of the healthy donor exosomes or exosomes alone, formed tumors ( FIG. 8G ).
  • exosomes that formed tumors were also shown to have the highest fold-change increase in the amount of mature miRNAs after 72 h culture ( FIGS. 8E-F ).
  • Exosomes were further isolated from a new set of serum samples obtained from 5 healthy individuals (C46, C45, C44, C43, and C41) and 4 patients with metastatic breast carcinoma (Met219, Met354, Met299 and Met356). Dicer expression in exosomes was observed only in metastatic breast carcinoma samples and not in exosomes from serum of healthy individuals ( FIG. 8H ).
  • miRNA p Value mmu-miR-709 1.30E ⁇ 06 hsa-miR-1308 3.71E ⁇ 06 mmu-miR-615-3p 9.08E ⁇ 06 hsa-miR-1260b 1.06E ⁇ 05 mmu-miR-1937a 1.36E ⁇ 05 mmu-mir-321-A 1.54E ⁇ 05 hsa-miR-615-3p 1.80E ⁇ 05 hsa-miR-1979 2.10E ⁇ 05 mmu-miR-1937b 2.72E ⁇ 05 hsa-mir-373 3.15E ⁇ 05 mmu-miR-1937c 3.28E ⁇ 05 hsa-miR-1273d-P 3.68E ⁇ 05 mmu-miR-720 4.08E ⁇ 05 mmu-miR-1274a 4.45E ⁇ 05

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