WO2019144183A1

WO2019144183A1 - Biomarkers of colorectal cancer

Info

Publication number: WO2019144183A1
Application number: PCT/AU2019/050043
Authority: WO
Inventors: Richard J. Simpson; Maoshan CHEN
Original assignee: La Trobe University
Priority date: 2018-01-23
Filing date: 2019-01-23
Publication date: 2019-08-01

Abstract

Provided herein are methods and kits for the diagnosis and prognosis of colorectal cancer based on microRNA signatures in extracellular vesicles such as exosomes and shed microvesicles. In an embodiment, a method provided herein comprises executing the step of determining the expression of one or more miRNAs disclosed herein in a biological sample obtained from the subject, wherein the biological sample comprises one or both of exosomes and shed microvesicles, wherein the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the exosomes or shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

Description

BIOMARKERS OF COLORECTAL CANCER

Technical Field

[0001] The present disclosure relates generally to methods and protocols for the diagnosis and prognosis of colorectal cancer. More particularly the methods and protocols of the present disclosure are based on microRNA signatures in extracellular vesicles.

Background of the Disclosure

[0002] Despite advances in our understanding and therapeutic treatment of many forms of cancer, cancer remains one of leading causes of death around the world, with prevalence of many cancers on the increase. Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females. Metastatic CRC is the principal cause of death.

[0003] Early detection is the single most important factor influencing outcome for CRC patients. For example, according to the 2016 NCI Surveillance, Epidemiology, and End Results (SEER) study (Siegel et al, CA Cancer J Clin 20l7;67(3): 177-93), for CRC cases diagnosed between 2006 and 2012 inclusive, the projected 5-year survival rate if all tumours were still localised when detected is -91%. However current community-based methods (e.g., annual faecal occult blood test (FOBT) and 5-yearly sigmoidoscopy) for detecting CRC are inadequate. While the FOBT is sensitive for faecal blood, it is not specific for CRC. Although FOBT in conjunction with flexible sigmoidoscopy has the potential to reduce CRC mortality, these approaches have not been found to be acceptable for large-scale population screening.

[0004] The sensitivity of currently available molecular markers of CRC, such as carcinoembryonic antigen (CEA) and the carbohydrate antigen CA19.9, is low and, coupled with the fact only a small proportion of CRCs express elevated CEA and CA19.9 levels at the time of diagnosis, they are not useful for early detection of the disease. However, CEA and CA19.9 are useful to monitor disease progression in patients after primary therapy or to detect disease recurrence at an early stage. In the context of population screening for CRC, more reliable non-invasive and specific markers that meet the criteria outlined by the Tumour Marker Utility Grading System (TMUGS) are needed to improve diagnosis and to monitor CRC progression.

[0005] In recent years there has been much interest in non-coding RNAs (ncRNAs), the best understood of which are microRNAs (miRNAs). miRNAs are a class of short, endogenous, single- stranded, non-coding RNA molecules that bind with imperfect complementarity to the 3’ untranslated regions (3’-UTRs) of target mRNAs. miRNAs are initially transcribed as long primary transcripts (pri-miRNAs or pri-miRs). These are typically processed in the nucleus by the Drosha-DGCR8 complex, producing a 60-70 nucleotide (nt) stem loop structure known as precursor miRNA (pre-miRNA). The pre-miRNA is then exported to the cytoplasm and further processed into an intermediate miRNA duplex before association with the RNA-induced silencing complex (RISC) and maturation to single stranded miRNA. Mature miRNAs interact with sites of imperfect complementarity in 3' untranslated regions (UTRs) of target mRNAs. These targeted transcripts subsequently undergo accelerated turnover and translational down regulation.

[0003] Although miRNAs represent less than 0.1% of the entire mammalian transcriptome, they can control up to two thirds of gene expression in mammalian cells. There is now overwhelming evidence that many miRNAs are dysregulated in common cancers such as those originating in the breast, lung, colon, liver, and the prostate. They are regarded as key regulators in the process of tumourigenesis and many studies have suggested the use of specific miRNAs as potential biomarkers for cancer.

[0004] Circulating miRNAs have been detected in most human bodily fluids, including plasma, serum, saliva, sweat, tears, breast milk and urine. As such miRNA levels can be readily determined using non-invasive techniques using standard techniques and methods well known to those skilled in the art. Circulating miRNAs are also extremely stable and are RNase-resistant. These characteristics make circulating miRNAs excellent candidates as biomarkers of disease. Summary of the Disclosure

[0005] In one aspect, the present disclosure provides a method for detecting colorectal cancer in a subject, the method comprising executing the step of determining the expression of one or more miRNAs in a biological sample obtained from the subject, wherein the biological sample comprises one or both of exosomes and shed microvesicles, and wherein the one or more miRNAs are selected from the group consisting of hsa-let- 7b-3p, hsa-miR-l00-5p, hsa-miR-l06b-3p, hsa-miR-l07, hsa-miR-l0a-5p, hsa-miR- l0b-5p, hsa-miR-l224-5p, hsa-miR-l246, hsa-miR-l247-3p, hsa-miR-l247-5p, hsa- miR-l25a-5p, hsa-miR-l25b-2-3p, hsa-miR-l25b-5p, hsa-miR-l266-5p, hsa-miR- l268a, hsa-miR-l268b, hsa-miR-l307-5p, hsa-miR-l39-5p, hsa-miR-l46a-5p, hsa- miR-l46b-5p, hsa-miR-l50-5p, hsa-miR-l5la-3p, hsa-miR-l5lb, hsa-miR-l8la-5p, hsa-miR-l82-5p, hsa-miR-l83-5p, hsa-miR-l9l-5p, hsa-miR-l84, hsa-miR-l92-5p, hsa-miR-l93b-3p, hsa-miR-l94-3p, hsa-miR-l97-3p, hsa-miR-203b-3p, hsa-miR-204- 5p, hsa-miR-2lO-5p, hsa-miR-2l0-3p, hsa-miR-22l-5p, hsa-miR-222-5p, hsa-miR-22- 5p, hsa-miR-27a-5p, hsa-miR-28-3p, hsa-miR-30e-3p, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, hsa-miR-324-5p, hsa-miR-339-5p, hsa-miR-342-3p, hsa- miR-3605-5p, hsa-miR-36l3-3p, hsa-miR-36l-5p, hsa-miR-365l, hsa-miR-365a-3p, hsa-miR-365b-3p, hsa-miR-3687, hsa-miR-369l-5p, hsa-miR-375, and wherein the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the exosomes or shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

[0006] Typically, the expression of the one or more miRNAs are upregulated in exosomes or shed microvesicles in the biological sample from the subject.

[0007] In a particular embodiment, the comparison between the expression of the miRNA(s) in the exosomes or shed microvesicles from the subject and the expression of corresponding miRNA(s) in the reference sample(s) is assessed by calculating the Pearson’s correlation coefficient values for the miRNA(s). Thus, in an embodiment, the statistical value derived from the expression level of the at least one miRNA is the Pearson’s correlation coefficient (PCC) value.

[0008] The colorectal cancer may be colon cancer, bowel cancer or rectal cancer. Typically the colorectal cancer is an adenocarcinoma.

[0009] In particular embodiments the biological sample obtained from the subject is a blood sample, more typically a serum or plasma sample. Typically the reference sample(s) is a blood sample, more typically a serum or plasma sample. The reference sample(s) may be derived from one or more individuals known not to have colorectal cancer.

[00010] In an exemplary embodiment, the level of expression of all of said miRNAs listed above are determined and the level of expression of said miRNAs, or a statistical value(s) derived therefrom, in the exosomes or shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

[00011] In one embodiment, the biological sample comprises exosomes, the one or more miRNAs are selected from hsa-let-7b-3p, hsa-miR-l00-5p, hsa- miR-l06b-3p, hsa-miR-l07, hsa-miR-l0a-5p, hsa-miR-l0b-5p, hsa-miR-l224-5p, hsa- miR-l246, hsa-miR-l247-3p, hsa-miR-l247-5p, hsa-miR-l25a-5p, hsa-miR-l25b-5p, hsa-miR-l266-5p, hsa-miR-l268a, hsa-miR-l268b, hsa-miR-l307-5p, hsa-miR-l39-5p, hsa-miR-l46a-5p, hsa-miR-l46b-5p, hsa-miR-l50-5p, hsa-miR-l5la-3p, hsa-miR- 15 lb, hsa-miR-l8la-5p, hsa-miR-l82-5p, hsa-miR-l83-5p, hsa-miR-l9l-5p, hsa-miR- l92-5p, hsa-miR-l93b-3p, hsa-miR-l94-3p, hsa-miR-l97-3p, hsa-miR-203b-3p, hsa- miR-204-5p, hsa-miR-2lO-5p, hsa-miR-22l-5p, hsa-miR-222-5p, hsa-miR-27a-5p, hsa- miR-28-3p, hsa-miR-30e-3p, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c, hsa-miR- 320d, hsa-miR-339-5p, hsa-miR-342-3p, hsa-miR-3605-5p, hsa-miR-36l3-3p, hsa- miR-36l-5p, hsa-miR-365l, hsa-miR-365a-3p, hsa-miR-365b-3p, hsa-miR-369l-5p, hsa-miR-375, and the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the exosomes derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

[00012] In accordance with the above embodiment, the level of expression of all of said miRNAs may be determined and the level of expression of said miRNAs, or a statistical value(s) derived therefrom, in the exosomes derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

[00013] In one embodiment, the biological sample comprises shed microvesicles, the one or more miRNAs are selected from hsa-let-7b-3p, hsa-miR-l06b-3p, hsa- miR-l07, hsa-miR-l0a-5p, hsa-miR-l0b-5p, hsa-miR-l224-5p, hsa-miR-l246, hsa- miR-l247-3p, hsa-miR-l247-5p, hsa-miR-l25a-5p, hsa-miR-l25b-2-3p, hsa-miR-l25b- 5p, hsa-miR-l268a, hsa-miR-l268b, hsa-miR-l307-5p, hsa-miR-l39-5p, hsa-miR- l46a-5p, hsa-miR-l46b-5p, hsa-miR-l50-5p, hsa-miR-l5lb, hsa-miR-l82-5p, hsa-miR- l83-5p, hsa-miR-l84, hsa-miR-l92-5p, hsa-miR-l93b-3p, hsa-miR-l97-3p, hsa-miR- 203b-3p, hsa-miR-204-5p, hsa-miR-2l0-3p, hsa-miR-222-5p, hsa-miR-22-5p, hsa-miR- 320a, hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, hsa-miR-324-5p, hsa-miR-339-5p, hsa-miR-342-3p, hsa-miR-3605-5p, hsa-miR-365l, hsa-miR-3687, hsa-miR-375, and the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject. [00014] In accordance with the above embodiment, the level of expression of all of said miRNAs may be determined and the level of expression of said miRNAs, or a statistical value(s) derived therefrom, in the shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

[00015] In one embodiment, the biological sample comprises exosomes and shed microvesicles, the one or more miRNAs are selected from hsa-let-7b-3p, hsa-miR-l5lb, hsa-miR- l06b-3p, hsa-miR-l82-5p, hsa-miR-l07, hsa-miR-l83-5p, hsa-miR-l0a-5p, hsa-miR- l92-5p, hsa-miR-l0b-5p, hsa-miR-l93b-3p, hsa-miR-l224-5p, hsa-miR-l97-3p, hsa- miR-l246, hsa-miR-203b-3p, hsa-miR-l247-3p, hsa-miR-204-5p, hsa-miR-l247-5p, hsa-miR-222-5p, hsa-miR-l25a-5p, hsa-miR-320a, hsa-miR-l25b-5p, hsa-miR-320b, hsa-miR-l268a, hsa-miR-320c, hsa-miR-l268b, hsa-miR-320d, hsa-miR-l307-5p, hsa- miR-339-5p, hsa-miR-l39-5p, hsa-miR-342-3p, hsa-miR-l46a-5p, hsa-miR-3605-5p, hsa-miR-l46b-5p, hsa-miR-365l, hsa-miR-l50-5p, hsa-miR-375, and the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the exosomes and shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

[00016] In accordance with the above embodiment, the level of expression of all of said miRNAs may be determined and the level of expression of said miRNAs, or a statistical value(s) derived therefrom, in the exosomes and shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

[00017] Also provided herein are kits for use in screening for colorectal cancer, wherein the kits comprise one or more reagents for determining the expression of one or more miRNAs as defined in the above embodiments. [00018] Also provided herein is a computer system or apparatus, configured to aid in the detection or diagnosis of colorectal cancer, wherein computer software is employed to analyze data relating to the expression of one or more miRNAs as defined in the above embodiments, and to provide a diagnostic prediction with respect to a subject. Typically, the computational software is also employed to compare said data to data relating to the expression of the one or more miRNAs in one or more cancer-free reference samples.

[00019] Also provided herein is a method for selecting a subject for treatment for colorectal cancer, the method comprising:

(a) executing a step of determining the level of expression of one or more miRNAs as defined in the above embodiments, in a biological sample derived from a subject, wherein the biological sample comprises one or both of exosomes and shed microvesicles derived from colorectal cancer cells from the subject, wherein the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the exosomes and/or shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject; and

(b) selecting a subject, identified in (a) as having colorectal cancer, for treatment for said cancer.

[00020] Also provided herein is a protocol for monitoring the efficacy of a therapeutic treatment for colorectal cancer, the protocol comprising:

(a) obtaining from a subject a first biological sample, wherein the first biological sample is obtained before or after commencement of treatment and comprises one or both of exosomes and shed microvesicles derived from colorectal cancer cells from the subject;

(b) obtaining from the same subject a second biological sample, wherein the second biological sample is obtained at a time point after commencement of treatment and after the first biological sample is obtained, and comprises one or both of exosomes and shed microvesicles derived from colorectal cancer cells from the subject;

(c) executing the step of determining the level of expression of one or more miRNAs as defined in the above embodiments in the first and second biological samples; and

(d) comparing the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the first biological sample with the level of expression of the same one or more miRNAs, or a statistical value(s) derived therefrom, in the second biological sample;

wherein a change in the level of expression, or statistical value(s) derived therefrom, between the first and second biological samples is indicative of whether or not the therapeutic treatment is effective.

[00021] The protocol may further comprise obtaining and executing steps in respect of a third or subsequent sample.

[00022] The above described protocol may also be used in the screening of candidate agents for treating the cancer.

Brief Description of the Drawings

[00023] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the following drawings.

[00024] Figure 1. Purification and characterization of sMVs and exosomes secreted from human colorectal cancer cell lines SW480 and SW620. A, A combination of differential centrifugation and density gradient ultracentrifugation (OptiPrep™) was used to purify shed microvesicles (sMVs) and exosomes (Exos) in high yield. B, Western blot analysis of EV markers. Antibodies for stereotypic exosomal markers (ALIX, TSG101, and CD9) revealed enrichment of exosomes in fractions 6-7 (buoyant density, 1.08-1.12 g/mL) (up panel); antibodies of TSG101, ALIX, KIF23 and GAPDH showed KIF23 enriched in sMVs (below panel). C, Cryo-electron microscopy was used to determine median particle diameters of purified sMVs and Exos: SW480-sMVs 266 nm, SW480-Exos - 119 nm, SW620-sMVs - 486 nm, and SW620-Exos - 127 nm (scale bar: 200 nm). Images representative of 2 biological replicates with n=l2 images obtained for each replicate; vesicle diameters determined using ImageJ software. D, NanoSight Tracking Analysis was used to calculate size distribution of sMVs and exosomes: SW480-sMVs, 339 nm; SW480-Exos, 138 nm; SW620-sMVs, 327 nm; SW620-Exos, 201 nm (representative of a single biological replicate, with 3 technical replicates performed, and data averaged and merged).

[00025] Figure 2. Analysis of cellular miRNAs in human colon cancer cell models. A, Venn diagrams of miRNAs identified in SW480/ SW620 cell lysates (biological replicates combined) and for each cell line the number of unique miRNAs that selectively distribute to secreted EVs: sMVs and Exos. B, Differential miRNA expression analysis (log₂ FC >1 or <-l, p-value <0.05, FDR <0.05) in SW480/SW620 cell lines by edgeR revealed 134 dysregulated miRNAs. Of these, 73 are miRNAs down-regulated in SW620-CL and 61 up-regulated, relative to SW480-CL; the Venn diagrams show the number of dysregulated miRNAs that selectively distribute to SW480/SW620 secreted EVs.

[00026] Figure 3. Selective packaging of SW480/SW620 cellular miRNAs into sMVs and exosomes. A, Of the 292 total SW480 cellular miRNAs detected, 227 and 222 distribute to sMVs and Exos, respectively (based on normalized expression >5 TPM); 214 miRNAs are common to both SW480-sMVs and -Exos, of which 48 are enriched according to differential miRNA expression analysis by edgeR (log2FC > 1, p- value <0.05, FDR <0.05); the bar plot below lists 32 common to sMVs and Exos (*), and 1 (#) and 15 (+) miRNAs enriched in sMVs and Exos, respectively. B, Of the 315 detected SW620 cellular miRNAs, 261 and 242 were distributed into sMVs and exosomes, respectively (based on normalized expression >5 TPM). 227 cellular miRNAs are common to both sMVs and exosomes. Differential miRNA expression analysis by edgeR (log2FC > 1, p-value <0.05, FDR <0.05) revealed 36 cellular miRNAs are selectively enriched in both EV subtypes; the bar plot below lists 16 miRNAs common to both EV types (*), and 1 (#) and 19 (+) miRNAs selectively enriched in sMVs and Exos, respectively. C, Venn diagram showing distribution of 186 miRNAs that are common to all SW480/SW620-secreted EVs into their respective cell line-derived sMVs and Exos and miRNAs identified specifically in each EV subtype. D, Venn diagram showing distribution of 13 significantly enriched (log2FC > 1, p-value <0.05, FDR <0.05) miRNAs in all SW480/SW620 EV types and miRNAs enriched specifically in each EV subtype. [00027] Figure 4. Correlation of miRNAs in SW480/SW620 cell lines and their secreted EVs with miRNAs expressed in non-tumoral colon tissues and CRC tumors. A, Pearson correlation coefficients of SW480/SW620 cellular miRNAs and dysregulated cellular miRNAs with miRNA expression profiles of normal colon tissue (11 samples) and 594 tumor samples from different pathological stages; tissue samples were obtained from TCGA. B, Volcano plot for comparing 97 EV-enriched miRNAs (from this study) in different CRC tumor stages with normal colon tissue; x-axis: log2FC, y-axis: -loglO (FDR). The vertical dash lines represent the cut-off levels of log2FCs <-l (down-regulated miRNAs in tumors, relative to normal colon tissue) or >1 (up-regulated miRNAs in tumors, relative to normal colon tissues), respectfully. The horizontal dash line shows the cut-off for FDR <0.05. C, Venn diagrams of the number of up-regulated (left) /down-regulated (right) miRNAs identified in CRC tumors of different pathological stages. D, Pearson correlation coefficients of SW480/SW620-EV miRNA profiles with miRNA expression profile of different stage colon tumors and normal colon samples.

[00028] Figure 5. Differences in PCC values between normal and tumour tissues. Margin = lowest PCC of tumour - highest PCC of normal. A. SW480-sMVs. B. SW480-Exos.

[00029] Figure 6. Box plot of PCC values of miRNA profiles of SW480 EVs (left: SW480-sMVs; right: SW480-Exos).

Detailed Description

[00030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the disclosure belongs. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information.

[00031] As used herein, the singular forms "a", "an" and "the" also include plural aspects (i.e. at least one or more than one) unless the context clearly dictates otherwise. Thus, for example, reference to "a miRNA" includes a single miRNA, as well as two or more miRNAs.

[00032] In the context of this specification, the term "about," is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[00033] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[00034] As used herein, "microRNA" or "miRNA" refers to a non-coding RNA, typically between about 18 and 25 nucleotides in length that hybridizes to and regulates the expression of a coding RNA. In certain embodiments, a miRNA is the product of cleavage of a precursor (pre-miRNA), for example by the enzyme Dicer. As used herein, "pre-miRNA" refers to a non-coding RNA having a hairpin structure, which contains a miRNA. Typically the term“pre-miRNA” refers to a precursor molecule, the processing and cleavage of which gives rise to a mature miRNA. In certain embodiments, a pre-miRNA is the product of cleavage of a pri-miR by a double- stranded RNA-specific ribonuclease.

[00035] As used herein, the term“derived from” means originates from or obtained from. The terms“derived from” and“obtained from” may be used interchangeably herein.

[00036] The term“subject” as used herein refers to mammals and includes humans, primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys), laboratory test animals (eg. mice, rabbits, rats, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (eg. foxes, kangaroos, deer). Typically, the mammal is human or a laboratory test animal. More typically, the mammal is a human.

[00037] EVs are a heterogeneous population of endogenous nano-membraneous vesicles that range in diameter from 50-1500 nm and can be classified into two broad classes based upon their protein profiles and biogenesis pathways: exosomes (approximately 50 - 120 nm diameter); and shed microvesicles (sMVs of approximately 50 - 1500 nm diameter; also referred to as microvesicles and microparticles). EVs from a variety of cancer types have been found circulating in body fluids such as blood, and the present inventors have previously described robust procedures for isolating EV subtypes from colorectal cancer cells (Mathivanan et ah, Mol Cell Proteomics 20l0;9(2): 197-208; Tauro et al, Mol Cell Proteomics 20l3;l2(3):587-598; Greening et ah, Methods Mol Biol 2015; 1295:179-209).

[00038] As exemplified herein, using RNA-Seq analysis the inventors compared the miRNA profiles of exosomes and sMVs secreted from two isogenic colorectal cell lines, primary carcinoma-derived SW480 cells and its lymph node metastatic variant SW620 cells. The comparison of the miRNA profiles of EVs secreted from SW480/SW620 cells revealed a subset of miRNAs critical in the aetiology of colorectal cancer and capable of being used as biomarkers of the disease.

[00039] Thus, the present disclosure is predicated on the inventors' findings that miRNA profiles (or signatures) in exosomes and sMVs secreted from human colorectal cancer cells can be used to predict colorectal tumours with high accuracy (approximately 96% to 100%).

[00040] The present disclosure provides, for the first time, a suite of biomarkers suitable for the rapid and early detection and diagnosis of colorectal cancers, thereby enabling appropriate treatment and patient management strategies to be put into place before progression of the disease to later stages less amenable to treatment. The present disclosure thereby also provides means of improving the prognosis of sufferers of colorectal cancer by early intervention facilitated by the employment of the biomarkers and suites of biomarkers disclosed herein. [00041] In one aspect, disclosed herein is a method for detecting colorectal cancer in a subject, the method comprising executing the step of determining the expression of one or more miRNAs in a biological sample obtained from the subject, wherein the biological sample comprises one or both of exosomes and shed microvesicles, and wherein the one or more miRNAs are selected from the group consisting of hsa-let- 7b-3p, hsa-miR-l00-5p, hsa-miR-l06b-3p, hsa-miR-l07, hsa-miR-l0a-5p, hsa-miR- l0b-5p, hsa-miR-l224-5p, hsa-miR-l246, hsa-miR-l247-3p, hsa-miR-l247-5p, hsa- miR-l25a-5p, hsa-miR-l25b-2-3p, hsa-miR-l25b-5p, hsa-miR-l266-5p, hsa-miR- l268a, hsa-miR-l268b, hsa-miR-l307-5p, hsa-miR-l39-5p, hsa-miR-l46a-5p, hsa- miR-l46b-5p, hsa-miR-l50-5p, hsa-miR-l5la-3p, hsa-miR-l5lb, hsa-miR-l8la-5p, hsa-miR-l82-5p, hsa-miR-l83-5p, hsa-miR-l9l-5p, hsa-miR-l84, hsa-miR-l92-5p, hsa-miR-l93b-3p, hsa-miR-l94-3p, hsa-miR-l97-3p, hsa-miR-203b-3p, hsa-miR-204- 5p, hsa-miR-2lO-5p, hsa-miR-2l0-3p, hsa-miR-22l-5p, hsa-miR-222-5p, hsa-miR-22- 5p, hsa-miR-27a-5p, hsa-miR-28-3p, hsa-miR-30e-3p, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, hsa-miR-324-5p, hsa-miR-339-5p, hsa-miR-342-3p, hsa- miR-3605-5p, hsa-miR-36l3-3p, hsa-miR-36l-5p, hsa-miR-365l, hsa-miR-365a-3p, hsa-miR-365b-3p, hsa-miR-3687, hsa-miR-369l-5p, hsa-miR-375, and wherein the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the exosomes and/or shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

[00042] In another aspect, disclosed herein is a method for detecting colorectal cancer in a subject, the method comprising executing the step of determining the expression of one or more miRNAs in a biological sample obtained from the subject, wherein the biological sample comprises exosomes, and wherein the one or more miRNAs are selected from hsa-let-7b-3p, hsa-miR-l00-5p, hsa-miR-l06b-3p, hsa-miR-l07, hsa-miR-l0a-5p, hsa-miR-l0b-5p, hsa-miR-l224-5p, hsa-miR-l246, hsa-miR-l247-3p, hsa-miR-l247-5p, hsa-miR-l25a-5p, hsa-miR-l25b- 5p, hsa-miR-l266-5p, hsa-miR-l268a, hsa-miR-l268b, hsa-miR-l307-5p, hsa-miR- l39-5p, hsa-miR-l46a-5p, hsa-miR-l46b-5p, hsa-miR-l50-5p, hsa-miR-l5la-3p, hsa- miR-l5lb, hsa-miR-l8la-5p, hsa-miR-l82-5p, hsa-miR-l83-5p, hsa-miR-l9l-5p, hsa- miR-l92-5p, hsa-miR-l93b-3p, hsa-miR-l94-3p, hsa-miR-l97-3p, hsa-miR-203b-3p, hsa-miR-204-5p, hsa-miR-2lO-5p, hsa-miR-22l-5p, hsa-miR-222-5p, hsa-miR-27a-5p, hsa-miR-28-3p, hsa-miR-30e-3p, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c, hsa- miR-320d, hsa-miR-339-5p, hsa-miR-342-3p, hsa-miR-3605-5p, hsa-miR-36l3-3p, hsa-miR-36l-5p, hsa-miR-365l, hsa-miR-365a-3p, hsa-miR-365b-3p, hsa-miR-369l- 5p, hsa-miR-375, and wherein the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the exosomes derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

[00043] In another aspect, disclosed herein is a method for detecting colorectal cancer in a subject, the method comprising executing the step of determining the expression of one or more miRNAs in a biological sample obtained from the subject, wherein the biological sample comprises shed microvesicles, and wherein the one or more miRNAs are selected from hsa-let-7b-3p, hsa-miR-l06b- 3p, hsa-miR-l07, hsa-miR-l0a-5p, hsa-miR-l0b-5p, hsa-miR-l224-5p, hsa-miR-l246, hsa-miR-l247-3p, hsa-miR-l247-5p, hsa-miR-l25a-5p, hsa-miR-l25b-2-3p, hsa-miR- l25b-5p, hsa-miR-l268a, hsa-miR-l268b, hsa-miR-l307-5p, hsa-miR-l39-5p, hsa- miR-l46a-5p, hsa-miR-l46b-5p, hsa-miR-l50-5p, hsa-miR-l5lb, hsa-miR-l82-5p, hsa- miR-l83-5p, hsa-miR-l84, hsa-miR-l92-5p, hsa-miR-l93b-3p, hsa-miR-l97-3p, hsa- miR-203b-3p, hsa-miR-204-5p, hsa-miR-2l0-3p, hsa-miR-222-5p, hsa-miR-22-5p, hsa- miR-320a, hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, hsa-miR-324-5p, hsa-miR- 339-5p, hsa-miR-342-3p, hsa-miR-3605-5p, hsa-miR-365l, hsa-miR-3687, hsa-miR- 375, and wherein the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

[00044] In another aspect, disclosed herein is a method for detecting colorectal cancer in a subject, the method comprising executing the step of determining the expression of one or more miRNAs in a biological sample obtained from the subject, wherein the biological sample comprises exosomes and shed microvesicles, and wherein the one or more miRNAs are selected from hsa-let-7b-3p, hsa-miR-l5lb, hsa-miR-l06b-3p, hsa-miR-l82-5p, hsa-miR-l07, hsa-miR-l83-5p, hsa-miR-l0a-5p, hsa-miR-l92-5p, hsa-miR-l0b-5p, hsa-miR-l93b-3p, hsa-miR-l224-5p, hsa-miR-l97- 3p, hsa-miR-l246, hsa-miR-203b-3p, hsa-miR-l247-3p, hsa-miR-204-5p, hsa-miR- l247-5p, hsa-miR-222-5p, hsa-miR-l25a-5p, hsa-miR-320a, hsa-miR-l25b-5p, hsa- miR-320b, hsa-miR-l268a, hsa-miR-320c, hsa-miR-l268b, hsa-miR-320d, hsa-miR- l307-5p, hsa-miR-339-5p, hsa-miR-l39-5p, hsa-miR-342-3p, hsa-miR-l46a-5p, hsa- miR-3605-5p, hsa-miR-l46b-5p, hsa-miR-365l, hsa-miR-l50-5p, hsa-miR-375, and wherein the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the exosomes and shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

[00045] The methods of the present disclosure are applicable to any form or colorectal cancer. In particular embodiments, the colorectal cancer may be colon cancer, bowel cancer or rectal cancer. Typically the cancer is an adenocarcinoma. Other exemplary forms of colorectal cancer which may be diagnosed in accordance with the present disclosure include carcinoid tumours, stromal tumours, sarcomas and lymphomas.

[00046] In particular embodiments, the methods disclosed herein comprise determining the expression of at least two or up to all of the miRNAs disclosed herein above. For example, the methods may comprise determining the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58 miRNAs disclosed herein above. The use of combinations of miRNA biomarkers can serve to improve the sensitivity and/or specificity of cancer detection and diagnosis. Combinations of any of the miRNAs disclosed herein may be employed.

[00047] The term "expression" is used herein in its broadest context to denote a measurable presence of the biomarker miRNA. As described hereinbelow, a variety of methods of determining or measuring expression of miRNAs are contemplated. In some embodiments, measuring the expression of a miRNA comprises determining the level of the miRNA. As used herein the terms "level" and "amount" may be used interchangeably to refer to a quantitative amount, a semi-quantitative amount, a relative amount, a concentration, or the like. Thus, these terms encompass absolute or relative amounts or concentrations of a miRNA in a sample, including levels in a population of subjects represented as mean levels and standard deviations.

[00048] The sequences of the mature miRNAs the subject of the present disclosure, and of corresponding pre-miRNAs, are publicly available through the miRBase database (www.mirbase.org) .

[00049] The expression of miRNA in accordance with the present disclosure is determined from extracellular vesicles, specifically exosomes and/or shed microvesicles. It is within the skill and capability of those of ordinary skill in the art to determine suitable biological samples that may be obtained from subjects that contain exosomes and/or shed microvesicles suitable for use in the methods of the present disclosure, for detecting a particular colorectal cancer. The biological sample may be any sample in which exosomes and/or shed microvesicles are found, and in which the expression of the biomarker miRNA(s) can be detected or measured. In particular embodiments the biological sample comprises whole blood. In other embodiments, biological samples may comprise, for example, saliva, urine, faecal matter or ascitic fluid.

[00050] The biological sample may be processed and analyzed for the purpose of determining the presence of colorectal cancer in accordance with the present disclosure, almost immediately following collection (i.e., as a fresh sample), or it may be stored for subsequent analysis. If storage of the biological sample is desired or required, it would be understood by persons skilled in the art that it should ideally be stored under conditions that preserve the integrity of the biomarker of interest within the sample (e.g., at -80°C).

[00051] Exosomes and shed microvesicles may be isolated from biological samples using any suitable methodology or protocol known in the art. For example, as noted above present inventors have previously described robust procedures for isolating EV subtypes from colorectal cancer cells (Mathivanan et ah, Mol Cell Proteomics 20l0;9(2): 197-208; Tauro et al, Mol Cell Proteomics 20l3;l2(3):587-598; Greening et ah, Methods Mol Biol 2015; 1295:179-209; the disclosures of which are incorporated herein in its entirety). Other suitable procedures will be known to those skilled in the art. For example, exemplary isolation procedures may utilize size-exclusion chromatography (see, for example, Welton et ah, J Extracellular Vesicles 2015; 4:27269; Lobb et ah, J Extracellular Vesicles 2015; 4:27031). An exemplary methodology for the isolation, identification and characterization of EVs in peripheral blood is also described in Jayachandran et al, J Immunol Methods 2012; 375:207-214. Further, commercial kits may be used for EV isolation, such as, for example, Total Exosome Isolation Kit (ThermoFisher), miRCETRY Exosome Kits (QIAGEN), ExoQuick (System Biosciences Institute) and PureExo (lOlbio).

[00052] Typically, miRNA detection and determination of expression requires isolation of nucleic acid from a sample. Nucleic acids, including RNA and specifically miRNA, can be isolated using any suitable technique known in the art. For example, phenol-based isolation procedures can recover RNA species in the 10-200-nucleotide range (e.g., precursor and mature miRNAs). Extraction procedures such as those using Trizol™ or Tri-Reagent™ can be used to purify all RNAs, large and small, and are efficient methods for isolating total RNA from biological samples that contain miRNAs. Any number of suitable RNA extraction techniques and commercially available RNA extraction kits (e.g. Qiagen RNeasy kits) are well known to those skilled in the art and may be employed in accordance with the present disclosure.

[00053] Any method of detecting and measuring miRNA expression in sample can be used in the methods disclosed herein, with illustrative examples described below. In particular embodiments, determining or measuring the expression of a miRNA comprises determining or measuring the level of the mature miRNA. Alternatively, the expression of the corresponding pre-miRNA or encoding gene may be determined or measured.

[00054] In some embodiments biochip-based techniques, such as microarrays, may be desirable for determining and measuring expression (such as are described in Hacia et ah, 1996, Nature Genetics 14: 441-447). By tagging nucleic acids with oligonucleotides or using fixed probe arrays, one can employ biochip technology to segregate target molecules as high-density arrays and screen these molecules on the basis of hybridization. The design of suitable nucleic acid probes or oligonucleotides is well within the capabilities and expertise of those skilled in the art. Microarrays can be fabricated using a variety of technologies and microarray analysis of miRNA can be accomplished according to any method known in the art. Several types of microarrays, known to those skilled in the art, can be employed including spotted oligonucleotide microarrays, pre-fabricated oligonucleotide microarrays, long oligonucleotide arrays and short oligonucleotide arrays.

[00055] Particles (e.g. beads) in suspension or in planar arrays can also be used as the basis of assays. Biomolecules such as oligonucleotides can be conjugated to the surface of beads to capture miRNAs of interest. A range of detection methods, such as flow cytometric or other suitable imaging technologies, known to persons skilled in the art can then be used for characterization of the beads and detection of miRNA presence.

[00056] In particular embodiments of the present disclosure PCR methodologies or other template-dependent amplification techniques may be desirable. For example, any method of PCR that can determine the expression of a nucleic acid molecule, including a miRNA, falls within the scope of the present disclosure. Exemplary PCR methods include, but are not limited to, reverse transcriptase PCR, real time PCR, quantitative PCR (qPCR), quantitative real time PCR (qRT-PCR), and multiplex PCR. The skilled addressee will be able determine the appropriate means of measuring expression in any given circumstance, for any given miRNA(s), without undue burden or experimentation. [00057] Using the known sequences for the miRNAs disclosed herein, specific probes and primers can be designed for use in the detection methods described as appropriate.

[00058] It will be understood by those skilled in the art that the method of determining or measuring expression of a miRNA in a biological sample can be quantitative, semi-quantitative or qualitative in nature. For example, quantitative analyses will typically provide a concentration of a miRNA in the sample within an appropriate error margin ( e.g mean +/- standard deviation). By contrast, semi- quantitative or qualitative analyses will typically provide an indication of the relative amount of a miRNA in a sample. This may involve a comparison of an amount of miRNA in a first sample with an amount of the same miRNA in a second sample, and making a determination as to the relative amount between the first and second samples.

[00059] Methods of the present disclosure may be employed to detect or diagnose colorectal cancer in a subject where no diagnosis, or confirmed diagnosis, previously existed. Typically, the expression levels of the miRNAs disclosed herein are compared to reference levels, where the reference levels represent the absence of colorectal cancer. The reference levels may be from one or more reference samples. In this context the term "reference" or "reference sample" means one or more biological samples from individuals or groups of individuals diagnosed as not having colorectal cancer. A "reference sample" may comprise the compilation of data from one or more individuals whose diagnosis as a "reference" or“control” for the purposes of the present disclosure has been confirmed. That is, samples to be used as reference samples or controls need not be specifically or immediately obtained for the purpose of comparison with the sample(s) obtained from a subject under assessment.

[00060] Thus, reference levels of miRNAs can be pre-determined using biological samples from a cohort of healthy subjects (i.e. free of colorectal cancer) to obtain an accurate median or mean. Reference levels can be determined for various samples, such as various cell and tissue types and various body fluids. For the most accurate detection, the reference sample used for comparison comprises the same type of sample as taken from the subject under assessment in the provided methods. Reference levels also can be matched by age, sex or other factors. [00061] In accordance with the present disclosure, expression data or profiles for the selected miRNAs are typically subjected to one or more statistical analyses to determine a miRNA signature profile, thereby facilitating the diagnostic or prognostic method. The statistical analysis may comprise, for example, calculation of Pearson’s correlation coefficient (PCC) values, logistical regression, logistical regression with k-fold validation, machine learning or machine learning with k-fold validation. The statistical analysis may also comprise determining one or more of ACt or Cq values for the selected miRNAs.

[00062] Diagnoses made in accordance with embodiments disclosed herein may be correlated with other means of diagnosing colorectal cancer. Thus, methods of the present disclosure may be used alone or in conjunction with, or as an adjunct to, one or more other diagnostic methods and tests to diagnose colorectal cancer. Such other diagnostic methods and tests will be well known to those skilled in the art and include, for example, fine needle aspiration biopsy, faecal occult blood test (FOBT), and the determination of one or more additional markers of colorectal cancer, such as carcinoembryonic antigen (CEA) and the carbohydrate antigen CA19.9.

Kits

[00063] All essential materials and reagents required for measuring for the expression of the at least one biomarker as disclosed herein may be assembled together in a kit. Thus, the present disclosure provides diagnostic and test kits for detecting or determining the level of expression of one or more of the miRNAs disclosed herein in a biological sample, in order to facilitate the detection or diagnosis of colorectal cancer. Such kits typically comprise one or more reagents and/or devices for use in performing the methods disclosed herein. For example, the kits may contain reagents for isolating exosomes and/or shed microvesicles from biological samples, reagents for extracting or isolating RNA, and/or for measuring the expression of one or more miRNA. As such, kits may comprise one or more agents for detecting and to facilitate measurement of miRNAs, including primers, probes or other agents, and/or may comprise suitable reagents for determining or measuring expression of the miRNAs (such as diluents, reaction buffers, wash buffers, labelling reagents, enzymes etc). Kits may also, for example, include components for making an array comprising oligonucleotides complementary to miRNAs, and thus, may include, for example, a solid support.

[00064] Kits may also include suitable software, or access to suitable software, to facilitate comparisons between reference levels of expression of miRNAs and expression levels from subjects to be diagnosed, and to facilitate statistical analysis that may be employed in such comparisons. Accordingly, also provided herein is a computer system or apparatus, configured to aid in the detection or diagnosis of colorectal cancer, wherein computer software is employed to analyse data relating to the expression of one or more miRNAs as defined in the above embodiments, and to provide a diagnostic prediction with respect to a subject. Typically, the computational software is also employed to compare said data to data relating to the expression of the one or more miRNAs in one or more cancer-free reference samples.

[00065] Kits for carrying out the methods of the present disclosure may include, in suitable container means comprising, or adapted to receive, reagents required. The container means may include at least one vial, test tube, flask, bottle, syringe and/or other container. The kits may also include means for containing the reagents in close confinement for commercial sale. Such containers may include injection and/or blow- moulded plastic containers.

[00066] Kits may also include suitable means to receive a biological sample, one or more containers or vessels for carrying out methods described herein, positive and negative controls, including a reference sample, and instructions for the use of kit components contained therein, in accordance with the methods disclosed herein.

Therapeutic regimens

[00067] A subject who is identified, in accordance with the methods of the present disclosure described hereinbefore as having colorectal cancer, can be selected for treatment, or stratified into a treatment group, wherein an appropriate therapeutic regimen can be adopted or prescribed with a view to treating the cancer.

[00068] Thus, in an embodiment, the methods disclosed herein may comprise the step of exposing (i.e., subjecting) a subject identified as having colorectal cancer to a therapeutic regimen for treating said cancer. [00069] An aspect of the present disclosure provides a method for selecting a subject for treatment for colorectal cancer, the method comprising:

[00070] The nature of the therapeutic treatment or regimen to be employed can be determined by persons skilled in the art and will typically depend on factors such as, but not limited to, the age, weight and general health of the subject. Suitable therapeutic treatments and regimens would be known to persons skilled in the art, non-limiting examples of which include chemotherapeutic agents and/or radiotherapy.

[00071] As used herein the terms "treating" and“treatment” refer to any and all uses which remedy a condition or symptoms, or disease, or otherwise prevent, hinder, retard, or reverse the progression of a condition or disease or other undesirable symptoms in any way whatsoever. Thus the term "treating" and the like are to be considered in their broadest context. For example, treatment does not necessarily imply that a patient is treated until total recovery. In conditions which display or a characterized by multiple symptoms, the treatment or prevention need not necessarily remedy, prevent, hinder, retard, or reverse all of said symptoms, but may prevent, hinder, retard, or reverse one or more of said symptoms.

[00072] Without being bound by theory or a particular mode of practice, it also follows from the present disclosure that the methods disclosed herein can be used to monitor the efficacy of treatment of colorectal cancer, whereby the expression of one or more miRNAs disclosed herein is determined (e.g., measured) in biological samples obtained from a subject at two or more separate time points, including before commencement of treatment, during the course of treatment and after cessation of treatment, to determine whether said treatment is effective, for example, in inhibiting the progression of the cancer.

[00073] Also provided herein is a protocol for monitoring the efficacy of a therapeutic treatment for colorectal cancer, the protocol comprising:

[00074] The protocol may further comprise obtaining and executing steps in respect of a third or subsequent sample.

[00075] In an embodiment, a change of expression of a miRNA between the first and second (or subsequent) sample may be indicative of an effective therapeutic regimen. Where the protocol disclosed herein indicates that the therapeutic regimen is ineffective (i.e. no change in expression of one or more miRNAs between the first and second, or subsequent, sample), the protocol may further comprise altering or otherwise modifying the therapeutic regimen with a view to providing a more efficacious or aggressive treatment. This may comprise administering to the subject additional doses of the same agent with which they are being treated or changing the dose and/or type of medication.

[00076] Also provided herein are screening methods for candidate compounds and compositions as therapeutic agents to treat colorectal cancer. For example, a suitable therapeutic agent may be obtained by selecting a compound or composition capable of modulating the expression level of one or more miRNAs disclosed herein.

[00077] Such methods of screening for a therapeutic agent can be carried out either in vivo or in vitro. For example, a screening method may be performed by administering a candidate compound or composition to a subject, such as a laboratory test animal subject; measuring the expression level of a miRNA in a biological sample from the subject; and selecting a compound or composition that increases or decreases the expression level of the miRNA, as compared to that in a control with which the candidate compound or composition has not been contacted.

[00078] Methods for selecting a compound or composition for treating colorectal cancer, for monitoring the efficacy of such a treatment or for screening candidate agents may also be employed by, for example: obtaining a biological sample from a subject, such as a laboratory test animal subject; separately maintaining aliquots of the sample in the presence of a plurality of compounds or compositions; comparing expression of a miRNA in each of the aliquots; and selecting one of the compounds or compositions which significantly alters the level of expression of the miRNA in the aliquot containing that compound or composition, relative to the levels of expression of the mRNA in the presence of other compounds or compositions.

[00079] It will be appreciated that the above described terms and associated definitions are used for the purpose of explanation only and are not intended to be limiting. [00080] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

[00081] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Examples

General methods

Cell culture and exosome isolation

[00082] Human CRC cell lines SW480 (CCL-228) and SW620 (CCL-227) were from ATCC (Manassas, VA, USA). Cells were initially cultured in l75-cm flasks in RPMI-1640 medium (Life Technologies, CA) supplemented with 10% (v/v) foetal bovine serum (Thermo Fisher Scientific, CA) and 1% (v/v) Penicillin Streptomycin (Pen/Strep, Thermo Fisher Scientific) at 37°C and 10% C0₂ and then transferred to CELLine™ AD- 1000 Bioreactor adherent flasks (Integra Biosciences, NH) and continuous culture performed over a period of several weeks as previously described (Ji et al, Proteomics 2013 ; 13(10-11): 1672-86). Cell culture medium (CM) from two separate bioreactor flasks (150 ml) was collected for EV isolation (in duplicate, biological replicates), also as described by Ji et al. Protein quantification was performed by protein staining (SYPRO^® Ruby) densitometry following 1D-SDS-PAGE (Ji et al., supra).

Cell lysate preparation

[00083] The cell lysate was prepared as previously described (Ji et al, supra).

Cryo-electron microscopy

[00084] Cryo-EM imaging of EV preparations was performed essentially as described (Tauro et al, Mol Cell Proteomics 20l3;l2(3):587-98; Tauro et al, Mol Cell Proteomics 2013;12(8):2148-59), with minor modifications. Briefly, EV preparations (~2 μg protein, non-frozen samples prepared within 2 days of analysis) were transferred onto glow-discharged C-flat holey carbon grids (ProSciTech Pty Ftd). Excess liquid was blotted and grids were plunge-frozen in liquid ethane. Grids were mounted in a Gatan cryoholder (Gatan, Inc., Warrendale, PA, USA) in liquid nitrogen. Images were acquired at 300 kV using a Tecnai G2 F30 (FEI, Eidhoven, NL) in low dose mode. Size distribution of vesicles (range 30->l000 nm) was calculated using ImageJ for the 12 fields of view (~200 different vesicles for each EV preparation).

Nanoparticle Tracking Analysis

[00085] EV diameter (size) and concentration was determined using the NanoSight NS300 system (Malvern, UK) equipped with a blue laser (488 nm). Briefly, EVs were diluted in water (~8 x 10 particles/ml) and loaded into a flow-cell top plate using a syringe pump. Three videos (1 min) were recorded for each sample and analysed by NTA software (Build 3.1.45).

Western blotting

[00086] Western blot analyses of cell/EV samples (10 μg protein) were performed as described (Ji et al, supra). Briefly, membranes were probed with primary mouse anti-TSGlOl (1:500, BD Biosciences, San Jose, CA), mouse anti-Alix (1:1000, Cell Signalling Technology Danvers, MA), mouse anti-CD9 antibody (1:1000, Abeam, Cambridge, MA), mouse anti-KIF23 (1:1000, Thermo Fisher Scientific, CA), and rabbit anti-GAPDH (1:1000, Abeam, Cambridge, UK) in TTBS (Tris buffered- saline containing 0.1% Tween-20) for 1 h. After washing with TTBS (3 x 10 min), membranes were probed with secondary antibody (IRDye 800 goat anti-mouse or IRDye 700 goat anti-rabbit IgG, 1: 15,000). The fluorescent signals were detected using the Odyssey Infrared Imaging System, v3.0 (Fi-COR Biosciences, Nebraska, USA).

Total RNA isolation

[00087] Total RNA extraction from samples was performed (in duplicate), as described previously (Chen et al, Sci Rep 20l6;6:38397). Small RNA library construction and deep sequencing

[00088] Small RNA (18-30 nt) libraries for SW480/SW620 cells and derived EVs (sMVs and exosomes) were constructed using Illumina^® TruSeq™ Small RNA Sample Preparation Kit v2 and sequenced on a HiSeq 2000 platform (Illumina), according to the manufacturer’s protocols. Briefly, small RNAs (18-30 nt) were fractioned on a 15% Tris-borate-EDTA (TBE) polyacrylamide gel (Life Technologies, CA) from total RNA (200 ng), purified by centrifugation, and ligated with adaptors. Small RNAs were then reverse transcribed into cDNAs and amplified using the adaptor primers for 14 cycles. The cDNA fragments (-150 bp) were isolated from a 6% TBE PAGE-gel and directly used for cluster generation by using TruSeq PE Cluster Kit v3 (Illumina). Using TruSeq SBS Kit v3 (Illumina) biological replicates (n=2) were sequenced for each sample. Raw data (FASTQ formatted files) can be accessed in NCBI SRA database under the accession numbers SRA440609 and SRA448517.

Small RNA annotation

[00089] Clean reads were obtained for subsequent analyses by removing low- quality, adapter, and short (<18 nt) reads from the raw reads. Clean reads were first aligned to the human reference genome (GRCh38, http://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/) by SOAP2 without mismatch. Then, miRNAs were identified and profiled by mapping the clean reads to human miRNA precursors obtained from miRBase (v2l, http://www.mirbase.org). Other noncoding RNA types (rRNA, tRNA, snRNA, snoRNA, srcRNA, srpRNA) in the clean reads were annotated using Rfam and GenBank databases by BLAST software. Repeat-associated small RNAs and mRNA degraded fragments were characterized by mapping clean reads to repeat, exon and intron regions on the human genome. Target prediction and pathway analyses were performed by using Ingenuity Pathway Analysis (IPA).

qRT-PCR validation

[00090] Quantitative real-time PCR (qRT-PCR) was used to validate expression levels of 11 candidate miRNAs (let-7a-5p, miR-l0a-5p, miR-l82-5p, miR-l83-5p, miR-l92-5p, miR-l93b-3p, miR-l9b-3p, miR-222-5p, miR-28-3p, miR-339-5p and miR-486-5p) and a housekeeping gene RNU43 was used as the internal control. Briefly, total RNA (100 ng) from cells/ derived-EVs was obtained as described above, transcribed into cDNAs by reverse transcription (RT) primers using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad Laboratories Inc., USA). Then, cDNA templates (2 μl), forward and universal reverse primers (2 mΐ), l xSsoAdvanced™ Universal SYBR Green Supermix (10 mΐ, Bio-Rad Laboratories Inc., USA) and ddH₂0 (6 mΐ) were mixed together to make a super qRT-PCR mix (final concentration of primers: 0.3 mM). Three amplifications for every candidate miRNA in each sample were ran on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories Inc., USA). All miRNA expression levels were normalized to RNU43 using CLX Manager™ Software v3.l (Bio-Rad Laboratories Inc., USA).

Clinical sample miRNA data

[00091] Clinical sample miRNA data used in this study were obtained from The Cancer Genome Atlas (TCGA). miRNA expression profile data for normal and tumour colon tissues were obtained from two colorectal cancer projects (TCGA-COAD and TCGA-READ) of TCGA, which recruit miRNA expression profiles of 11 normal colon tissues, and CRC tumours of known pathological stage - 103 stage-I, 223 stage-II, 179 stage-III, and 89 stage-IV tumour tissues. Original miRNA counts and normalized miRNA expression files were downloaded for subsequent analysis.

Statistical analysis

[00092] edgeR was used to identify miRNAs enriched in the EVs and dysregulated in the tumour samples in comparison with normal tissues. The inventors first filtered miRNAs expressed no more than 5 transcripts per million reads (TPM). Raw miRNA read counts were then used in edgeR as input for differential expression analysis. Strict criteria were used to select differentially-expressed miRNAs as follows: log2 fold change (log2LC) >1 (for up-regulated miRNAs) or log2LC <-l (for down-regulated miRNAs), p-value <0.05, and false discovery rate (LDR) <0.05. The Pearson correlation coefficient calculated by‘cor’ (an R package) was used to evaluate the correlation between cell/EVs and clinical samples.

Example 1— Extracellular vesicle isolation, purification and characterization

[00093] SW480 and SW620 cells were characterized as previously described (Ji et al, supra). Cells were maintained and cultured in two CELLine AD- 1000 Bioreactor classic flasks and 10 collections of culture media (total volume 150 ml) were made over a period of 5 days. As shown in Fig. 1A, shed microvesicles (sMVs) and crude exosomes were isolated from the CM by sequential centrifugation. The presence of sMVs in the 10,000 x g pellet was confirmed by the stereotypic sMV marker KIF23 (Fig. IB). The yields of SW480- and SW620-derived sMVs were 1.85 mg and 1.41 mg protein, respectively. Next, the crude exosomes (reconstituted in 500 μl PBS) were further purified by density gradient centrifugation using a 5-40% iodixanol (OptiPrep™) density gradient. Exosomes were enriched in factions 6 and 7 (buoyant density 1.08-1.12 μg/ml) based on western blot analysis of the stereotypic exosomal marker proteins Alix, TSG101 and CD9 (Fig. IB). The yields of SW480- and SW620- derived exosomes were 0.83 mg and 0.69 mg protein, respectively. Both cryo-EM and NTA analyses (Fig. 1C, ID) showed the particle size of SW480 and SW620 exosomes to be in the range 40-200 nm (mean size: SW480 - 119 nm and SW620 - 127 nm), while those of sMVs were in the range 50-1500 nm (mean size: SW480 - 266 nm and SW620 - 486 nm). Total RNA was extracted from SW480 and SW620 cell lysates and their purified EVs using Trizol reagent (Life Technologies, CA). RNA quality analysis, evaluated using an Agilent 2100 Bioanalyzer (data not shown), revealed that small RNAs (<50 nt) were enriched in the EVs.

Example 2 - Differential expression of miRNAs in SW480 and SW620 cell lysates

[00094] Small RNA sequencing was performed to profile miRNA expression in SW480 and SW620 cell lysates (CLs) and their released EVs. Initially, a total of 160.46 million raw reads were generated by an Illumina HiSeq-2000 system for all the samples yielding an average 12.77 million clean reads. Clean reads were aligned to miRBase (v2l) to profile known human miRNAs in each sample. The inventors used total miRNA mapped reads for normalization purposes, and then filtered lowly-expressed miRNAs (<5 TPM) to obtain a total of 345 miRNAs in SW480 and SW620 CLs; 292 and 315 being identified in SW480 and SW620 CLs, respectively. Among these, 102 (29.6%) were identified as passenger (star) miRNAs and 262 cellular miRNAs are common to both cell lines (Fig. 2A). A heat map (data not shown) of sample correlations, based on their miRNA profiles, indicated sMVs, exosomes and cells contain distinct miRNA signatures (clusters); 30 and 53 miRNAs were found to be present exclusively in SW480 and SW620 CLs, respectively. Of these, the most abundant miRNAs found uniquely in SW480 CLs included miR-371a-5p, miR372-3p, miR-373-3p, and miR-509-3p. In the case of SW620 CLs, miR-1224-5p and miR-125b- 5p were the most abundant.

[00095] Next, the statistical software package edgeR was used to identify miRNAs differentially expressed in SW480/ SW620 CLs. The inventors identified 134 dysregulated miRNAs (Fig. 2B, Table 1). Of these, 61 SW480 cellular miRNAs were found to be upregulated in SW620 CLs, and 73 downregulated. The most highly- dysregulated miRNAs in SW480/SW620 CLs are listed in Table 2. Most prominent of these are four miRNAs in SW480 CLs ( miR-371a-5p , miR-509-3p, miR-200c-3p, and miR-141-3p) whose levels are >28-fold higher when compared with SW620 CLs and 6 members of the miR-l7~92a cluster ( miR-17-5p , miR-18a-5p, miR-19a/b-3p, miR-20a- 5p, and miR-92a-l-5p ) that are significantly upregulated in SW620 relative to SW480 CLs (Tables 1 and 2). Using qRT-PCR, and RNU43 as the internal control, the inventors validated 11 miRNAs in all samples. Then, the inventors normalized the miRNA expression by using SW480-CL as the control. Normalized expression by both qRT-PCR and miRNA sequencing revealed 69.7% of the comparisons to be consistent (data not shown). IPA analysis revealed cellular dysregulated miRNAs were involved in the pathways associated with CRC progression and metastasis, such as ‘colorectal cancer metastasis signaling’, ‘p53 signaling’ and ‘Wnt/p-catenin signaling’. Experimentally validated target genes of the cellular dysregulated miRNAs revealed known oncogenes and tumour suppressors from these three pathways, including APC, TP53, KRAS, CD44 and TGFBR2. Table 1. Differentially expressed miRNAs between SW480 and SW620 cell lysates

(CLs)

^cp-value<0.05 and FDR <0.05.

dmiRNAs dysregulated in CRC tumors compared to normal colon tissues (source, TCGA) according to dbDEMC 2.0; UP - upregulated, DOWN- down regulated.

Example 3 Cellular miRNAs selectively traffic into sMVs and exosomes

[00096] Next, the inventors asked how many of the unique, as well as dysregulated, miRNAs found in SW480/ SW620 CLs sort into their cognate EVs. Of the 30 unique miRNAs found in SW480-CLs, 14 distribute to both sMVs and Exos while an additional miRNA ( miR-1262 ) uniquely distributes to sMVs. In the case of the 53 unique SW620 CL miRNAs, 31 and 23 were found to distribute into sMVs and Exos, respectively; 20 of these sorts to both EV subtypes (Fig. 2A). Concerning those miRNAs in SW480-CL whose expression levels are dysregulated (relative to their expression in SW620-CL), 55/73 of the up-regulated miRNAs are common to both SW480-derived sMVs /Exos, while 46/61 of the down-regulated miRNAs in SW480- CL distribute to both SW620-derived EV subtypes; interestingly, 6 and 4 of the up- regulated miRNAs in SW620-CL were observed to uniquely distribute to SW620- derived sMVs and Exos, respectively (Fig. 2B).

Example 4 - Cross cell line comparison of miRN A profiles in sMVs and Exos

[00097] In addition to comparing miRNA profiles of SW480/SW620 CLs, a further goal of this study was to compare the miRNA profiles of EVs derived from these isogenic cell lines to ascertain whether they reveal a subset of miRNAs critical in CRC progression from primary carcinoma (SW480 cell line surrogate) to metastasis (SW620 cell line surrogate) - information that might be used as markers to assist in the clinical staging of the disease. To address this question, the inventors performed a cross cell-line comparison of miRNA profiles of the two major EV subtypes isolated from SW480/SW620 cell culture media.

[00098] Of the 292 miRNAs (expression levels >5 TPM) in SW480-CL, 227 and 222 were detected in derived sMVs and Exos, respectively (Fig. 3A). 214 of these EV miRNAs are common to both EV subtypes while 13 and 8 are exclusively detected in derived sMVs and Exos, respectively. edgeR analysis of the 214 common miRNAs revealed 32 /214 to be significantly (log2FC>l) enriched in both EV subtypes; one ( miR-210-3p ) and 15 miRNAs (including the passenger miRNA of miR-210, miR-2lO- 5p) are selectively enriched in SW480-sMVs and -Exos, respectively. Next, the inventors applied this analysis to the SW620 cell model. Of the identified 315 SW620- CL miRNAs, a combined total of 276 miRNAs are found in SW620-sMVs (261) and SW620-Exos (242). 227/276 of these miRNAs are common to both EV subtypes; 34 and 15 are unique to SW620-sMVs and -Exos, respectively (Fig. 3B). 16/227 miRNAs were significantly enriched in both SW620-sMVs/ -Exos (middle panel of bar chart); while one (left panel of bar chart) and 19 (right panel of bar chart) were enriched only in SW620-sMVs and SW620-Exos, respectively.

[00099] Next, the inventors performed a cross cell-line (SW480/SW620) analysis of dysregulated sMVs and Exos miRNAs (see 4-way Venn diagram Fig. 3C). It can be seen that a total of 186 miRNAs are common to all sMVs and Exos from these two cell lines (Table 3). Interestingly, with the exception of miR-365a-3p and miR-365b-3p, 17 miRNAs observed in SW480 EVs are down-regulated in SW620-CLs, relative to SW480-CLs. In the case of the 33 miRNAs detected in SW620 EVs (Table 4), including 17 SW620-CL up-regulated miRNAs; in addition, 1 (miR-l262), 18 and 8 miRNAs were found exclusively in SW620-sMVs and SW620-Exos, respectively. 6 miRNAs (miR-l5b-3p, miR-29c-3p, miR-34a-5p, miR-449c-5p, miR-9-3p, and miR- 95-3p) were found exclusively in sMVs and 4 miRNAs (miR-l9l-3p, miR-l97-5p, miR-589-3p, miR-93-3p) are unique to Exos.

[00100] In another analysis, the inventors examined miRNAs enriched in the four EV subtypes relative to their respective parental CLs (Fig. 3D). It can be seen that 13 miRNAs were commonly enriched in all four EV subtypes. Interestingly, only one miRNA ( miR-7641 ) is specifically enriched in both SW620 EV subtypes. Also, there are 7 EV-enriched miRNAs common to exosomes released by the two cell lines, including the only SW480-CL up-regulated miRNA ( miR-210-5p ). Enrichment of miR- 210-3p (mature miRNA of miR-210) is common to SW480-sMVs, SW620-sMVs and SW620-Exos.

Example 5 - Low-abundance cellular SW480/SW620 miRNAs are significantly enriched in EVs

[00101] A salient finding in this study was the significant enrichment of many low- abundance cellular miRNAs (<5 TPM) in secreted EVs. In some cases, these non- detectable cellular miRNAs were found in EVs at levels in excess of 1,000 TPM (data not shown). In the case of SW480 cells, 35 non-detectable cellular miRNAs were significantly enriched in SW480-derived EVs (9 exclusively found in sMVs, 7 in Exos and 19 common to both EVs). For SW620 cells, 73 non-detectable cellular miRNAs were found to be enriched in EVs from these cells (55 in sMVs, 4 in Exos and 14 common to both EV subtypes). Further examination of these data reveals striking miRNA distribution patterns. For example, 7 miRNAs non-detectable in SW480-CL (miR- 1224-5/_?, miR-125b-5p, miR-7641, miR-99a-5p, miR-1266-5p, miR-194-3p, and miR-125b-2-3p), but enriched in SW480-derived EVs (log2FC in the range 1.36 to 7.94), were found to be up-regulated in SW620-CL (log2FC in the range 1.25 to 4.80); of these, miR-125b-5p, miR-7641 and miR-99a-5p are also enriched in SW620 EVs (relative to SW620-CL). Another 7 miRNAs that are highly abundant in SW480-CL yet non-detectable in SW620-CL ( miR-6716-3p , miR-26a-l-3p, miR-203b-3p, miR-891a- 5p, miR-143-3p, miR-371a-5p, and miR-509-3p ) are significantly enriched in SW620- derived EVs (log2FC in the range 1.03 to 5.40). Example 6 Correlation of miRNA profiles of SW480/SW620 cells and derived EVs with colorectal normal/tumour tissues

[00102] To investigate the relationship of SW480/SW620 cellular miRNA expression patterns and those reported for primary CRC tumours, the inventors performed a correlation analysis of the cell line miRNA data from the present study with CRC tumour miRNA profiles obtained from The Cancer Genome Atlas, TCGA - 594 tumours representing 11 normal colon tissues and 103 CRC stage I tumours, 223 stage II, 179 stage III, and 89 stage IV tumours. First, the inventors examined correlations between SW480-CL and SW620-CL and CRC tumours based on 292 and 315 miRNAs, respectively, observed in these cell lines. The mean Pearson correlation coefficients (PCC) for SW480-CL range from 0.05 (normal colon tissue) to 0.41 (for CRC tumours) and, in the case of SW620-CL 0.22 (for normal tissue) to 0.55 for tumours. Based on these correlation coefficients, the miRNA profiles for SW480-CL can be used to predict 592/594 CRC tumours and SW620-CL miRNA profiles, 564/594 (94.9%) of CRC tumours. These predictions improve marginally if the correlations are analysed using the 134 dysregulated miRNAs observed in SW480-/SW620-CLs (see Fig. 4A, Table 5) - for example, 593/594 and 572/594 (96.3%) for SW480-/SW620- CLs, respectively. Correlations between SW480/SW620 cell lines and CRC tumours are further illustrated in Fig. 4A.

Table 5. Correlation of SW480/SW620 miRNA profiles with normal colon tissue and CRC tumors of different pathological stages

[00103] Next, in line with the recognition that functional miRNAs in EVs are potential candidates as stable blood-based biomarkers, the inventors performed PCC analyses between SW480/SW620 cell-derived EVs (sMVs and Exos) and CRC tumours. A total of 97 miRNAs significantly enriched in SW480/SW620 cell-secreted EVs (sMVs and Exos) were used for these analyses. As shown in the volcano plots in Fig. 4B more than half of the EV-enriched miRNAs were dysregulated in different CRC tumour stages compared to normal colon tissue. The number of miRNAs up-regulated and down-regulated revealed in different CRC tumour stages is given in the Venn diagrams shown in Fig. 4C. For the EV-enriched miRNAs, the inventors next calculated their PCC values between normal colon tissue and different tumour stage tissues (Table 5). A salient finding of this study is that the mean PCC values between EV miRNA profiles and normal colon tissue miRNA profiles are striking lower than the mean PCC values between cellular miRNA profiles and normal colon tissue miRNA profiles, thereby enhancing CRC tumour prediction accuracy (Fig. 4D, Table 5).

Example 7— Identification of minimal miRNA set for diagnosis of colorectal cancer

[00104] The inventors used 97 miRNAs (above; 60 miRNAs enriched in SW480- sMVs, 72 miRNAs enriched in SW480-Exos, 34 miRNAs enriched in SW620-sMVs and 51 miRNAs enriched in SW620-Exos) to predict colorectal cancer. Using these data, the Pearson correlation coefficient (PCC) values of miRNA profiles of CRC tumour tissues with SW480/SW620 EVs were clearly distinguishable from the PCC values of miRNA profiles of normal colon tissues.

[00105] The inventors evaluated the prediction accuracy of CRC tumour using these 97 miRNAs. Table 6 lists the largest PCC value of the EV miRNA profiles and CRC normal tissues, the smallest PCC value of the EV miRNA profiles and CRC tumour tissues, and the margins between them. The PCC values of the EV miRNA profiles and normal/tumour tissues do have overlaps, indicating some false positives. Notwithstanding, the accuracy of using 97 miRNAs for CRC tumour prediction is -96%.

Table 6

[00106] Next, the inventors evaluated the prediction accuracy of CRC tumour using the EV-enriched miRNAs (Table 7). This analysis shows that SW480-EV miRNA profiles can distinguish CRC tumour tissues from normal tissues with 100% accuracy. The PCC values of miRNA profiles of SW620 EVs (sMVs and Exos) and TCGA dataset are high with a large overlap between normal colon tissues and CRC tumour tissues. Alone, these SW620 miRNA EV datasets cannot predict CRC tumours and must be used in combination with SW480 EV datasets; for this reason these data were not considered further.)

Table 7

[00107] The inventors then tried to reduce the number of miRNAs to predict CRC tumour. As shown in Fig. 5A, the PCC margin becomes smaller and smaller if the 60 SW480-sMVs enriched miRNAs are reduced for PCC calculation. It was calculated that 42 SW480-sMVs enriched miRNAs (Table 8) can predict CRC tumour with 100% accuracy.

Table 8. Minimal suite of 42 miRNAs enriched in SW480-sMVs for CRC prediction

[00108] Next, the inventors applied this method to SW480-Exos enriched miRNAs. Considering the 72 miRNAs significantly enriched in SW480-Exos relative to SW480- CL (SW480 cell lysates), the PCC margin between CRC tumour and normal tissues was decreased when one miRNA was removed (Fig. 5B). The inventors calculated that a minimum of 52 SW480-Exos enriched miRNAs can predict CRC with 100% accuracy (Table 9).

Table 9. Minimal suite of 52 miRNAs enriched in SW480-Exos for CRC prediction

[00109] The inventors also used box plot figures to show the PCC values of miRNA profiles of SW480-EVs and TCGA dataset (Fig. 6). It is clear that using miRNAs enriched in SW480 EVs can distinguish CRC tumours from normal colon tissue. Interestingly, the mean PCC value correlates with tumour stage

[00110] Table 10 below shows the PCC margins using 42 SW480-sMVs miRNAs and 52 SW480-Exos miRNAs to predict CRC.

Table 10

[00111] To employ this model in the diagnosis or prognosis of disease progression, the miRNA profile in a biological sample is first estimated, for example using deep sequencing, microarray or PCR analysis, and PCC values (PCC1, PCC2, PCC3 and PCC4) of this miRNA profile are calculated using the SW480-/SW620-EV values herein, according to the predictions shown in Table 11 below. PCC1 is the higher PCC value of the patient miRNA profile and SW480-sMVs (60 miRNAs). PCC2 is the higher PCC value of the patient miRNA profile and SW480-Exos (60 miRNAs). PCC3 is the higher PCC value of the patient miRNA profile and SW620-sMVs (60 miRNAs). PCC4 is the higher PCC value of the patient miRNA profile and SW620-Exos (60 miRNAs).

Table 11

[00112] Four‘Y’s (Ύ for each of PCC1 to PCC4) indicates a healthy (non-CRC tumour) individual, while four‘N’s (‘N’ for each of PCC 1 to PCC4) indicates potential CRC tumour in an individual. All other combinations of Ύ and‘N’ indicate a subject requiring further analysis.

[00113] Finally, a comparison of the 42 SW480-sMVs-enriched with 52 SW480- Exos-enriched miRNAs, reveals 36 that are common (Table 12). Analysis of 36 SW480-EV-enriched miRNAs show a clear, statistically sound, margin between PCC values allowing distinction of normal and CRC tumour tissues (Table 13). Table 12. Suite of 36 miRNAs commonly enriched in SW480-sMVs and SW480-Exos for CRC prediction

Table 13

Claims

1. A method for detecting colorectal cancer in a subject, the method comprising executing the steps of:

(a) obtaining a biological sample from the subject, wherein the biological sample comprises one or both of exosomes and shed microvesicles; and

(b) determining the expression of the miRNAs hsa-let-7b-3p, hsa-miR-l5lb, hsa-miR-l06b-3p, hsa-miR-l82-5p, hsa-miR-l07, hsa-miR-l83-5p, hsa-miR-l0a-5p, hsa-miR-l92-5p, hsa-miR-l0b-5p, hsa-miR-l93b-3p, hsa-miR-l224-5p, hsa-miR-l97- 3p, hsa-miR-l246, hsa-miR-203b-3p, hsa-miR-l247-3p, hsa-miR-204-5p, hsa-miR- l247-5p, hsa-miR-222-5p, hsa-miR-l25a-5p, hsa-miR-320a, hsa-miR-l25b-5p, hsa- miR-320b, hsa-miR-l268a, hsa-miR-320c, hsa-miR-l268b, hsa-miR-320d, hsa-miR- l307-5p, hsa-miR-339-5p, hsa-miR-l39-5p, hsa-miR-342-3p, hsa-miR-l46a-5p, hsa- miR-3605-5p, hsa-miR-l46b-5p, hsa-miR-365l, hsa-miR-l50-5p, and hsa-miR-375 in the exosomes and/or shed microvesicles in the biological sample, and optionally determining a statistical value(s) derived from the expression levels;

wherein an upregulated level of expression of said miRNAs, relative to the level of expression of the corresponding miRNAs in one or more cancer-free reference samples, is indicative of the presence of colorectal cancer in the subject.

2. A method according to claim 1, wherein the biological sample comprises exosomes and shed microvesicles and the expression of said miRNAs is determined in the exosomes and shed microvesicles.

3. A method according to claim 1 or 2, wherein the comparison between the expression of the miRNA in the exosomes and/or shed microvesicles from the subject and the expression of corresponding miRNAs in the reference sample(s) is assessed by calculating the Pearson’s correlation coefficient values for the miRNAs.

4. A method according to claim 3, wherein the statistical value derived from the expression level of the miRNAs is the Pearson’s correlation coefficient (PCC) value.

5. A method according to any one of claims 1 to 4, wherein the colorectal cancer is colon cancer, bowel cancer or rectal cancer.

6. A method according to any one of claims 1 to 5, wherein the colorectal cancer is an adenocarcinoma.

7. A method according to any one of claims 1 to 6, wherein the biological sample obtained from the subject is a blood sample.

8. A method according to claim 7, wherein the sample is a serum or plasma sample.

9. A method according to any one of claims 1 to 8, wherein the reference sample(s) is a blood sample.

10. A method according to claim 9, wherein the reference sample(s) is a serum or plasma sample.

11. A method according to any one of claims 1 to 10, wherein the reference sample(s) are derived from one or more individuals known not to have colorectal cancer.

12. A method for detecting colorectal cancer in a subject, the method comprising executing the step of determining the expression of one or more miRNAs in a biological sample obtained from the subject, wherein the biological sample comprises one or both of exosomes and shed microvesicles, and wherein the one or more miRNAs are selected from the group consisting of hsa-let- 7b-3p, hsa-miR-l00-5p, hsa-miR-l06b-3p, hsa-miR-l07, hsa-miR-l0a-5p, hsa-miR- l0b-5p, hsa-miR-l224-5p, hsa-miR-l246, hsa-miR-l247-3p, hsa-miR-l247-5p, hsa- miR-l25a-5p, hsa-miR-l25b-2-3p, hsa-miR-l25b-5p, hsa-miR-l266-5p, hsa-miR- l268a, hsa-miR-l268b, hsa-miR-l307-5p, hsa-miR-l39-5p, hsa-miR-l46a-5p, hsa- miR-l46b-5p, hsa-miR-l50-5p, hsa-miR-l5la-3p, hsa-miR-l5lb, hsa-miR-l8la-5p, hsa-miR-l82-5p, hsa-miR-l83-5p, hsa-miR-l9l-5p, hsa-miR-l84, hsa-miR-l92-5p, hsa-miR-l93b-3p, hsa-miR-l94-3p, hsa-miR-l97-3p, hsa-miR-203b-3p, hsa-miR-204- 5p, hsa-miR-2lO-5p, hsa-miR-2l0-3p, hsa-miR-22l-5p, hsa-miR-222-5p, hsa-miR-22- 5p, hsa-miR-27a-5p, hsa-miR-28-3p, hsa-miR-30e-3p, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, hsa-miR-324-5p, hsa-miR-339-5p, hsa-miR-342-3p, hsa- miR-3605-5p, hsa-miR-36l3-3p, hsa-miR-36l-5p, hsa-miR-365l, hsa-miR-365a-3p, hsa-miR-365b-3p, hsa-miR-3687, hsa-miR-369l-5p, hsa-miR-375, and wherein the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the exosomes or shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

13. A method according to claim 12, wherein the level of expression of all of said miRNAs defined in claim 12 are determined and the level of expression of said miRNAs, or a statistical value(s) derived therefrom, in the exosomes or shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

14. A method according claim 12, wherein the biological sample comprises exosomes, the one or more miRNAs are selected from hsa-let-7b-3p, hsa-miR-l00-5p, hsa- miR-l06b-3p, hsa-miR-l07, hsa-miR-l0a-5p, hsa-miR-l0b-5p, hsa-miR-l224-5p, hsa- miR-l246, hsa-miR-l247-3p, hsa-miR-l247-5p, hsa-miR-l25a-5p, hsa-miR-l25b-5p, hsa-miR-l266-5p, hsa-miR-l268a, hsa-miR-l268b, hsa-miR-l307-5p, hsa-miR-l39-5p, hsa-miR-l46a-5p, hsa-miR-l46b-5p, hsa-miR-l50-5p, hsa-miR-l5la-3p, hsa-miR- 15 lb, hsa-miR-l8la-5p, hsa-miR-l82-5p, hsa-miR-l83-5p, hsa-miR-l9l-5p, hsa-miR- l92-5p, hsa-miR-l93b-3p, hsa-miR-l94-3p, hsa-miR-l97-3p, hsa-miR-203b-3p, hsa- miR-204-5p, hsa-miR-2lO-5p, hsa-miR-22l-5p, hsa-miR-222-5p, hsa-miR-27a-5p, hsa- miR-28-3p, hsa-miR-30e-3p, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c, hsa-miR- 320d, hsa-miR-339-5p, hsa-miR-342-3p, hsa-miR-3605-5p, hsa-miR-36l3-3p, hsa- miR-36l-5p, hsa-miR-365l, hsa-miR-365a-3p, hsa-miR-365b-3p, hsa-miR-369l-5p, hsa-miR-375, and the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the exosomes derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

15. A method according to claim 14, wherein the level of expression of all of said miRNAs defined in claim 14 are determined and the level of expression of said miRNAs, or a statistical value(s) derived therefrom, in the exosomes derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

16. A method according to claim 12, wherein the biological sample comprises shed microvesicles, the one or more miRNAs are selected from hsa-let-7b-3p, hsa-miR-l06b-3p, hsa- miR-l07, hsa-miR-l0a-5p, hsa-miR-l0b-5p, hsa-miR-l224-5p, hsa-miR-l246, hsa- miR-l247-3p, hsa-miR-l247-5p, hsa-miR-l25a-5p, hsa-miR-l25b-2-3p, hsa-miR-l25b- 5p, hsa-miR-l268a, hsa-miR-l268b, hsa-miR-l307-5p, hsa-miR-l39-5p, hsa-miR- l46a-5p, hsa-miR-l46b-5p, hsa-miR-l50-5p, hsa-miR-l5lb, hsa-miR-l82-5p, hsa-miR- l83-5p, hsa-miR-l84, hsa-miR-l92-5p, hsa-miR-l93b-3p, hsa-miR-l97-3p, hsa-miR- 203b-3p, hsa-miR-204-5p, hsa-miR-2l0-3p, hsa-miR-222-5p, hsa-miR-22-5p, hsa-miR- 320a, hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, hsa-miR-324-5p, hsa-miR-339-5p, hsa-miR-342-3p, hsa-miR-3605-5p, hsa-miR-365l, hsa-miR-3687, hsa-miR-375, and the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

17. A method according to claim 16, wherein the level of expression of all of said miRNAs defined in claim 16 are determined and the level of expression of said miRNAs, or a statistical value(s) derived therefrom, in the shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

18. A method according to claim 12, wherein the biological sample comprises exosomes and shed microvesicles, the one or more miRNAs are selected from hsa-let-7b-3p, hsa-miR-l5lb, hsa-miR- l06b-3p, hsa-miR-l82-5p, hsa-miR-l07, hsa-miR-l83-5p, hsa-miR-l0a-5p, hsa-miR- l92-5p, hsa-miR-l0b-5p, hsa-miR-l93b-3p, hsa-miR-l224-5p, hsa-miR-l97-3p, hsa- miR-l246, hsa-miR-203b-3p, hsa-miR-l247-3p, hsa-miR-204-5p, hsa-miR-l247-5p, hsa-miR-222-5p, hsa-miR-l25a-5p, hsa-miR-320a, hsa-miR-l25b-5p, hsa-miR-320b, hsa-miR-l268a, hsa-miR-320c, hsa-miR-l268b, hsa-miR-320d, hsa-miR-l307-5p, hsa- miR-339-5p, hsa-miR-l39-5p, hsa-miR-342-3p, hsa-miR-l46a-5p, hsa-miR-3605-5p, hsa-miR-l46b-5p, hsa-miR-365l, hsa-miR-l50-5p, hsa-miR-375, and the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the exosomes and shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

19. A method according to claim 18, wherein the level of expression of all of said miRNAs defined in claim 18 are determined and the level of expression of said miRNAs, or a statistical value(s) derived therefrom, in the exosomes and shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject.

20. A method according to any one of claims 12 to 19, wherein the expression of the one or more miRNAs are upregulated in exosomes and/or shed microvesicles in the biological sample from the subject.

21. A method according to any one of claims 12 to 20, wherein the comparison between the expression of the miRNA(s) in the exosomes and/or shed microvesicles from the subject and the expression of corresponding miRNA(s) in the reference sample(s) is assessed by calculating the Pearson’s correlation coefficient values for the miRNA(s).

22. A method according to any one of claims 12 to 21, wherein the statistical value derived from the expression level of the at least one miRNA is the Pearson’s correlation coefficient (PCC) value.

23. A method according to any one of claims 12 to 22, wherein the colorectal cancer is colon cancer, bowel cancer or rectal cancer.

24. A method according to any one of claims 12 to 23, wherein the colorectal cancer is an adenocarcinoma.

25. A method according to any one of claims 12 to 24, wherein the biological sample obtained from the subject is a blood sample.

26. A method according to claim 25, wherein the sample is a serum or plasma sample.

27. A method according to any one of claims 12 to 26, wherein the reference sample(s) is a blood sample.

28. A method according to claim 27, wherein the reference sample(s) is a serum or plasma sample.

29. A method according to any one of claims 12 to 28, wherein the reference sample(s) are derived from one or more individuals known not to have colorectal cancer.

30. A kit for use in screening for colorectal cancer, wherein the kit comprises one or more reagents for determining the expression of one or more miRNAs as defined in any one of claims 1 or 12 to 19.

31. A computer system or apparatus, configured to aid in the detection or diagnosis of colorectal cancer, wherein computer software is employed to analyze data relating to the expression of one or more miRNAs as defined in any one of claims 1 or 12 to 19, and to provide a diagnostic prediction with respect to a subject.

32. A computer system or apparatus according to claim 31, wherein the computer software is employed to compare said data to data relating to the expression of the one or more miRNAs in one or more cancer-free reference samples.

33. A method for selecting a subject for treatment for colorectal cancer, the method comprising:

(a) executing a step of determining the level of expression of one or more miRNAs as defined in any one of claims 1 or 12 to 19, in a biological sample derived from a subject, wherein the biological sample comprises one or both of exosomes and shed microvesicles derived from colorectal cancer cells from the subject, wherein the level of expression of the one or more miRNAs, or a statistical value(s) derived therefrom, in the exosomes and/or shed microvesicles derived from the subject, relative to the level of expression, or a statistical value(s) derived therefrom, of the corresponding one or more miRNAs in one or more cancer-free reference samples is indicative of the presence of colorectal cancer in the subject; and

34. A protocol for monitoring the efficacy of a therapeutic treatment for colorectal cancer, the protocol comprising:

(c) executing the step of determining the level of expression of one or more miRNAs as defined in any one of claims 1 or 12 to 19 in the first and second biological samples; and