Method for determining cancer prognosis

Abstract

To provide a method for determining the prognosis of cancer, especially lung cancer.SOLUTION: The invention is based, at least in part, on the surprising discovery that hypoxia-induced exosomal proteins identified in vitro are accurate prognostic biomarkers of cancer progression and aggressiveness in patients. Provided herein are methods of determining aggressiveness, prognosis and therapeutic response to cancer, such as non-small cell lung cancer (NSCLC), said methods comprising determining the expression level of one or more differentially expressed protein markers in an exosome sample from a subject. Methods and agents for treating cancer are also provided.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] The present invention relates to cancer. More particularly, the present invention relates to a method for determining the prognosis of cancer, especially lung cancer. [Background technology]

[0002] Lung cancer is the leading cause of cancer mortality and disease burden in many countries. As an example, lung cancer in Australia accounts for 1 in 14 deaths in men and 1 in 25 deaths in women from any cause. Classification of patients into responding and non-responding categories is currently not possible for lung cancer.

[0003] Surgery is considered the optimal treatment for early-stage lung cancer in people well suited for surgical resection. Nevertheless, clinical staging is imperfect, as people treated with curative intent still have a significant chance of recurrence. For example, in stage I, II, or IIIA non-small cell lung cancer (NSCLC), approximately 40-50% of stage IB patients, 55-70% of stage II patients, and an even larger proportion of stage IIIA NSCLC patients will recur their disease and die despite potentially curative surgery. Recently, more active platinum-based combination therapy and numerous large-scale clinical trials demonstrating the efficacy of adjuvant chemotherapy for resected NSCLC have led to the use of adjuvant chemotherapy to improve outcomes for patients with completely resected NSCLC.

[0004] Currently, pathologic (TNM) staging is the most important prognostic factor determining the likelihood of lung cancer recurrence. Genomic biomarkers are being investigated for their potential prognostic value (3-5); however, at present, they are not routinely used in the clinic, unlike breast cancer, where FDA-approved tests are increasingly available for patients (e.g., Oncotype DX). Similarly, other biomarkers, including protein expression and proteomics, have been proposed for use in lung cancer but have not yet been routinely applied clinically.

[0005] Therefore, as a significant proportion of NSCLC patients who undergo complete resection or chemoradiotherapy as primary treatment for apparently treatable lung cancer ultimately relapse and recur, there is an urgent need for accurate prognostic biomarkers after treatment with curative intent. Prognostic factors are needed to guide clinicians in determining which patients will benefit from adjuvant chemotherapy and who will not receive any benefit but will suffer potential chemotherapy-related adverse effects.

[0006] In addition to the above, conventional confirmed prognostic biomarkers generally require invasive biopsy. However, in NSCLC patients, 20% of patients are unsuitable for such biopsy due to comorbidities and general health problems. Furthermore, the biopsy itself can cause injury and inflammation, contributing to the morbidity and mortality of NSCLC patients. For this reason, improved methods for assessing patient outcomes from minimally invasive sampling, such as blood tests, are needed. Summary of the Invention

[0007] The present invention generally relates to determining the expression level of one or more exosomal proteins as a prognostic marker for cancer progression in a subject. In some embodiments, the present invention also generally relates to treating cancer using such exosomal proteins to inform treatment selection or decision-making. In a particular embodiment, the cancer is lung cancer, such as non-small cell lung cancer.

[0008] In a first aspect, the present invention provides a method of determining the aggressiveness of cancer in a subject, comprising determining the expression level of one or more markers in an exosome sample of the subject, wherein said markers comprise one or more of those proteins listed in Table 1 and / or Table 2, and wherein the expression level of the one or more markers is indicative of or correlates with the level of aggressiveness of the cancer.

[0009] In a second aspect, the present invention provides a method of determining the prognosis of cancer in a subject, comprising the step of determining the expression level of one or more markers in an exosome sample of the subject, wherein said markers comprise one or more of those proteins listed in Table 1 and / or Table 2, and wherein the expression level of said one or more markers is indicative of or correlates with a less or more favorable prognosis for said cancer.

[0010] In one embodiment of the method of the above aspect, a relatively decreased expression level of one or more markers indicates or correlates with a more favorable prognosis and / or a less aggressive cancer; and / or a relatively increased expression level of one or more markers indicates or correlates with a less favorable prognosis and / or a more aggressive cancer.

[0011] Suitably, the methods of the first and second aspects further comprise the further step of diagnosing said subject as having (i) a highly aggressive cancer or a less aggressive cancer; and / or (ii) a less favorable prognosis or a more favorable prognosis.

[0012] In one embodiment of the method of the foregoing aspect, the prognosis or aggressiveness of the cancer is used, at least in part, to determine the likelihood of metastasis of the cancer in the subject. Suitably, a relatively decreased expression level of the one or more markers indicates or correlates with a decreased likelihood of metastasis of the cancer; and / or a relatively increased expression level of the one or more markers indicates or correlates with an increased likelihood of metastasis of the cancer.

[0013] In a third aspect, the present invention provides a method of predicting responsiveness of a cancer to an anti-cancer treatment in a subject, comprising determining the expression level of one or more markers in an exosome sample from the subject, wherein said markers comprise one or more of those proteins listed in Table 1 and / or Table 2, and wherein altered or modulated expression levels of said one or more markers are indicative of or correlate with a relatively increased or decreased responsiveness of the cancer to the anti-cancer treatment.

[0014] With respect to the first, second and third aspects of the invention, the method suitably comprises the further step of treating cancer in the subject.

[0015] In a fourth aspect, the present invention provides a method of treating cancer in a subject, comprising determining the expression level of one or more markers in an exosome sample from the subject, wherein said markers comprise one or more of those proteins listed in Table 1 and / or Table 2, and based on the determination made, initiating, continuing, modifying or discontinuing anti-cancer treatment.

[0016] Suitably, with respect to the methods of the third and fourth aspects, the anti-cancer treatment comprises administering to the subject a therapeutically effective amount of an anti-cancer agent that reduces the expression and / or activity of one or more markers.

[0017] According to one embodiment of the method of the third and fourth aspects, the anti-cancer treatment comprises administering to the subject a therapeutically effective amount of an anti-cancer agent that prevents or inhibits metastasis of the cancer.

[0018] For the methods of the third and fourth aspects, the anti-cancer agent is suitably an antibody or a small molecule (eg, an organic or inorganic small molecule antagonist).

[0019] Suitably, the method of the foregoing embodiment further comprises the step of obtaining an exosome sample from the subject.

[0020] For the methods of the foregoing embodiments, the one or more markers are suitably selected from the group consisting of galectin-3-binding protein, transitional endoplasmic reticulum ATPase, neutral α-glucosidase AB, 60 kDa heat shock protein, lysyl oxidase homolog 2, tenascin C, fatty acid synthase, agrin, aspartyl aminopeptidase, proteasome subunit α1, proteasome subunit α2, proteasome subunit α3, proteasome subunit α4, proteasome subunit In one particular embodiment, the one or more markers are selected from the group consisting of galectin-3-binding protein, transitional endoplasmic reticulum ATPase, tenascin-C, proteasome subunit alpha 2, thrombospondin-1, and any combination thereof.

[0021] Suitably, the method of the above embodiment further comprises the step of comparing the expression level of the one or more markers in the exosome sample with a reference exosomal expression level of each of the one or more markers.

[0022] In a fifth aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) contacting a cell expressing a marker listed in Table 1 and / or Table 2 with a candidate agent; and (b) a method for identifying or producing an agent for use in treating cancer in a subject, comprising determining whether the candidate agent modulates the expression and / or activity of a marker.

[0023] In certain embodiments, the candidate agent at least partially reduces, eliminates, suppresses or inhibits the expression and / or activity of the marker.

[0024] Suitably, the cancer of the foregoing aspects is or comprises lung cancer. Preferably, lung cancer comprises squamous cell carcinoma, adenocarcinoma, large cell carcinoma, small cell carcinoma and mesothelioma. Even more preferably, the lung cancer is non-small cell lung cancer.

[0025] Suitably, the subject of the above aspects is a mammal, preferably a human.

[0026] Unless otherwise specified, the terms "comprise," "comprises," and "comprising," or similar terms, refer to a non-exclusive inclusion, such that a list of enumerated elements or features does not merely include those stated or enumerated elements, but may include other elements or features not listed or described.

[0027] The indefinite articles "a" and "an" are used herein to denote or encompass singular or plural elements or features and should not be construed as meaning or defining "one" or "single" element or feature; for example, "a" cell includes one cell, one or more cells, and plural cells. [Brief explanation of the drawings]

[0028] [Figure 1-1] Figure 1 shows that exosomes are secreted from NSCLC cells. A: Protein characterization of exosomes demonstrates the presence of exosomal markers and the absence of non-exosomal calnexin. B: Exosomes secreted by NSCLC have the expected size distribution. C: Hypoxia increases exosome secretion but does not alter the exosome size range. D: Hypoxia significantly increases exosome secretion in NSCLC cells. CL: cell lysate; E: exosome lysate. [Figure 1-2]Figure 1 shows that exosomes are secreted from NSCLC cells. A: Protein characterization of exosomes demonstrates the presence of exosomal markers and the absence of non-exosomal calnexin. B: Exosomes secreted by NSCLC have the expected size distribution. C: Hypoxia increases exosome secretion but does not alter the exosome size range. D: Hypoxia significantly increases exosome secretion in NSCLC cells. CL: cell lysate; E: exosome lysate. [Figure 1-3] Figure 1 shows that exosomes are secreted from NSCLC cells. A: Protein characterization of exosomes demonstrates the presence of exosomal markers and the absence of non-exosomal calnexin. B: Exosomes secreted by NSCLC have the expected size distribution. C: Hypoxia increases exosome secretion but does not alter the exosome size range. D: Hypoxia significantly increases exosome secretion in NSCLC cells. CL: cell lysate; E: exosome lysate. [Figure 2] Figure 2 shows that hypoxia alters exosome content. Exosomes were harvested from conditioned medium from cells cultured under normoxic (21% CO2) or hypoxic (2% CO2) conditions for 24 hours. A: Scanning electron microscopy demonstrates conventional exosome morphology. B: Quantitative mass spectrometry revealed 55 proteins upregulated under hypoxia, n=5, FDR 1%. C, D: Protein targets were validated by Western blot and ELISA. [Figure 3-1]Figure 3 shows that upregulated proteins correlate with patient disease progression. A: Exosomes isolated from NSCLC patients show the expected size range and morphology. B, C: In vitro identified hypoxia protein markers are upregulated in patients who relapse within the first 18 months. D: ROC curve of a combined protein signature (GANAB, VCP, and galectin-3-binding protein) to identify patients who will relapse within 12 months. E: Patient disease-free survival in relation to exosome content. Patients who had at least two of the above markers progressed more rapidly compared to patients who expressed only one or no markers in their exosomes. [Figure 3-2] Figure 3 shows that upregulated proteins correlate with patient disease progression. A: Exosomes isolated from NSCLC patients show the expected size range and morphology. B, C: In vitro identified hypoxia protein markers are upregulated in patients who relapse within the first 18 months. D: ROC curve of a combined protein signature (GANAB, VCP, and galectin-3-binding protein) to identify patients who will relapse within 12 months. E: Patient disease-free survival in relation to exosome content. Patients who had at least two of the above markers progressed more rapidly compared to patients who expressed only one or no markers in their exosomes. [Figure 3-3] Figure 3 shows that upregulated proteins correlate with patient disease progression. A: Exosomes isolated from NSCLC patients show the expected size range and morphology. B, C: In vitro identified hypoxia protein markers are upregulated in patients who relapse within the first 18 months. D: ROC curve of a combined protein signature (GANAB, VCP, and galectin-3-binding protein) to identify patients who will relapse within 12 months. E: Patient disease-free survival in relation to exosome content. Patients who had at least two of the above markers progressed more rapidly compared to patients who expressed only one or no markers in their exosomes. [Figure 4] Figure 4 shows that other upregulated proteins identified in hypoxic exosomes have prognostic value: TNC is upregulated under hypoxia and is more abundant in exosomes of rapidly progressing NSCLC patients. [Figure 5] FIG. 5 shows the individual ROC and survival curves for the proteins used in the patient signature. [Figure 6-1] Figure 6 shows hypoxia-induced changes in the protein composition of exosomes derived from NSCLC cells. a: The morphology of isolated exosomes was assessed using transmission electron microscopy. Images of normoxic and hypoxic SKMES1-derived exosomes (size bar 200 nm) also show a clear upregulation of exosome concentration. b: Nanoparticle analysis using TRPS of exosomes isolated from four different NSCLC cell lines demonstrates that the majority of exosomes have a size range of 30-150 nm. c: Quantitative mass spectrometry identified 32 proteins commonly upregulated in H358 and SKMES1 exosomes (FDR < 0.1%; n = 5). d, e: Mass spectrometry results were confirmed using Western blot analysis of VCP (FLOT1 was used as a loading control) and ELISA for MAC2BP, TNC, PSMA2, and THBS1 in H358, SKMES1, H23, and H1975 NSCLC cell lines (● - H358, black square - SKMES1, ▲ - H23, ◆ - H1975). *p<0.05, **p<0.01. [Figure 6-2]Figure 6 shows hypoxia-induced changes in the protein composition of exosomes derived from NSCLC cells. a: The morphology of isolated exosomes was assessed using transmission electron microscopy. Images of normoxic and hypoxic SKMES1-derived exosomes (size bar 200 nm) also show a clear upregulation of exosome concentration. b: Nanoparticle analysis using TRPS of exosomes isolated from four different NSCLC cell lines demonstrates that the majority of exosomes have a size range of 30-150 nm. c: Quantitative mass spectrometry identified 32 proteins commonly upregulated in H358 and SKMES1 exosomes (FDR < 0.1%; n = 5). d, e: Mass spectrometry results were confirmed using Western blot analysis of VCP (FLOT1 was used as a loading control) and ELISA for MAC2BP, TNC, PSMA2, and THBS1 in H358, SKMES1, H23, and H1975 NSCLC cell lines (● - H358, black square - SKMES1, ▲ - H23, ◆ - H1975). *p<0.05, **p<0.01. [Figure 6-3]Figure 6 shows hypoxia-induced changes in the protein composition of exosomes derived from NSCLC cells. a: The morphology of isolated exosomes was assessed using transmission electron microscopy. Images of normoxic and hypoxic SKMES1-derived exosomes (size bar 200 nm) also show a clear upregulation of exosome concentration. b: Nanoparticle analysis using TRPS of exosomes isolated from four different NSCLC cell lines demonstrates that the majority of exosomes have a size range of 30-150 nm. c: Quantitative mass spectrometry identified 32 proteins commonly upregulated in H358 and SKMES1 exosomes (FDR < 0.1%; n = 5). d, e: Mass spectrometry results were confirmed using Western blot analysis of VCP (FLOT1 was used as a loading control) and ELISA for MAC2BP, TNC, PSMA2, and THBS1 in H358, SKMES1, H23, and H1975 NSCLC cell lines (● - H358, black square - SKMES1, ▲ - H23, ◆ - H1975). *p<0.05, **p<0.01. [Figure 7-1]Figure 7 shows that the hypoxic exosome signature predicts disease progression in NSCLC patients. a, b: Exosomes can be isolated from NSCLC plasma based on morphology, as shown by TEM (size bar 200 nm) and a size distribution ranging from 20 to 150 nm. c: TRPS demonstrates no difference in exosome concentrations in plasma from healthy controls, patients who progress within 18 months, or patients who do not relapse at 18 months. d: Exosomes isolated from NSCLC patients show enrichment of VCP in patients who progress at 18 months compared to patients who do not relapse and healthy controls (FLOT1 is used as a loading target). e: The hypoxic exosome signature is upregulated in exosomes derived from patients who progress at 18 months. f: The number of hypoxic protein markers above the Youden index threshold demonstrates a clear separation between patients who progress within 18 months and those who relapse at 18 months. g: Kaplan-Meier shows a clear separation of patient DFS based on the abundance of proteins from the hypoxic exosome signature (≥3 markers above the Youden index value). h: ROC curve demonstrates that the hypoxic exosome signature is a perfect prognostic marker for disease progression (<18 months) in NSCLC patients, whereas exosome signature concentration has no prognostic value. i: Kaplan-Meier curve demonstrates that the hypoxic exosome signature also correlates with overall survival in NSCLC patients. [Figure 7-2]Figure 7 shows that the hypoxic exosome signature predicts disease progression in NSCLC patients. a, b: Exosomes can be isolated from NSCLC plasma based on morphology, as shown by TEM (size bar 200 nm) and a size distribution ranging from 20 to 150 nm. c: TRPS demonstrates no difference in exosome concentrations in plasma from healthy controls, patients who progress within 18 months, or patients who do not relapse at 18 months. d: Exosomes isolated from NSCLC patients show enrichment of VCP in patients who progress at 18 months compared to patients who do not relapse and healthy controls (FLOT1 is used as a loading target). e: The hypoxic exosome signature is upregulated in exosomes derived from patients who progress at 18 months. f: The number of hypoxic protein markers above the Youden index threshold demonstrates a clear separation between patients who progress within 18 months and those who relapse at 18 months. g: Kaplan-Meier shows a clear separation of patient DFS based on the abundance of proteins from the hypoxic exosome signature (≥3 markers above the Youden index value). h: ROC curve demonstrates that the hypoxic exosome signature is a perfect prognostic marker for disease progression (<18 months) in NSCLC patients, whereas exosome signature concentration has no prognostic value. i: Kaplan-Meier curve demonstrates that the hypoxic exosome signature also correlates with overall survival in NSCLC patients. [Figure 7-3]Figure 7 shows that the hypoxic exosome signature predicts disease progression in NSCLC patients. a, b: Exosomes can be isolated from NSCLC plasma based on morphology, as shown by TEM (size bar 200 nm) and a size distribution ranging from 20 to 150 nm. c: TRPS demonstrates no difference in exosome concentrations in plasma from healthy controls, patients who progress within 18 months, or patients who do not relapse at 18 months. d: Exosomes isolated from NSCLC patients show enrichment of VCP in patients who progress at 18 months compared to patients who do not relapse and healthy controls (FLOT1 is used as a loading target). e: The hypoxic exosome signature is upregulated in exosomes derived from patients who progress at 18 months. f: The number of hypoxic protein markers above the Youden index threshold demonstrates a clear separation between patients who progress within 18 months and those who relapse at 18 months. g: Kaplan-Meier shows a clear separation of patient DFS based on the abundance of proteins from the hypoxic exosome signature (≥3 markers above the Youden index value). h: ROC curve demonstrates that the hypoxic exosome signature is a perfect prognostic marker for disease progression (<18 months) in NSCLC patients, whereas exosome signature concentration has no prognostic value. i: Kaplan-Meier curve demonstrates that the hypoxic exosome signature also correlates with overall survival in NSCLC patients. [Figure 7-4]Figure 7 shows that the hypoxic exosome signature predicts disease progression in NSCLC patients. a, b: Exosomes can be isolated from NSCLC plasma based on morphology, as shown by TEM (size bar 200 nm) and a size distribution ranging from 20 to 150 nm. c: TRPS demonstrates no difference in exosome concentrations in plasma from healthy controls, patients who progress within 18 months, or patients who do not relapse at 18 months. d: Exosomes isolated from NSCLC patients show enrichment of VCP in patients who progress at 18 months compared to patients who do not relapse and healthy controls (FLOT1 is used as a loading target). e: The hypoxic exosome signature is upregulated in exosomes derived from patients who progress at 18 months. f: The number of hypoxic protein markers above the Youden index threshold demonstrates a clear separation between patients who progress within 18 months and those who relapse at 18 months. g: Kaplan-Meier shows a clear separation of patient DFS based on the abundance of proteins from the hypoxic exosome signature (≥3 markers above the Youden index value). h: ROC curve demonstrates that the hypoxic exosome signature is a perfect prognostic marker for disease progression (<18 months) in NSCLC patients, whereas exosome signature concentration has no prognostic value. i: Kaplan-Meier curve demonstrates that the hypoxic exosome signature also correlates with overall survival in NSCLC patients. [Figure 8-1]Figure 8 shows that the hypoxic exosome signature originates from lung cells undergoing EMT. a: GSEA identified a prominent epithelial-mesenchymal transition gene set significantly associated with exosomes derived from hypoxic NSCLC cells. b: Immunofluorescence of normal lung epithelial cells (30KT) and transformed lung mesenchymal cells (30KTp53 / KRAS / LKB1) demonstrating a pro-oncogenic phenotypic transition to a stromal phenotype. c: Western blot of cell-like structures demonstrates the loss of the epithelial marker E-cadherin and the gain of the mesenchymal marker vimentin in 30KTp53 / KRAS / LKB1 cells. d: Western blot of VCP in exosomes derived from epithelial (30KT) and mesenchymal (30KTp53 / KRAS / LKB1) lung cells (CD9 was used as a loading control). e: ELISA of MAC2BP, TNC, PSMA2, and THBS1 in exosomes derived from epithelial (30KT) and mesenchymal (30KTp53 / KRAS / LKB1) lung cells. *p<0.05, **p<0.01, ***p<0.001. f: Immunohistochemistry of primary tumors demonstrates that loss of E-cadherin expression correlates with patients classified into the high signature group (≥3 markers above Youden's index). [Figure 8-2]Figure 8 shows that the hypoxic exosome signature originates from lung cells undergoing EMT. a: GSEA identified a prominent epithelial-mesenchymal transition gene set significantly associated with exosomes derived from hypoxic NSCLC cells. b: Immunofluorescence of normal lung epithelial cells (30KT) and transformed lung mesenchymal cells (30KTp53 / KRAS / LKB1) demonstrating a pro-oncogenic phenotypic transition to a stromal phenotype. c: Western blot of cell-like structures demonstrates the loss of the epithelial marker E-cadherin and the gain of the mesenchymal marker vimentin in 30KTp53 / KRAS / LKB1 cells. d: Western blot of VCP in exosomes derived from epithelial (30KT) and mesenchymal (30KTp53 / KRAS / LKB1) lung cells (CD9 was used as a loading control). e: ELISA of MAC2BP, TNC, PSMA2, and THBS1 in exosomes derived from epithelial (30KT) and mesenchymal (30KTp53 / KRAS / LKB1) lung cells. *p<0.05, **p<0.01, ***p<0.001. f: Immunohistochemistry of primary tumors demonstrates that loss of E-cadherin expression correlates with patients classified into the high signature group (≥3 markers above Youden's index). [Figure 9-1] Figure 9 shows confirmation that the hypoxic exosome signature predicts disease recurrence in NSCLC patients. a, b: 18F-FDG PET / CT images of two patients (validation cohort) followed at the indicated points in c. c: In support of the discovery cohort, exosome concentrations are similar in patients who relapse within 18 months compared to patients who relapse after 18 months, particularly in patients 44 and 53. d: The number of hypoxic proteins exceeding Youden's index threshold demonstrates a clear distinction between patients who progress within 18 months and those who do not relapse at 18 months. e: Kaplan-Meier plot of DFS for NSCLC patients with low or high abundance hypoxic exosomal proteins shows a clear distinction in DFS. f: ROC curve analysis also shows the complete classification of patients who will progress within 18 months. g: Kaplan-Meier plot confirms that this signature is also a prognostic marker for overall survival in NSCLC patients. [Figure 9-2] Figure 9 shows confirmation that the hypoxic exosome signature predicts disease recurrence in NSCLC patients. a, b: 18F-FDG PET / CT images of two patients (validation cohort) followed at the indicated points in c. c: In support of the discovery cohort, exosome concentrations are similar in patients who relapse within 18 months compared to patients who relapse after 18 months, particularly in patients 44 and 53. d: The number of hypoxic proteins exceeding Youden's index threshold demonstrates a clear distinction between patients who progress within 18 months and those who do not relapse at 18 months. e: Kaplan-Meier plot of DFS for NSCLC patients with low or high abundance hypoxic exosomal proteins shows a clear distinction in DFS. f: ROC curve analysis also shows the complete classification of patients who will progress within 18 months. g: Kaplan-Meier plot confirms that this signature is also a prognostic marker for overall survival in NSCLC patients. [Figure 10] Figure 10 shows that hypoxia increases exosome secretion from NSCLC cells. a: Exosomes isolated from NSCLC cells express standard exosome markers HSP70, FLOT1, and CD63. The cellular marker CANX is found only in cell lysates, not exosome lysates. b: Hypoxia increases exosome secretion from NSCLC cell lines. n = 3 ± SEM, *p<0.05, **p<0.01, ***p<0.001. [Figure 11-1] Figure 11 shows that the discovery cohort demonstrates that exosomal proteins are associated with disease progression in NSCLC patients. ae: Individual Kaplan-Meier and ROC curves for each protein in the hypoxic exosome signature. [Figure 11-2] Figure 11 shows that the discovery cohort demonstrates that exosomal proteins are associated with disease progression in NSCLC patients. ae: Individual Kaplan-Meier and ROC curves for each protein in the hypoxic exosome signature. [Figure 11-3]Figure 11 shows that the discovery cohort demonstrates that exosomal proteins are associated with disease progression in NSCLC patients. ae: Individual Kaplan-Meier and ROC curves for each protein in the hypoxic exosome signature. [Figure 11-4] Figure 11 shows that the discovery cohort demonstrates that exosomal proteins are associated with disease progression in NSCLC patients. ae: Individual Kaplan-Meier and ROC curves for each protein in the hypoxic exosome signature. [Figure 12-1] Figure 12 shows that gene set enrichment analysis (GSEA) identified gene sets significantly increased in exosomes from hypoxic NSCLC cells. a: Heatmap of proteins identified in the EMT gene set. be: GSEA using the entire exosomal protein expression dataset against the signature gene set reveals that hypoxic exosomes are enriched in proteins related to glycolysis, MYC targets, E2F targets, and xenobiotic metabolism (FDR<0.05). NES-normalized enrichment scores. [Figure 12-2] Figure 12 shows that gene set enrichment analysis (GSEA) identified gene sets significantly increased in exosomes from hypoxic NSCLC cells. a: Heatmap of proteins identified in the EMT gene set. be: GSEA using the entire exosomal protein expression dataset against the signature gene set reveals that hypoxic exosomes are enriched in proteins related to glycolysis, MYC targets, E2F targets, and xenobiotic metabolism (FDR<0.05). NES-normalized enrichment scores. [Figure 12-3]Figure 12 shows that gene set enrichment analysis (GSEA) identified gene sets significantly increased in exosomes from hypoxic NSCLC cells. a: Heatmap of proteins identified in the EMT gene set. be: GSEA using the entire exosomal protein expression dataset against the signature gene set reveals that hypoxic exosomes are enriched in proteins related to glycolysis, MYC targets, E2F targets, and xenobiotic metabolism (FDR<0.05). NES-normalized enrichment scores. [Figure 13] Figure 13 shows that reduced E-cadherin expression correlates with the number of signature proteins above Youden's index threshold. a: Table of IHC scores against signature scores. b: Low E-cadherin IHC scores are significantly more associated with patients relapsing within 18 months. [Figure 14] Figure 14 shows that upregulated signature proteins correlate with DFS in the validation cohort. a: Western blot of VCP demonstrates upregulation in patients progressing within 18 months compared to patients progressing after 18 months (FLOT1 is used as a loading control). b: Individual signature values ​​for patients 44 and 53 show that patient 53, who progresses within 18 months, has significantly increased baseline levels of signature protein compared to patient 44. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention is based, at least in part, on the surprising discovery that hypoxia-induced exosomal proteins identified in vitro are accurate prognostic biomarkers of cancer progression and aggressiveness in patients.

[0030] In one aspect, the present invention provides a method of determining the aggressiveness of cancer in a subject, comprising determining the expression level of one or more markers in an exosome sample of the subject, wherein said markers comprise one or more of those proteins listed in Table 1 and / or Table 2, and wherein the expression level of the one or more markers is indicative of or correlates with the level of aggressiveness of the cancer.

[0031] In a related aspect, the present invention provides a method of determining the prognosis of cancer in a subject, comprising determining the expression level of one or more markers in an exosome sample from the subject, wherein said markers comprise one or more of those proteins listed in Table 1 and / or Table 2, and wherein the expression level of said one or more markers is indicative of or correlates with a less or more favorable prognosis for said cancer.

[0032] For the above aspects, the one or more markers are suitably selected from the group consisting of galectin-3-binding protein, transitional endoplasmic reticulum ATPase, neutral α-glucosidase AB, 60 kDa heat shock protein, lysyl oxidase homolog 2, tenascin C, fatty acid synthase, agrin, aspartyl aminopeptidase, proteasome subunit α1, proteasome subunit α2, proteasome subunit α3, proteasome subunit α4, and proteasome subunit α5. , proteasome subunit alpha 6, proteasome subunit beta 1, proteasome subunit beta 2, proteasome subunit beta 3, proteasome subunit beta 4, proteasome subunit beta 5, proteasome subunit beta 6, proteasome subunit beta 7, proteasome subunit beta 8, thrombospondin-1, latent transforming growth factor beta binding protein 3, and any combination thereof. According to one particular embodiment, the one or more markers are selected from the group consisting of galectin-3-binding protein, transitional endoplasmic reticulum ATPase, tenascin C, proteasome subunit alpha 2, thrombospondin-1, and any combination thereof.

[0033] As generally used herein, the expression level of one or more of (a) the 55 marker proteins identified as upregulated in Table 1; and (b) the 32 marker proteins identified as upregulated in Table 2 can refer to the expression level of the nucleic acid (e.g., RNA, mRNA, and cDNA) encoding the protein, the protein itself, or both, unless otherwise specified.

[0034] As generally used herein, the terms "cancer" and "tumor." "Malignant" and "malignancy" refer to a disease or condition, or cells or tissues associated with that disease or condition, characterized by aberrant or abnormal cell proliferation, differentiation, and / or metastasis, often accompanied by one or more gene mutations or other genetic changes associated with oncogenesis, tumor marker expression, loss of tumor suppressor expression or activity, and / or aberrant or abnormal cell surface marker expression.

[0035] "Aggressiveness" and "aggressive" refer to the quality or tendency of a cancer to have a relatively poor prognosis due to one or more of the following characteristics or combinations of factors, including, but not limited to: at least partial resistance to available therapies for the treatment of the cancer; invasiveness; potential for metastasis; recurrence after treatment; and a low likelihood of patient survival.

[0036] In certain embodiments, the proteins provided herein, such as those provided in Tables 1 and 2, are prognostic for aggressive disease, and particularly for shorter time to pathological recurrence and / or shorter patient survival. In further embodiments, the proteins provided herein, such as those provided in Tables 1 and 2, correlate with or are indicative of metastatic cancer, and more particularly, metastatic NSCLC. In this regard, it will be apparent that, for example, with the exception of LTBP3, the 32 proteins provided in Table 2 are also listed in Table 1.

[0037] Cancer includes any aggressive or potentially aggressive cancer, tumor, or other malignancy as listed in the NCI Cancer Index at http: / / www.cancer.gov / cancertopics / alphalist, including all major forms of cancer, such as, but not limited to, sarcoma, carcinoma, lymphoma, leukemia, and blastoma, including, but not limited to, breast cancer, lung cancer, including lung adenocarcinoma and mesothelioma, cancers of the reproductive system, including ovarian cancer, cervical cancer, uterine cancer, and prostate cancer, cancers of the brain and nervous system, head and neck cancer, gastrointestinal cancer, including colon cancer, colorectal cancer, and stomach cancer, liver cancer, kidney cancer, skin cancer, e.g., melanoma and skin carcinoma, blood cancer, including lymphatic cancer and myelomonocytic cancer, cancers of the endocrine system, e.g., pancreatic cancer and pituitary cancer, and musculoskeletal system cancer, including bone and soft tissue cancer.

[0038] In certain embodiments, the cancer comprises breast cancer, lung cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, cancer of the brain or nervous system, head and neck cancer, colon cancer, colorectal cancer, stomach cancer, liver cancer, kidney cancer, bladder cancer, skin cancer, pancreatic cancer, pituitary cancer or adrenal cancer. More preferably, the cancer is or comprises lung cancer, e.g., NSCLC.

[0039] According to certain embodiments, the cancer of the aspects disclosed herein is or comprises lung cancer. For purposes herein, it will be apparent that lung cancer can include any aggressive lung cancer and subtypes known in the art, such as non-small cell carcinoma (i.e., squamous cell carcinoma, adenocarcinoma, and large cell carcinoma), small cell carcinoma, and mesothelioma. According to one preferred embodiment, the lung cancer is or comprises non-small cell lung cancer (NSCLC).

[0040] The terms "prognosis" and "prognostic" are used herein to include making a prognosis, which can provide for the prediction of clinical outcome (with or without medical treatment), the selection of an appropriate course of treatment (or whether treatment is effective), and / or the monitoring of current treatment and substantial changes in treatment. This may be combined with the determination of the expression levels of additional proteins and / or other nucleic acid biomarkers. It may be based, at least in part, on the determination of the gene and / or protein expression levels of one or more markers by the methods of the present invention. Prognosis may also include the prediction, forecast, or expectation of any lasting or permanent physical or psychological effects of cancer suffered by a subject after the cancer has been successfully treated or otherwise resolved. Furthermore, prognosis may include one or more of the following: determining the likelihood or recurrence of metastasis, therapeutic responsiveness, implementation of an appropriate treatment plan, determining the likelihood, propensity, or likelihood of cancer recurrence after treatment, and predicting the development of resistance to established treatments (e.g., chemotherapy). It will be understood that a positive prognosis typically refers to a favorable clinical outcome or outlook, such as long-term survival without recurrence of the subject's cancer, whereas a negative prognosis typically refers to a negative clinical outcome or outlook, such as cancer recurrence or progression.

[0041] In one embodiment of the methods of the two foregoing aspects, a relatively decreased expression level of one or more markers is indicative of or correlates with a more favorable prognosis and / or a less aggressive cancer; and / or a relatively increased expression level of one or more markers is indicative of or correlates with a less favorable prognosis and / or a more aggressive cancer.

[0042] According to one particular embodiment, the prognosis or aggressiveness of the cancer is used at least in part to determine the likelihood of metastasis of the cancer in said subject.

[0043] As used herein, "metastasis" or "metastatic" refers to the migration or metastasis of malignant cells or neoplasms from the primary focus of a tumor, cancer, or neoplasm to a distant site in the body, typically via the circulatory or lymphatic system or via a natural body cavity, and the subsequent development of one or more secondary tumors or colonies in one or more new locations. "Metastases" refers to secondary tumors or colonies formed as a result of metastasis, and includes micrometastases, and regional metastases, including lymph nodes, and distant metastases.

[0044] Suitably, a relatively decreased expression level of one or more markers indicates or correlates with a decreased likelihood of metastasis of said cancer; and / or a relatively increased expression level of one or more markers indicates or correlates with an increased likelihood of metastasis of said cancer.

[0045] According to one embodiment, cancer prognosis or aggressiveness is used, at least in part, to determine whether a subject will benefit from cancer treatment. For example, patients with a favorable prognosis and / or less aggressive cancer may be less likely to suffer from rapid local progression and / or metastasis of the cancer and / or may be spared more aggressive monitoring and / or treatment.

[0046] According to another embodiment, the cancer prognosis or aggressiveness is used, at least in part, to develop a treatment strategy for the subject.

[0047] According to one embodiment, cancer prognosis or aggressiveness is used, at least in part, to determine the progression or recurrence of disease in a subject.

[0048] According to one embodiment, cancer prognosis or aggressiveness is used, at least in part, to determine expected survival time.

[0049] For purposes of this invention, "isolated" means material that has been removed from its natural state or that has otherwise been subjected to human manipulation. Isolated material is substantially or essentially free from components that normally accompany it in its natural state, or it may be manipulated so as to exist in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in natural, chemically synthesized, or recombinant form.

[0050] As used herein, a "gene" is a nucleic acid that is a structural hereditary unit of a genome that can contain one or more amino acid-encoding nucleotide sequences and one or more non-coding nucleotide sequences, including, but not limited to, a promoter, its 5' untranslated sequences, introns, polyadenylation sequences, and other 3' untranslated sequences. In most cellular organisms, a gene is a nucleic acid that comprises double-stranded DNA.

[0051] The term "nucleic acid" as used herein refers to single-stranded or double-stranded DNA or RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA, and autocatalytic RNA. Nucleic acids can also be DNA-RNA hybrids. Nucleic acids typically include a nucleotide sequence that includes nucleotides containing A, G, C, T, or U bases. However, the nucleotide sequence can include other bases, such as, but not limited to, inosine, methylcytosine, methylinosine, methyladenosine, and / or thiouridine.

[0052] Also included are "variant" nucleic acids that include naturally occurring (e.g., allelic) variant nucleotide sequences, and orthologs (e.g., from different species) of nucleic acids that respectively encode one or more markers provided herein. Preferably, nucleic acid variants share at least 70% or 75%, preferably at least 80% or 85%, or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequences disclosed herein.

[0053] Nucleic acid fragments are also included. A "fragment" is a segment, domain, portion, or region of a nucleic acid, each of which constitutes less than 100% of the nucleotide sequence. Non-limiting examples are amplification products or primers or probes. According to certain embodiments, a nucleic acid fragment can comprise, for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, and 7500 consecutive nucleotides of said nucleic acid.

[0054] As used herein, a "polynucleotide" is a nucleic acid having 80 or more consecutive nucleotides, while an "oligonucleotide" has fewer than 80 consecutive nucleotides. A "probe" can be a single- or double-stranded oligonucleotide or polynucleotide that is appropriately labeled for detecting complementary sequences, for example, in Northern or Southern blots. A "primer" is typically a single-stranded oligonucleotide, preferably having 15 to 50 consecutive nucleotides, that can anneal to a complementary nucleic acid "template" and be extended in a template-dependent manner by the action of a DNA polymerase, such as Taq polymerase, RNA-dependent DNA polymerase, or Sequenase™. A "template" nucleic acid is a nucleic acid that is subjected to nucleic acid amplification.

[0055] "Protein" refers to an amino acid polymer. The amino acids may be natural or unnatural amino acids, D- or L-amino acids, as is well understood in the art. As will be understood by those skilled in the art, the term "protein" also includes within its scope phosphorylated forms of proteins (i.e., phosphoproteins) and / or glycosylated forms of proteins (i.e., glycoproteins). A "peptide" is a protein having 50 or fewer amino acids. A "polypeptide" is a protein having more than 50 amino acids.

[0056] Also provided are protein "variants," such as naturally occurring variants (e.g., allelic variants), and their orthologs or isoforms, of one or more markers provided herein, e.g., those listed in Tables 1 and 2. Preferably, protein variants share at least 70% or 75%, preferably at least 80% or 85%, or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of one or more markers disclosed herein or known in the art. To this end, Tables 1 and 2 also include accession numbers that refer to exemplary protein sequences of the listed protein markers, as is well understood in the art and incorporated by reference herein.

[0057] Protein fragments are also provided, including peptide fragments that comprise less than 100% of the entire amino acid sequence. According to certain embodiments, the protein fragment can comprise, for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, and 1200 consecutive amino acids of the protein.

[0058] Those skilled in the art will recognize that exosomes are small (i.e., typically 30-150 nm), cell-derived membrane vesicles of endocytic origin. They contain lipids, nucleic acids, and proteins and are released into the extracellular environment upon fusion with the plasma membrane. They are generally characterized by the presence of marker proteins, including CD63, CD9, HSP70, flotillin-1, and TSG101, as well as their morphology and size.

[0059] In the methods of the present invention, the exosome sample comprising one or more exosomes may comprise or be obtained from most biological fluids, including, but not limited to, blood, serum, plasma, ascites, cyst fluid, pleural effusion, ascites, cerebrospinal fluid, tears, urine, saliva, sputum, nipple aspirate, lymphatic fluid, fluids of the respiratory tract, intestinal tract, genitourinary tract, breast milk, intraorgan system fluids, or combinations thereof. To this end, the exosome sample may be isolated or purified from biological fluids or samples, such as those provided above, to facilitate removal of contaminating proteins, lipoproteins, etc.

[0060] To this end, exosomes or exosomal proteins may be isolated by any means known in the art, including but not limited to, ultracentrifugation, size exclusion chromatography, exosome precipitation (e.g., ExoQuick from System Biosciences), affinity-based capture of exosomes (e.g., affinity purification using antibodies against CD63, CD81, CD82, CD9, Alix, annexin, EpCAM, and Rab5), and combinations thereof.

[0061] As will be understood by those skilled in the art, the gene and / or protein expression level of one or more proteins provided herein may be relatively (i) higher or greater; or (ii) lower, reduced, or attenuated compared to the expression level in a control or reference sample, or a threshold expression level. In one embodiment, an expression level may be classified as higher, increased, or greater if it exceeds the mean and / or median expression level of a reference population. According to one embodiment, an expression level may be classified as lower, reduced, or attenuated if it is less than the mean and / or median expression level of a reference population. In this regard, the reference population may be a control group having the same cancer type, subgroup, stage, and / or grade as the mammal for which the expression level is determined.

[0062] As used herein, the terms "higher," "increased," and "greater" refer to an increased amount or level of a nucleic acid and / or protein in an exosome sample when compared to a control or reference level or amount. The nucleic acid and / or protein expression level of one or more markers can be relative or absolute. In some embodiments, the gene and / or protein expression of one or more markers is An expression level is higher, increased or greater if the expression level is about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, or at least about 500% higher than the level of gene and / or protein expression of the respective or corresponding protein at a control or reference level or amount.

[0063] As used herein, the terms "lower," "reduced," and "decreased" refer to a lower amount or level of a nucleic acid and / or protein, for example, in an exosome sample, when compared to a control or reference level or amount. The nucleic acid and / or protein expression levels of one or more markers provided herein can be relative or absolute. According to some embodiments, gene and / or protein expression of one or more markers is lower, decreased, or attenuated if its expression level is less than about 1%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, or 0.0001%, of the gene and / or protein expression level or amount of the respective or corresponding protein at the control or reference level or amount.

[0064] The term "control sample" typically refers to a biological sample, such as an exosome sample, from a non-diseased (healthy) individual who does not have cancer. In one embodiment, the control sample may be from a subject known to be cancer-free, or a sample obtained from the subject at an earlier time point. Alternatively, the control sample may be from a subject in remission from cancer. The control sample may be a pooled sample, an average sample, or an individual sample. An internal control is a marker from the same biological sample (e.g., an exosome sample) being tested.

[0065] As used herein, gene and / or protein expression levels may be absolute or relative amounts. Thus, according to some embodiments, the gene and / or protein expression levels of one or more markers provided herein are compared to a control level of expression, such as the gene and / or protein expression levels of one or more "housekeeping" genes and / or proteins in a subject's exosome sample.

[0066] According to further embodiments, the gene and / or protein expression levels of one or more markers are compared to a threshold level of expression, e.g., the level of gene and / or protein expression in an exosome sample. The threshold level of expression is generally a quantified level of gene and / or protein expression of one or more markers of the invention. Typically, gene and / or protein expression levels of one or more markers in an exosome sample that are above or below the threshold level of expression are predictive of a particular disease state or outcome. The nature of expression and the value (if any) of the threshold level will typically vary based on the method selected for determining the expression of one or more genes or their products, for example, to determine prognosis and / or response to anti-cancer therapy in a subject.

[0067] Those skilled in the art will be able to use any method for measuring gene or protein expression known in the art, such as those described herein, to determine a threshold level of gene and / or protein expression in an exosome sample that can be used to determine prognosis and / or response to anti-cancer therapy. In one embodiment, the threshold level is the average and / or median gene and / or protein expression level (median or absolute value) of one or more markers in a reference population having the same cancer type, subgroup, stage, and / or grade as the subject whose expression level is being determined. Furthermore, the concept of a threshold level of expression should not be limited to a single value or result. In this regard, a threshold level of expression can encompass multiple threshold expression levels that may indicate, for example, a high, medium, or low likelihood of metastasis of the subject's cancer.

[0068] In one embodiment, lower gene and / or protein expression levels of one or more markers provided herein are indicative of or correlate with a relatively increased responsiveness of the cancer to anti-cancer treatment, hi another embodiment, closer gene and / or protein expression levels of one or more markers provided herein are indicative of or correlate with a relatively decreased responsiveness of the cancer to anti-cancer treatment.

[0069] The terms "determining," "measuring," "evaluating," "assessing," and "assaying" are used interchangeably herein and can include any form of measurement known in the art, such as those described hereinafter.

[0070] Determining, assessing, evaluating, assaying, or measuring the nucleic acids, such as RNA, mRNA, and cDNA, corresponding to one or more markers provided herein can be performed by any technique known in the art, including nucleic acid sequence amplification, nucleic acid hybridization, nucleotide sequencing, mass spectrometry, and any combination thereof.

[0071] Nucleic acid amplification techniques typically involve repeated cycles of annealing one or more primers to a "template" nucleotide sequence under appropriate conditions and using a polymerase to synthesize a nucleotide sequence complementary to the target, thereby amplifying the target nucleotide sequence. Nucleic acid amplification techniques are well known to those skilled in the art and include, but are not limited to: polymerase chain reaction (PCR); strand displacement amplification (SDA); rolling circle replication (RCR); nucleic acid sequence-based amplification (NASBA); Qβ replicase amplification; helicase-dependent amplification (HAD); loop-mediated isothermal amplification (LAMP); nicking enzyme amplification reaction (NEAR) and recombinase polymerase amplification (RPA). As generally used herein, "amplification product" refers to a nucleic acid product produced by a nucleic acid amplification technique.

[0072] PCR includes: quantitative and semi-quantitative PCR, real-time PCR, allele-specific PCR, methylation-specific PCR, asymmetric PCR, nested PCR, multiplex PCR, touchdown PCR, digital PCR, and other variations and modifications to the "basic" PCR amplification.

[0073] Nucleic acid amplification techniques can be performed using DNA or RNA extracted, isolated, or otherwise obtained from a cell or tissue source. According to other embodiments, nucleic acid amplification can be performed directly on appropriately processed cell or tissue samples.

[0074] Nucleic acid hybridization typically involves hybridizing a nucleotide sequence, typically in the form of a probe, to a target nucleotide sequence under appropriate conditions, whereby the hybridized probe-target nucleotide sequence is subsequently detected. Non-limiting examples include, but are not limited to, Northern blotting, slot blotting, in situ hybridization, and fluorescence resonance energy transfer (FRET) detection. Nucleic acid hybridization can be performed using DNA or RNA extracted, isolated, amplified, or otherwise obtained from a cell or tissue source, or directly on an appropriately processed cell or tissue sample.

[0075] It will also be appreciated that a combination of nucleic acid amplification and nucleic acid hybridization may be utilized.

[0076] Determining, evaluating, assessing, assaying, or measuring the protein level of one or more exosomal proteins can be performed by any technique known in the art that can detect such proteins, whether expressed on the surface of exosomes or internally, or proteins isolated, extracted, or otherwise obtained from a subject's exosome sample. These techniques include, but are not limited to, antibody-based detection using one or more antibodies that bind the protein, electrophoresis, isoelectric focusing, protein sequencing, chromatographic techniques, and mass spectrometry, as well as combinations thereof. Antibody-based detection can include, but is not limited to, flow cytometry using fluorescently labeled antibodies, ELISA, immunoblotting, immunoprecipitation, radioimmunoassay (RIA), and immunocytochemistry.

[0077] It will be understood that expression of one or more markers provided herein can include determination of both their nucleic acid levels, such as by nucleic acid amplification and / or nucleic acid hybridization, and their protein levels. Thus, detecting and / or measuring expression of one or more markers from a subject's exosome sample can be performed by any of the methods described herein or a combination thereof (for example, but not limited to, by measuring mRNA levels or amplified cDNA copies thereof, and / or their protein products).

[0078] In view of the above, it will be further understood that the expression level of one or more markers provided herein may be the absolute or relative amount of the expressed gene or its gene product, including nucleic acids, e.g., RNA, mRNA and cDNA, and / or protein.

[0079] Suitably, the method of the foregoing aspect further comprises the step of diagnosing said subject as having (i) a highly aggressive cancer or a less aggressive cancer; and / or (ii) a less favorable prognosis or a more favorable prognosis.

[0080] In a further aspect, the present invention provides a method of predicting responsiveness of a cancer to an anti-cancer treatment in a subject, comprising determining the expression level of one or more markers in an exosome sample from the subject, wherein said markers comprise one or more of those proteins listed in Table 1 and / or Table 2, and wherein altered or regulated expression levels of said one or more markers are indicative of or correlate with a relatively increased or decreased responsiveness of the cancer to the anti-cancer treatment.

[0081] As will be understood by those skilled in the art, the expression level of a gene or protein may be considered "altered" or "medulated" if the expression level is higher / increased or lower / reduced compared to a control or reference sample, or expression level, e.g., a threshold level. In one embodiment, an expression level may be classified as high if it is greater than the mean and / or median relative expression level of a reference population, and an expression level may be classified as low if it is less than the mean and / or median relative expression level of a reference population. In this regard, a reference population may be a group of subjects having the same cancer type, subgroup, stage, and / or grade as the mammal for which the expression level is determined. Furthermore, expression levels may be relative or absolute.

[0082] Suitably, the one or more markers are galectin-3-binding protein, transitional endoplasmic reticulum ATPase, neutral α-glucosidase AB, 60 kDa heat shock protein, lysyl oxidase homolog 2, tenascin C, fatty acid synthase, agrin, aspartyl aminopeptidase, proteasome subunit α1, proteasome subunit α2, proteasome subunit α3, proteasome subunit α4, proteasome subunit α5, proteasome subunit α6, proteasome subunit α7, proteasome subunit α8, proteasome subunit α9, proteasome subunit α10, proteasome subunit α11, proteasome subunit α12, proteasome subunit α13, proteasome subunit α14, proteasome subunit α15, proteasome subunit α16, proteasome subunit α17, proteasome subunit α18, proteasome subunit α19 ...9, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, In one particular embodiment, the one or more markers are selected from the group consisting of proteasome subunit α6, proteasome subunit β1, proteasome subunit β2, proteasome subunit β3, proteasome subunit β4, proteasome subunit β5, proteasome subunit β6, proteasome subunit β7, proteasome subunit β8, thrombospondin-1, latent transforming growth factor β binding protein 3, and any combination thereof. According to one particular embodiment, the one or more markers are selected from the group consisting of galectin-3-binding protein, transitional endoplasmic reticulum ATPase, tenascin-C, proteasome subunit α2, thrombospondin-1, and any combination thereof.

[0083] In one embodiment, a higher expression level of one or more markers indicates or correlates with a relatively increased responsiveness of the cancer to anti-cancer treatment, hi another embodiment, a higher expression level of one or more markers indicates or correlates with a relatively decreased responsiveness of the cancer to anti-cancer treatment.

[0084] With respect to the above aspects of the invention, the method suitably includes the further step of treating cancer in the subject.

[0085] A further aspect of the invention relates to treating cancer in a subject.

[0086] In one particular embodiment, cancer treatment is carried out in combination with determining the expression level of one or more markers in an exosome sample of the subject, wherein said markers include one or more of those proteins listed in Table 1 and / or Table 2, and based on the determination made, anti-cancer treatment is initiated, continued, modified, or discontinued.

[0087] Suitably, the one or more markers are galectin-3-binding protein, transitional endoplasmic reticulum ATPase, neutral α-glucosidase AB, 60 kDa heat shock protein, lysyl oxidase homolog 2, tenascin C, fatty acid synthase, agrin, aspartyl aminopeptidase, proteasome subunit α1, proteasome subunit α2, proteasome subunit α3, proteasome subunit α4, proteasome subunit α5, proteasome subunit α6, proteasome subunit α7, proteasome subunit α8, proteasome subunit α9, proteasome subunit α10, proteasome subunit α11, proteasome subunit α12, proteasome subunit α13, proteasome subunit α14, proteasome subunit α15, proteasome subunit α16, proteasome subunit α17, proteasome subunit α18, proteasome subunit α19 ...9, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, In one particular embodiment, the one or more markers are selected from the group consisting of proteasome subunit α6, proteasome subunit β1, proteasome subunit β2, proteasome subunit β3, proteasome subunit β4, proteasome subunit β5, proteasome subunit β6, proteasome subunit β7, proteasome subunit β8, thrombospondin-1, latent transforming growth factor β binding protein 3, and any combination thereof. In one particular embodiment, the one or more markers are selected from the group consisting of galectin-3-binding protein, transitional endoplasmic reticulum ATPase, tenascin-C, proteasome subunit α2, thrombospondin-1, and any combination thereof.

[0088] In this regard, the methods described herein for predicting the responsiveness of a cancer to an anti-cancer agent can further include administering a therapeutically effective amount of an anti-cancer therapeutic agent, e.g., an anti-cancer agent, to the mammal. In a preferred embodiment, the anti-cancer therapeutic agent is administered when the gene and / or protein expression levels of one or more markers described herein indicate or correlate with a relatively increased responsiveness of the cancer to the anti-cancer agent.

[0089] Suitably, the agent is administered to the subject as a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient, In this regard, any dosage form and route of administration, such as those provided herein, may be used to provide the composition of the invention to the subject.

[0090] Cancer treatments include, but are not limited to, drug therapies such as small organic or inorganic molecules, radiation therapy, surgery, nutritional therapy, relaxation or meditation therapy, and other natural or holistic therapies. Generally, drugs (e.g., small organic or inorganic molecules), biomolecules (e.g., antibodies, inhibitory nucleic acids such as siRNA), or chemotherapeutic agents are referred to herein as "anti-cancer therapeutic agents" or "anti-cancer agents."

[0091] Cancer treatment methods may be prophylactic, preventative, or therapeutic, and are suitable for the treatment of cancer in mammals, particularly humans. As used herein, "treating" or "treatment" refers to a therapeutic intervention, course of action, or protocol that at least ameliorates the symptoms of cancer after cancer and / or its symptoms have at least begun to develop. As used herein, "preventing," "prevent," or "prevention" refers to a therapeutic intervention, course of action, or protocol that is initiated prior to the onset of cancer and / or cancer symptoms in order to prevent, inhibit, or delay the development or progression of cancer or symptoms.

[0092] The term "therapeutically effective amount" describes an amount of a particular drug sufficient to achieve a desired effect in a subject treated with that agent. For example, this may be the amount of a chemotherapeutic agent necessary to reduce, alleviate, and / or prevent cancer or a cancer-related disease, disorder, or condition. According to some embodiments, a "therapeutically effective amount" is sufficient to reduce or eliminate the symptoms of cancer. According to other embodiments, a "therapeutically effective amount" is an amount sufficient to achieve a desired biological effect, e.g., an amount that is effective in reducing or preventing cancer growth and / or metastasis.

[0093] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce a desired result without causing substantial cytotoxic effects in the subject. The effective amount of an agent useful for reducing, alleviating, and / or preventing cancer will depend on the subject being treated, the type and severity of any associated disease, disorder, and / or condition (e.g., the number and location of any associated metastases), and the method of administration of the therapeutic composition.

[0094] Suitably, the anti-cancer therapeutic agent is administered to the mammal as a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient.

[0095] "Pharmaceutically acceptable carrier, diluent, or excipient" means a solid or liquid filler, diluent, or encapsulating substance that can be used for complete systemic administration. A variety of carriers well known in the art can be used depending on the particular route of administration. These carriers can be selected from the group consisting of sugars, starch, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline, and salts, e.g., mineral acid salts including hydrochlorides, bromides, and sulfates, organic acid salts such as acetates, propionates, malonates, and pyrogen-free water.

[0096] A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ USA, 1991), which is incorporated herein by reference.

[0097] Any safe route of administration can be used to provide the compositions of the present invention to a patient, such as oral, rectal, parenteral, sublingual, buccal, intravenous, intraarticular, intramuscular, intradermal, subcutaneous, inhalation, intraocular, intraperitoneal, intracerebroventricular, transdermal, and the like. Intramuscular and subcutaneous injections are suitable, for example, for administering immunotherapeutic compositions, proteinaceous vaccines, and nucleic acid vaccines.

[0098] Dosage forms include tablets, dispersions, suspensions, injectables, solutions, syrups, lozenges, capsules, suppositories, aerosols, transdermal patches, and the like. These dosage forms may also include injectable or implantable controlled-release devices specifically designed for this purpose, or other forms of implants modified to further function in this manner. Controlled release of therapeutic agents may be achieved by coating the therapeutic agent with hydrophobic polymers, including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids, and certain cellulose derivatives, such as hydroxypropylmethylcellulose. Additionally, controlled release may be achieved using other polymer matrices, liposomes, and / or microspheres.

[0099] Compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets, each containing a predetermined amount of one or more therapeutic agents of the present invention; as a powder or granules; or as a solution or suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or an oil-in-water liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy, but all methods include the step of bringing into association one or more agents as described above with the carrier, which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing an agent of the present invention with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired presentation.

[0100] The compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically effective. In the present invention, the dose administered to a patient should be sufficient to effect a beneficial response in the patient over a reasonable period of time. The amount of drug administered will depend on the subject to be treated, including the age, sex, weight, and general health of the patient, factors that may depend on the judgment of the practitioner.

[0101] In certain embodiments, anti-cancer treatments and / or agents may be directed to inhibiting the action of and / or reducing the expression of one or more markers.

[0102] In other embodiments, the anti-cancer treatment and / or medicament may be directed to preventing or inhibiting the metastasis of cancer.

[0103] In another embodiment, anti-cancer treatments and / or agents may be directed to genes or gene products other than one or more markers of the invention. By way of example, anti-cancer treatments may target genes or gene products known to interact, directly or indirectly, with one or more markers.

[0104] According to certain embodiments, the present invention provides a "companion diagnostic" for a cancer treatment, whereby the expression level of one or more markers of the present invention provides clinicians and others with information to be used for the safe and / or effective administration of said cancer treatment.

[0105] Suitably, the cancer is of, but is not limited to, the types listed above.

[0106] With reference to the above embodiment, the method suitably comprises the initial step of obtaining an exosome sample from a subject, e.g., from a biological sample thereof, and / or using an isolation method as described above.

[0107] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) contacting a cell expressing a marker listed in Table 1 and / or Table 2 with a candidate agent; and (b) a method for identifying or producing an agent for use in treating cancer in a subject, comprising determining whether the candidate agent modulates the expression and / or activity of a marker.

[0108] In certain embodiments, the candidate agent at least partially reduces, eliminates, suppresses or inhibits the expression and / or activity of the marker.

[0109] Suitably, the agent has few or no significant off-target and / or non-specific effects.

[0110] Preferably, the agent is an antibody or a small molecule.

[0111] Suitably, the markers are galectin-3-binding protein, transitional endoplasmic reticulum ATPase, neutral α-glucosidase AB, 60 kDa heat shock protein, lysyl oxidase homolog 2, tenascin C, fatty acid synthase, agrin, aspartyl aminopeptidase, proteasome subunit α1, proteasome subunit α2, proteasome subunit α3, proteasome subunit α4, proteasome subunit α5, proteasome subunit α6, proteasome subunit α7, proteasome subunit α8, proteasome subunit α9, proteasome subunit α10, proteasome subunit α11, proteasome subunit α12, proteasome subunit α13, proteasome subunit α14, proteasome subunit α15, proteasome subunit α16, proteasome subunit α17, proteasome subunit α18, proteasome subunit α19 ...9, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome In one particular embodiment, the one or more markers are selected from the group consisting of proteasome subunit α6, proteasome subunit β1, proteasome subunit β2, proteasome subunit β3, proteasome subunit β4, proteasome subunit β5, proteasome subunit β6, proteasome subunit β7, proteasome subunit β8, thrombospondin-1, latent transforming growth factor β binding protein 3, and any combination thereof. According to one particular embodiment, the one or more markers are selected from the group consisting of galectin-3-binding protein, transitional endoplasmic reticulum ATPase, tenascin-C, proteasome subunit α2, thrombospondin-1, and any combination thereof.

[0112] According to embodiments relating to antibody inhibitors, the antibodies can be polyclonal or monoclonal, natural or recombinant. Well-known protocols applicable to antibody production, purification, and use can be found, for example, in Chapter 2 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991-1994), and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988, both of which are incorporated herein by reference.

[0113] Generally, the antibodies of the present invention bind to or conjugate with an isolated protein, fragment, variant, or derivative of a marker. For example, the antibody may be a polyclonal antibody. Such antibodies may be prepared by injecting an isolated protein, fragment, variant, or derivative of a marker protein into a production species, including mice or rabbits, to obtain polyclonal antisera. Methods for generating polyclonal antibodies are well known to those skilled in the art. Exemplary protocols that may be used are described in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra, and Harlow & Lane, 1988, supra.

[0114] Monoclonal antibodies can be produced using standard methods, e.g., as described in the article by Kohler & Milstein, 1975, Nature 256, 495, incorporated herein by reference, or by more recent modifications, such as those described in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra, by immortalizing spleen or other antibody-producing cells from a production species inoculated with one or more isolated marker protein products and / or fragments, variants and / or derivatives thereof.

[0115] Typically, the inhibitory activity of a candidate inhibitor antibody can be assessed by in vitro and / or in vivo assays that detect or measure the expression level and / or activity of a marker protein in the presence of the antibody.

[0116] In some embodiments, modulators, e.g., inhibitors, can be rationally designed. These methods can involve structural analysis of the marker and the design and / or construction of molecules that bind to, interact with, or otherwise modulate the activity of the marker. These methods can, inter alia, involve computer-assisted three-dimensional modeling of the interaction between a candidate modulator and the marker.

[0117] In other embodiments, modulators, e.g., small organic molecule inhibitors, can involve screening large compound libraries of tens to millions of candidate inhibitors (compounds including synthetic small organic molecules or natural products such as inhibitory peptides or proteins) that can be screened or tested for biological activity at any one of hundreds of molecular targets to find potential new drugs or lead compounds. Screening methods can include, but are not limited to, computer-based ("in silico") screening and high-throughput screening based on in vitro assays.

[0118] Typically, active compounds, or "hits," from this initial screening process are then sequentially tested through a series of other in vitro and / or in vivo tests to further characterize the active compounds. Successively fewer "successful" compounds at each stage are selected for subsequent testing, ultimately leading to one or more drug candidates that are selected to proceed to be tested in human clinical trials.

[0119] At the clinical level, screening of candidate drugs involves obtaining samples from test subjects before and after the subjects are exposed to a test compound. The level of a marker protein in the sample, such as an exosome sample, is then measured and analyzed to determine whether the level and / or activity of the marker protein changes after exposure to the candidate drug. By way of example, the level of a protein product in a sample can be determined by mass spectrometry, Western blot, ELISA, electrochemistry, and / or any other suitable means known to those skilled in the art.

[0120] In this regard, candidate agents identified as being capable of reducing, eliminating, suppressing, or inhibiting the expression level and / or activity of a marker can then be administered to a patient suffering from cancer. For example, administration of a candidate drug that inhibits or reduces the activity and / or expression of a marker can treat cancer and / or reduce the risk of cancer if increased activity of the biomarker is at least partially responsible for the progression and / or development of cancer.

[0121] With respect to the above aspects, the term "subject" includes, but is not limited to, mammals, including humans, performance animals (e.g., horses, camels, greyhounds), livestock (e.g., cattle, sheep, horses), and companion animals (e.g., cats and dogs). Preferably, the subject is a human.

[0122] All computer programs, algorithms, patent and scientific literature mentioned herein is incorporated by reference.

[0123] With respect to the present invention, database accession numbers or unique identifiers provided herein for genes or proteins, such as those provided in Tables 1 and 2, and gene and / or protein sequences or sequences related thereto, are incorporated herein by reference.

[0124] In order that preferred embodiments of the present invention may be more fully understood and put to practical use, reference is made to the following non-limiting examples. [Example]

[0125] Example 1 Recent data suggest that tumor hypoxia is a powerful driving force for the secretion of factors that promote metastatic dissemination 8,9 A key component of the secreted factors thought to be involved in promoting metastasis is the release of exosomes. Increasing evidence suggests that the rich array of proteomic and genomic information carried by tumor-derived exosomes represents a novel mechanism by which cancer cells alter the behavior of surrounding stroma and malignant cells. 10 Exosomes play a key role in cardiovascular development 11 , immunosuppression 12 affecting signaling processes related to β-glucan and inducing drug resistance and oncogenic metastasis 13-15 Furthermore, the ability of exosomes to induce systemic changes appears to promote metastatic dissemination, which accounts for a large proportion of patient deaths. 16 .

[0126] Transfer of oncogenic proteins via exosomes has also been reported. 14 It has recently been demonstrated that exosome transfer in glioma cells promotes tumorigenesis through the delivery of a mutant epidermal growth factor receptor (EGFRvIII) isoform, leading to increased expression of anti-apoptotic genes and enhanced proliferation. 14 Similarly, colon cancer cells with mutant KRAS can enhance the three-dimensional growth of wild-type KRAS colon cancer cells through exosome transfer to mutant KRAS wild-type cells. Furthermore, non-metastatic melanoma cells can be induced to become more metastatic by uptake of exosomes derived from highly metastatic melanoma cells. 17 However, it remains unclear whether this change in metastatic potential is permanent.

[0127] The protein and RNA content of exosomes typically varies significantly depending on the cell type, tissue, and microenvironment from which they are derived. For this reason, cancer-secreted exosomes and their molecular weight represent potential sources of biomarkers and therapeutic targets in cancer. Therefore, the overall goal of this example was to establish a means to noninvasively predict disease progression in NSCLC patients using exosomes from their blood.

[0128] Currently, there is a great unmet need to develop non-invasive and informative diagnostic markers for various solid malignancies. The proteomic and RNA information contained in tumor-derived exosomes has generated great interest in the use of exosomes as non-invasive diagnostic tools. Because exosome isolation techniques are now well established, and because exosomes are stable in body fluids, including serum, urine, and saliva, they show great potential as reliable biomarkers of disease progression. 23 Given that exosomes can provide a molecular signature of their cell of origin, proteomic and RNA analysis may also provide an efficient means for determining oncogenic mutations. Recently, it has been shown that the presence of an exosome-based protein, in this case, Glypican-1, can predict short disease-free survival in pancreatic cancer patients. 24 .

[0129] Furthermore, patient-derived exosomes may prove useful in understanding disease progression and treatment options. This has already been demonstrated with exosomes isolated from melanoma patients, which showed high protein content and increased expression of TYRP2, VLA4, and HSP70; these proteins were enriched in patients with poor prognosis. 16 Furthermore, a number of different groups have identified retrotransposon RNA transcripts, single-stranded DNA (ssDNA), mitochondrial DNA, and oncogene amplification (i.e., cMyc) in microvesicles, and double-stranded DNA (dsDNA) in exosomes. 25Among the oncogenes in exosomes, cMet (melanoma) 16 , mutated KRAS, and p53 in pancreatic cancer 26 Thus, given the presence of these specific exosomal biomolecules coupled with their known release by tumor cells, exosomes may be useful as simplexed or multiplexed diagnostic biomarkers. 27 This could prove to be a clinically useful enriched template for 28 It has been reconsidered by

[0130] Materials and Methods Cell lines and cell cultures Human non-small cell lung cancer (NSCLC) cell lines were purchased from the American Type Culture Collection (ATCC). All cell lines were confirmed by shoot tandem repeat (STR) profiling and found to be negative for mycoplasma. All cells were maintained at 37°C in a humidified incubator with 5% CO2. SKMES1 cells were cultured in DMEM supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) and penicillin-streptomycin. All other cells were cultured in RPMI supplemented with 10% FBS and penicillin-streptomycin. For hypoxia experiments, cells were cultured at 37°C in a humidified incubator with 2% CO2 and 5% CO2.

[0131] Exosome isolation The serum medium was removed by washing the cells twice with PBS and replacing it with 15 ml of serum-free medium. The medium was conditioned for 24 hours in normoxia (21% O2) or hypoxia (2% O2). The conditioned medium was aliquoted into Falcon tubes, and floating cells and debris were removed by centrifugation at 300 x g for 10 minutes at 4°C. The resulting supernatant was filtered through a 0.22 μm filter to remove remaining large particles. The clarified, conditioned medium was concentrated to 300-500 μl at 4°C using a Centricon Plus-70 Centrifugal Filter (Ultracel-PL Membrane, 100 kDa). Exosomes were then purified using an OptiPrep® density gradient. The concentrated medium was layered onto a discontinuous iodixanol gradient and centrifuged at 100,000 g. avg The exosome-containing fraction was identified by tunable resistive pulse sensing (TRPS) and diluted to 20 ml in PBS and centrifuged at 100,000 g for 2 hours at 4°C. The resulting pellet was resuspended in PBS for further analysis.

[0132] Electron microscopy Exosomes were visualized using transmission electron microscopy (TEM). 3 μl of the exosome suspension was fixed in 50–100 μl of 2% paraformaldehyde. A 2 μl microfilter aliquot was then transferred to each of two Formvar-carbon-coated electron microscope grids and then covered for 20 min. The grids were washed and then transferred to 50 μl of uranyl oxalate solution (pH 7) for 5 min, followed by 50 μl of methylcellulose UA (a mixture of 4% uranyl acetate and 2% methylcellulose at a ratio of 100 μl / 900 μl, respectively) for 10 min on ice. The grids were removed, dried, and then observed in a JEM 1.011 transmission electron microscope at 80 kV.

[0133] Adjustable Resistive Pulse Sensing (TRPS) Exosome concentration and size were analyzed by TRPS (qNano, Izon Science Ltd) using an NP100 nanopore at a stretch of 45 mm. Exosome concentration and size were standardized using multiple pressure calibration with 70 nm carboxylated polystyrene beads of known concentration.

[0134] Western blotting The following antibodies were used for Western blot: TSG101 (Santa Cruz, sc-6037), CD63 (Abcam, ab8219), flotillin-1 (BD Transduction Laboratories, 610821), HSP70 (Transduction Laboratories, 610608), calnexin (Cell Signaling Technology, 2679S), VCP (Abcam, ab11433), and GANAB (Abcam, ab179805). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Thermo Scientific. Samples were dissolved in reducing sample buffer (0.25 M Tris-HCl (pH 6.8), 40% glycerol, 8% SDS, 5% 2-mercaptoethanol, and 0.04% bromophenol blue) or nonreducing sample buffer (without 2-mercaptoethanol) and boiled at 95°C for 10 min. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes, blocked with 5% nonfat dry milk in PBS-T (0.5% Tween-20), and probed with antibodies. Proteins were detected using X-ray film and enhanced chemiluminescence reagent (Amersham ECL Select).

[0135] ELISA The Douset ELISA was purchased from R&D Systems and used according to the manufacturer's instructions. Briefly, the capture antibody was diluted to a working concentration in PBS and placed in a 96-well microplate overnight at room temperature. The capture antibody was then removed, and the plate was washed three times with wash buffer. The plate was then blocked with reagent diluent for 2 hours and then washed three times with wash buffer. Standards and samples were then incubated in the plate for 2 hours and then washed as before. The plate was then incubated with detection antibody for 2 hours and then washed as before. Streptavidin-HRP was then added for 20 minutes, and the plate was subsequently washed again. Color was developed by the addition of substrate solution for 20 minutes, after which the reaction was stopped by the addition of stop solution. The optical density of each well was determined using a microplate reader set at 450 nm and wavelength correction at 540 nm.

[0136] The TNC ELISA kit was purchased from RayBiotech and used according to the manufacturer's instructions.

[0137] plasma Plasma was thawed on ice and centrifuged at 1,500 g for 10 minutes at 4°C. The supernatant was removed, and large vesicles were removed by another centrifugation step at 10,000 g for 20 minutes at 4°C. Next, 500 μl was layered onto a qEV size-exclusion column (Izon) and subsequently eluted with PBS. Exosome-positive fractions were pooled and concentrated to a final volume of 50–100 μl using an Amicon® Ultra-4 10 kDa centrifugal filter unit.

[0138] mass spectrometry Proteins from disrupted exosomes were proteolytically digested and analyzed on an LTQ-OrbitrapElite instrument coupled to a Waters NanoAcquity UltraHighPressure liquid chromatography system. The number of distinct proteins identifiable within the quantitatively distinct exosomes was processed via a number of purpose-specific software packages.

[0139] statistical analysis GraphPad Prism version 6.0 and MedCalc version 16.8.4 were used for all calculations. An unpaired Student's t-test was used to calculate the difference in exosome-derived protein expression values. Receiver operating characteristic (ROC) curves were used to determine the sensitivity and specificity of the predictive values. Thresholds were selected using the Youden index. Univariate analysis using the locked rank test was used to assess disease-free survival (Kaplan-Meier curves).

[0140] result This study is the first to demonstrate that exosomes are secreted by the NSCLC cell lines H358, SKMES1, H23, and H1975. Figure 1a shows the presence of canonical exosomal proteins and the absence of the endoplasmic reticulum protein calnexin from exosomes isolated using the above protocol. Furthermore, the isolated exosomes exhibit the expected morphology and size profile consistent with a pure exosome preparation (Figures 1b and 2a).

[0141] Next, these NSCLC cell lines were cultured under hypoxic conditions and the effect on exosome secretion was monitored. As can be seen in Figures 1c, 1d, and 2a, hypoxic conditions induced exosome secretion from each of the four cell lines examined, but the size range and morphology of the exosomes remained unchanged.

[0142] Next, this study sought to determine whether hypoxia altered the protein content or signature of exosomes secreted by NSCLC cell lines. Quantitative mass spectrometry demonstrated that exosomes from H358 and SKMES-1 cell lines contained 83 and 156 upregulated proteins, respectively, under hypoxic conditions, with 55 upregulated proteins common to both cell lines (Figure 2b, Table 1). Next, this study sought to validate this mass spectrometry data. To this end, two upregulated proteins identified by mass spectrometry, namely, neutral α-glucosidase AB (GANAB) and transitional endoplasmic reticulum ATPase (VCP), were shown by Western blot and ELISA to be upregulated in hypoxic exosomes from four NSCLC cell lines (Figures 2c and 2d), thereby supporting the mass spectrometry data.

[0143] Next, this study attempted to determine whether these hypoxia-upregulated proteins correlated with patient disease progression in NSCLC. As seen in Figure 3a, exosomes isolated from the plasma of NSCLC patients exhibited a typical size range and morphology. Next, hypoxia exosome protein markers GANAB, VCP, galectin-3-binding protein, TNC, and PMSA 2 were shown to be significantly upregulated in NSCLC patients with poor prognosis (i.e., progression or recurrence within the first 12 months of treatment) (Figure 3c). The ROC curve in Figure 3d further demonstrates that the combined protein signature of GANAB, VCP, and galectin-3-binding protein has high overall accuracy for identifying NSCLC patients with poor prognosis. This is supported by Figure 3e, which shows that NSCLC patients with upregulated exosomal expression of at least two proteins, GANAB, VCP, and galectin-3-binding protein, demonstrate significantly shorter disease-free survival than those patients who have only one or none of these markers highly expressed in their exosomes.

[0144] In addition to the exosomal protein markers GANAB, VCP, and galectin-3-binding protein, additional proteins from the original 55 hypoxia protein signature identified in NSCLC cell lines may also be of prognostic value. For example, Figure 4 demonstrates that tenascin C (TNC) protein levels are also upregulated in exosomes of NSCLC patients who are likely to progress after treatment. Furthermore, the ROC curve in Figure 4 shows that the signature itself demonstrates considerable accuracy for identifying NSCLC patients with poor prognosis.

[0145] The individual protein ROC and survival curves for the patient exosomal proteins GANAB, VCP, and galectin-3-binding protein (MAC2BP) shown in Figures 3d and 3e are provided in Figure 5. The data, by themselves, confirm that each of the three proteins is an accurate prognostic marker for disease progression in NSCLC patients.

[0146] conclusion These data indicate that the above protein markers identified in hypoxic exosomes in vitro represent potential prognostic biomarkers for disease progression or recurrence in NSCLC cancer patients. Thus, such exosomal biomarkers may represent reliable and non-invasive prognostic markers for various solid malignancies.

[0147] [Table 1-1] [Table 1-2]

[0148] References 1. Thun MJ, Lally CA, Flannery JT, Calle EE, Flanders WD, Heath CW, Jr. Cigarette smoking and changes in the histopathology of lung cancer [see comments]. J Natl Cancer Inst 1997;89:1580-6. 2. Mathers C, Vos T. The burden of disease and injury in Australia-summary report. Canberra: Australian Institute of Health and Welfare ISBN 1 74024 022 7; 1999. 3. Larsen JE, Pavey SJ, Bowman R, et al. Gene expression of lung squamous cell carcinoma reflects mode of lymph node involvement. Eur Respir J 2007;30:21-5. 4. Larsen JE, Pavey SJ, Passmore LH, et al. Expression profiling defines a recurrence signature in lung squamous cell carcinoma. Carcinogenesis 2007;28:760-6. 5. Larsen JE, Pavey SJ, Passmore LH, Bowman RV, Hayward NK, Fong KM. Gene expression signature predicts recurrence in lung adenocarcinoma. Clin Cancer Res 2007;13:2946-54. 6. S ELA, Mager I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 2013;12:347-57. 7. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature cell biology 2007;9:654-9. 8. Sceneay J, Chow MT, Chen A, et al. Primary tumor hypoxia recruits CD11b+ / Ly6Cmed / Ly6G+ immune suppressor cells and compromises NK cell cytotoxicity in the premetastatic niche. Cancer Res 2012;72:3906-11. 9. Chafe SC, Lou Y, Sceneay J, et al. Carbonic anhydrase IX promotes myeloid-derived suppressor cell mobilization and establishment of a metastatic niche by stimulating G-CSF production. Cancer Res 2015;75:996-1008. 10. Martins VR, Dias MS, Hainaut P. Tumor-cell-derived microvesicles as carriers of molecular information in cancer. Curr Opin Oncol 2013;25:66-75. 11. Kucharzewska P, Christianson HC, Welch JE, et al. Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development. Proc Natl Acad Sci U S A 2013;110:7312-7. 12. Xiang X, Poliakov A, Liu C, et al. Induction of myeloid-derived suppressor cells by tumor exosomes. Int J Cancer 2009;124:2621-33. 13. Al-Nedawi K, Meehan B, Kerbel RS, Allison AC, Rak J. Endothelial expression of autocrine VEGF upon the uptake of tumor-derived microvesicles containing oncogenic EGFR. Proc Natl Acad Sci U S A 2009;106:3794-9. 14. Al-Nedawi K, Meehan B, Micallef J, et al. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol 2008;10:619-24. 15. Ciravolo V, Huber V, Ghedini GC, et al. Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy. J Cell Physiol 2012;227:658-67. 16. Peinado H, Aleckovic M, Lavotshkin S, et al. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nature medicine 2012;18:883-91. 17. Demory Beckler M, Higginbotham JN, Franklin JL, et al. Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS. Mol Cell Proteomics 2013;12:343-55. 18. Corcoran C, Rani S, O'Brien K, et al. Docetaxel-resistance in prostate cancer: evaluating associated phenotypic changes and potential for resistance transfer via exosomes. PLoS One 2012;7:e50999. 19. Wysoczynski M, Ratajczak MZ. Lung cancer secreted microvesicles: underappreciated modulators of microenvironment in expanding tumors. Int J Cancer 2009;125:1595-603. 20. Lv LH, Wan YL, Lin Y, et al. Anticancer drugs cause release of exosomes with heat shock proteins from human hepatocellular carcinoma cells that elicit effective natural killer cell antitumor responses in vitro. J Biol Chem 2012;287:15874-85. 21. Khan S, Jutzy JM, Aspe JR, McGregor DW, Neidigh JW, Wall NR. Survivin is released from cancer cells via exosomes. Apoptosis 2011;16:1-12. 22. Safaei R, Larson BJ, Cheng TC, et al. Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells. Mol Cancer Ther 2005;4:1595-604. 23. Vlassov AV, Magdaleno S, Setterquist R, Conrad R. Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta 2012;1820:940-8. 24. Melo SA, Luecke LB, Kahlert C, et al. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 2015;523:177-82. 25. Thakur BK, Zhang H, Becker A, et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell research 2014;24:766-9. 26. Kahlert C, Melo SA, Protopopov A, et al. Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer. J Biol Chem 2014;289:3869-75. 27. Roberson CD, Atay S, Gercel-Taylor C, Taylor DD. Tumor-derived exosomes as mediators of disease and potential diagnostic biomarkers. Cancer biomarkers : section A of Disease markers 2010;8:281-91. 28. Zocco D, Ferruzzi P, Cappello F, Kuo WP, Fais S. Extracellular vesicles as shuttles of tumor biomarkers and anti-tumor drugs. Frontiers in oncology 2014;4:267. 29. Wen SW, Everitt SJ, Bedo J, et al. Spleen Volume Variation in Patients with Locally Advanced Non-Small Cell Lung Cancer Receiving Platinum-Based Chemo-Radiotherapy. PLoS One 2015;10:e0142608. 30. Lobb RJ, Becker M, Wen SW, et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. Journal of extracellular vesicles 2015;4:27031.

[0149] Example 2 Despite significant therapeutic advances, lung cancer remains the leading cause of cancer-related deaths worldwide. 1 Patients with non-small cell lung cancer (NSCLC) have a very low 5-year survival rate of 15%. 2 Biopsy is used to diagnose and subtype NSCLC, and TNM staging is the most important factor for predicting survival and guiding clinical intervention. 2 However, a significant proportion of patients with early-stage and locoregional NSCLC have refractory disease or develop metastatic disease despite curative treatment with surgical radiotherapy or chemoradiotherapy, demonstrating that TNM staging alone is insufficient to guide disease management. Thus, there is an unmet need to identify those patients who respond poorly to current therapies and allow for tailoring of therapeutic interventions. Prognostic biomarkers, particularly noninvasive biomarkers, can enable clinicians to triage patients who require intensified treatment or adjuvant therapeutic interventions.

[0150] Small extracellular vesicles called exosomes have been shown to serve as a non-invasive method for identifying outcomes in pancreatic cancer. 3 Exosomes are secreted, directly membrane-enclosed vesicles ranging in size from 30 to 150 nm. 4 Exosomes, which result from the inward budding of multivesicular bodies, contain various nucleic acids, lipids, and proteins derived from their cell of origin. 4 Upon fusion with the plasma membrane, exosomes are released into the extracellular environment and can enter the environmental system. 4It is for this reason that exosome isolation from patient body fluids serves as a potential source of novel markers that may help characterize NSCLC in more detail compared to currently available clinical techniques.

[0151] It is well known that hypoxia occurs early in tumor development and causes aggressive, invasive, and metastatic phenotypes. 5,6 We hypothesized that NSCLC cells exposed to hypoxic conditions would secrete exosomes with distinct proteomic profiles indicative of the aggressive phenotype of the cells of origin. To address whether hypoxia causes changes in exosomal protein content, we used established methods to isolate exosomes secreted by human NSCLC cell lines (H358, SKMES1, H23, and H1975) cultured under normoxic (21% O) or hypoxic (2% O) conditions (Figures 6A & B and 10). 7、8 As measured by tunable resistive pulse sensing (TRPS), exosomes displayed a typical size distribution and contained the standard exosome markers HSP70, FLOT1, and CD63 (Figure 6B; Figure 10A). Interestingly, transmission electron microscopy (TEM) and TRPS nanoparticle analysis showed that NSCLC cells enhanced exosome secretion in response to hypoxia (Figure 6A&B; Figure 10B). The proteomes of normoxic and hypoxic exosomes from adenocarcinoma H358 and squamous cell carcinoma SKMES1 cells were assessed using mass spectrometry. Label-free quantification by spectral counting identified 32 proteins (16 cytoplasmic, 10 secreted, and 6 transmembrane) that were upregulated under hypoxia in both H358 and SKMES1 exosomes (Figure 6C; Tables 2&3). Five of these proteins (two cytoplasmic [VCP] and two cytoplasmic [VCP]) were upregulated under hypoxia based on their previous association with cancer progression. 9 , PSMA2 10 ], two secretory [TNC 11,12 , THBS1 13 ] and one transmembrane protein [MAC2BP 14We selected an exosome signature based on the 500kJ / 500kcal protein (500kJ / 500kcal) and confirmed that all five proteins were highly abundant in exosomes from additional hypoxic NSCLC cell lines (Figure 1D&E).

[0152] We next hypothesized that hypoxia-induced exosomal changes could be utilized as a prognostic marker for disease progression in early-stage NSCLC. Exosomes were isolated from the plasma of a discovery cohort of 32 treatment-naive, pre-stage I-III NSCLC patients sampled at the time of diagnosis (Figure 7A & B). Although hypoxia increases exosome secretion from NSCLC cells (Figure 10B), we surprisingly found that exosome concentration in the plasma of NSCLC patients, as a categorical variable, had no prognostic value for clinical recurrence within 18 months (Figure 7C). Interestingly, the combined five-protein exosome signature (VCP, MAC2BP, TNCPSMA2, and THBS1) was specifically increased in exosomes from relapsed NSCLC subjects (Figure 7D). Individually, each protein from the exosome signature was a prominent prognostic biomarker for disease recurrence (Figure 11). Interestingly, we were able to clearly distinguish disease-free survival (DFS) based on the abundance of those five exosomal proteins above Youden's threshold (≤=no recurrence; ≥3=recurrence) (Figure 7F&G). Importantly, receiver operating characteristic (ROC) curves demonstrated that those five exosomal proteins had the ability to predict disease progression with 100% specificity and sensitivity within this discovery cohort (Figure 7F). Furthermore, the exosomal signature could separate overall survival (OS) of patients in the discovery cohort (Figure 7I), suggesting that both recurrence and OS are associated with the abundance of the exosomal signature.

[0153] Based on the prognostic value of the exosome signature, we investigated the potential mechanisms underlying this exosome signature. We recently demonstrated that the protein content of exosomes can reflect the phenotype of the cell of origin. 15Gene set enrichment analysis (GSEA) was performed on the total protein abundance in exosomes derived from normoxic, normoxic, or hypoxic conditions. Numerous gene sets were significantly enriched in exosomes from NSCLC cells isolated under hypoxic conditions, including glycolysis, MYC targets, E2F targets, and xenobiotic metabolism (Figure 12). Interestingly, the top-ranked gene sets enriched in hypoxic exosomes were associated with EMT (Figure 3A; Figure 12A). Considering that hypoxia is a potent inducer of EMT in cancer cells, 16 We hypothesized that the mesenchymal phenotype alone might be sufficient to trigger the secretion of an exosome signature. To determine whether the five exosomal proteins are secreted by normal or transformed lung epithelial cells, we isolated exosomes from an isogenic human bronchial epithelial cell (HBEC) line. Surprisingly, p53 knockdown, Kras v12 overexpression, and LKB1 knockdown (30KT) were associated with the secretion of exosomes. p53 / KRAS / LKB1 ) 17 HBECs that underwent oncogenic EMT through EBV (Figure 8B&C) secreted increased exosome signature proteins even under normoxic conditions (Figure 8D&E). To validate the relevance of mesenchymal lung cancer cells secreting the exosome signature, we next analyzed E-cadherin expression in tumor biopsies from patients from the discovery cohort. Immunohistochemistry of tumor biopsies revealed a significant correlation (R ) of reduced E-cadherin expression in tumors from patients with a high exosome signature score of 3 or higher compared to patients with an exosome signature score of 2 or lower (Figure 8F). 2 =0.458, p<0.001) (Figure 13). These data support the idea that EMT in oncogenically transformed lung cells is responsible for the increased protein levels found in the exosome signature both in vivo and in NSCLC patients.

[0154] Depolarization of epithelial cell phenotype to extended mesenchymal cells promotes not only aggressive and metastatic phenotypes of cancer cells but also chemoresistance. 17,18Therefore, for independent validation, we evaluated 20 locally advanced NSCLC subjects (validation cohort) receiving standard-of-care chemoradiation therapy consisting of conformal RT (60 Gy / 30 fractions, 6 weeks) with combination chemotherapy (either cisplatin / etoposide or carboplatin / paclitaxel). Patients were followed-up at baseline, days 10, 24, and 90. 18 Patients were monitored at 12 months, 3-month intervals, and then 6-month intervals using F-FDG and PET / CT and standard CT scans (Figures 9A & B; Table 5). Exosome concentrations were measured at baseline using TRPS. Subjects who relapsed within 18 months did not have significantly different circulating exosome abundance (Figure 9C). Consistent with the discovery cohort, the exosome protein signature showed significant increase and prognostic value in subjects who relapsed within 18 months compared with those who did not (Figure 9D; Figure 14). Using the same threshold and algorithm established in the discovery cohort (≤ marker = low risk of relapse within 18 months; ≥ 3 = high risk of relapse within 18 months), the signature clearly separated patients who relapsed within 18 months from those who relapsed after 18 months (Figures 9D & E). ROC curve analysis further confirmed the specificity and sensitivity of the exosome signature for disease recurrence (Figure 9F). In further agreement with the expression cohort, the exosomal signature classified patients based on OS, suggesting that the exosomal protein signature is an ideal classification for subjects with early relapse and poor overall survival.

[0155] Considering the relationship between EMT and metastasis and chemotherapy resistance 16-20 These data identify a mechanism for the disease-free survival seen in both cohorts of NSCLC patients in this study. This study demonstrates that hypoxia / EMT-associated exosomal biomarkers hold great promise for identifying early-stage NSCLC patients at risk for early recurrence and poor clinical outcomes. Hypoxia may be involved in the regulation of the developing EMT program. 16 have multiple functions in promoting tumor growth and metastasis, including the induction of16-20、22 Importantly, the ability to noninvasively and reliably detect hypoxia and / or EMT in NSCLC could serve as a potential prognostic screen in early-stage NSCLC, facilitating curative treatments and reducing overall mortality. Our results provide strong initial evidence for the newly discovered exosomal protein signature as a marker of disease progression in NSCLC. Further studies will be conducted to determine whether the exosomal signature is a predictive biomarker in the setting of chemoradiotherapy or whether the exosomal signature is a prognostic biomarker in the setting of NSCLC in general. While TNM staging provides significant benefits in patient management and will continue to be important in the clinical management of NSCLC patients, the exosomal signature has the potential to complement TNM staging and enable specific tailoring of therapeutic interventions to improve clinical outcomes.

[0156] Materials and Methods cell culture Human non-small cell lung cancer (NSCLC) cell lines (adenosquamous carcinoma) H358, SKMES1, H23, and H1975 were purchased from ATCC. Cell line authentication was performed using short tandem repeat profiling. NSCLCs were maintained in DMEM or RPMI supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin and incubated at 37°C under 5% CO2. Isogenic normal human bronchial epithelial cells (HBECs) were a gift from Dr. Jill Larsen. 19,23 HBECs were cultured in keratinocyte serum-free medium (KSFM) supplemented with EGF (5 μg / L) and bovine pituitary extract (50 mg / L) at 37°C under 5% CO2. Conditioned cell medium (CCM) from NSCLC cell lines was collected from cells cultured in serum-free medium under normoxic (21% O2) or hypoxic (2% O2) conditions. CCM was collected from HBEC cells conditioned under normoxic or hypoxic conditions in bovine exosome-depleted KSFM through overnight centrifugation at 100,000 g / s.

[0157] Antibodies and reagents The following antibodies were used for Western blotting: calnexin (Cell Signaling Technology, 2679S), CD9 (Abcam, ab92726), CD63 (Abcam, ab8219), flotillin-1 (BD Transduction Laboratories, 610821), HSP70 (Transduction Laboratories, 610608), TSG101 (Santa Cruz, sc-6037), and VCP (Abcam, ab11433). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Thermo Scientific. AC2BP, PSMA2, and THBS1 ELISA DuoSets were purchased from R&D Systems, and TNC ELISA kits were purchased from Abcam. qEV columns were purchased from Izon and stored in PBS (0.1% sodium azide) at 4°C. OptiPrep® was purchased from Sigma-Aldrich. qPCR was performed as previously described 24 .

[0158] patient The independent validation cohort included 20 patients who provided informed consent to participate in an ERB-approved prospective study of sequential FDG PET / CT before, during, and after investigational chemotherapy-assisted resection of tumors. As previously reported, eligibility for this study was based on staging, along with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1. 18 F-FDG PET / CT, including histological or cytological confirmation of stage I-III NSCLC 25Exclusion criteria included previous thoracic radiotherapy and complete surgical tumor resection. Patients received concurrent chemoradiotherapy according to two standardized protocols. RT consisted of 60 Gy in 30 fractions over 6 weeks. One of two chemotherapy regimens was administered: carboplatin [area under the curve, 2 doses intravenously] and paclitaxel [45 mg / m ] weekly for elderly patients or those with significant comorbidities. 2 , intravenous]; or for younger patients, cisplatin 50 mg / m on days 1, 8, 29, and 36 2 , intravenous, and etoposide [50 mg / m between weeks 1 and 5] 2 , intravenous]. 18 F-FDG PET / CT scans were obtained at baseline, days 10, 24, and 90. Continuous monitoring was performed with standard CT imaging at 3-day intervals for 12 months, then at 6-month intervals.

[0159] Exosome isolation and analysis Exosomes were isolated and analyzed as previously described. 7,17,26For exosome isolation from in vitro cell cultures, CCM was centrifuged at 300 g for 10 minutes at 4°C and filtered through a 0.22 μm filter to remove floating cells and large extracellular vesicles. The clarified CCM was then concentrated to 500 μl and layered on a discontinuous iodixanol density gradient and centrifuged at 100,000 g for 16 hours at 4°C. The exosome-containing fraction was diluted to 20 ml with PBS and centrifuged at 100,000 g for 2 hours at 4°C. The resulting pellet was resuspended in PBS and stored at -80°C until use. For exosome isolation from human plasma, 3 ml of plasma was thawed at room temperature and centrifuged at 1,500 g and 10,000 g for 10 and 20 minutes, respectively, to remove the remaining pellet and large vesicles. The prepared plasma was then diluted to 20 ml with PBS containing 2 mM EDTA and centrifuged at 100,000 g for 2 hours at 4°C. The resulting pellet was resuspended in 500 μl of PBS and loaded onto a size-exclusion column, followed by elution with PBS. The exosome-containing fraction was collected and concentrated to 100 μl using an Amicom® Ultra-4 10 kDA nominal molecular weight centrifugal filter unit. The concentrated exosomes were stored at -80°C until use. Exosome isolation from cell culture and human plasma was confirmed using Western blot, variable resistive pulse sensing (TRPS), and transmission electron microscopy, as previously described. 7,17、26 .

[0160] Western blot analysis Western blots were performed as previously described 7,24Briefly, proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, blocked in 5% nonfat dry milk in PBS-T (0.5% Tween-20), and probed with antibodies. Protein bands were detected with enhanced chemiluminescence reagents (Amersham ECL Select). Protein bands were quantified using ImageJ and normalized to loading controls. To control for gel-to-gel variability, patient VCP levels were calibrated against 5 μg of hypoxia-derived SKMES1 exosomes from the same gel before normalizing to flotillin-1 as a loading control.

[0161] immunohistochemistry IHC analysis was performed on formalin-fixed, paraffin-embedded (FFPG) samples using automated staining and optimized methods. To assess E-cadherin expression in tumor cells, immunostained tumor cells were scored according to their staining intensity: 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong).

[0162] mass spectrometry Exosome preparations were reduced by the addition of 10 mM dithiothreitol (1 h at 4°C, 2 h at 22°C) in the presence of 2% SDS, protease inhibitors (Sigma-Aldrich, P8340), and 50 mM Tris·HCl (pH 8.9). The samples were then alkylated with iodoacetamide and methanol co-precipitated with trypsin (1:100 enzyme:substrate) at -20°C overnight. The pellet was resuspended in 10% acetonitrile, 40 mM ammonium bicarbonate, and digested for 8 h at 37°C with additional trypsin (1:100 enzyme:substrate) added after 2 h.

[0163] LCMS analysis of the acidified digest (trifluoroacetic acid) was performed using a NanoAcquity UPLC (Waters) coupled to an Elite Orbitrap ETD mass spectrometer (Thermo Fisher Scientific). 2 μg of the digest was loaded onto a 20 mm x 180 μm Symmetry C trap (Waters) and then loaded onto a 200 mm x 75 μm BEH130 1.7 μm column (Waters) using a series of wash gradients (Buffer A: aqueous 0.1% formic acid; Buffer B: acetonitrile in 0.1% formic acid), from 2% B to 5% B over 5 min, 30% B over 75 min, 50% B over 10 min, and 95% B over 5 min, followed by a 6-min hold and re-equilibration in 2% B. The column eluate was introduced into the mass spectrometer through a 100 μm P200P coated Millica emitter (New Objective) and a Nanospray-Flex source (Proxeon Biosystems A / S). The top 15 MS acquisitions were performed in an orbital trap with a power supply voltage of 1.8 kV, a heated capillary temperature of 275 °C, and a resolution of 120,000 s, using an AGC of 1E6. MS2 acquisitions were performed in an ion trap with an AGC of 1E4, and a maximum injection time of 50 ms. An MS1 ​​lock mass of 445.120024 was used.

[0164] Protein identification and label-free quantification were performed using MaxQuant (version 1.4.1.2). 27 Peak lists were extracted from Xcalibur raw files (Thermo Fisher Scientific, Germany) using MaxQuant and the built-in database search engine Andromeda 28Peptide-to-spectrum matches (PSMs) were assigned using the ELISA kit. The database searched consisted of the complete Homo sapiens proteome (88,378 standard sequences downloaded from www.uniprot.org in August 2013). Reverse sequences and the MaxQuant contaminant database were also searched. Label-free quantification was performed with the instrument type set to Orbitrap, precursor mass tolerance set to 20 ppm for the initial search and 4.5 ppm for the main search, fragment ion mass tolerance set to 0.5 Da, enzyme specificity set to trypsin / P, a maximum of two missed cleavages was allowed, carbamidomethylcysteine ​​was specified as a fixed modification, and protein N-terminal acetylation, asparagine / glutamine deamidation, and methionine oxidation were specified as variable modifications. Second-round peptide searches and inter-run matches were allowed with default settings. For identification, PSM and protein-level FDR were set to 0.01. Default settings were applied for all other parameters. Protein inference and label-free quantification by spectral counting (including normalization) were performed as previously described. 29 .

[0165] Gene set enrichment analysis Gene Set Enrichment Analysis (GSEA) 30 , version 2.2.3, was used to identify enriched pathways in exosomes isolated from hypoxic SKMES1 cells as previously described. 15 Non-log2 transformed protein intensity values ​​of all proteins in exosomes derived from normoxic or hypoxic SKMES1 exosomes were analyzed using the Molecular Signatures Database (MSigDB). Analysis was performed using the Hallmark gene set database (version 5.2), Signal2Noise ranking metrics, 1000 gene set permutations, and weighted enrichment statistics. Results were considered significant at a false discovery rate (FDR) <0.05.

[0166] statistical analysis GraphPad Prism version 6.0, EdgeR version 2.6.1031, MedCalc version 16.8.4, and SPSS statistics were used for all calculations. Differences in protein expression values ​​from in vitro exosomes were calculated using an unpaired Student's t-test. The Mann-Whitney test was used for patient-derived exosomes. Mass spectrometry-derived spectral counts were evaluated using a negative binomial exact test, where a Benjamini-Hochberg adjustment was applied to control for FDR. Sensitivity and specificity of prognostic values ​​were determined using receiver operator characteristic (ROC) curves. Thresholds were selected using the Youden index. Disease-free survival was assessed (Kaplan-Meier curves) using univariate analysis with the log-rank test. Differences with a p-value of less than 0.05 were considered significant ( * p<0.05, ** p<0.01, *** p<0.001), except for FDR thresholds of 0.001 and 0.05 for the number of spectra and GSEA data, respectively.

[0167] Throughout this specification, the objective has been to describe preferred embodiments of the invention without limiting the invention to any one embodiment or particular collection of features. Accordingly, those skilled in the art will appreciate in light of this disclosure that various modifications and changes can be made in the specific embodiments exemplified without departing from the scope of the invention.

[0168] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated by reference.

[0169] [Table 2]

[0170] [Table 3]

[0171]

Table 4

[0172]

Table 5

[0173] References 1. Torre, L.A., Bray, F., Siegel, R.L., Ferlay, J., Lortet-Tieulent, J. & Jemal, A. Global cancer statistics, 2012. CA: a cancer journal for clinicians 65, 87-108 (2015). 2. Molina, J.R., Yang, P., Cassivi, S.D., Schild, S.E. & Adjei, A.A. Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clinic proceedings 83, 584-594 (2008). 3. Melo, S.A., Luecke, L.B., Kahlert, C., Fernandez, A.F., Gammon, S.T., Kaye, J., et al. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523, 177-182 (2015). 4. Lobb, R.J., Lima, L.G. & Moller, A. Exosomes: Key mediators of metastasis and pre-metastatic niche formation. Seminars in cell & developmental biology (2017). 5. Vaupel, P. & Mayer, A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer metastasis reviews 26, 225-239 (2007). 6. Hockel, M. & Vaupel, P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. 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Global Oct4 target gene analysis reveals novel downstream PTEN and TNC genes required for drug-resistance and metastasis in lung cancer. Nucleic acids research 43, 1593-1608 (2015). 12. Oskarsson, T., Acharyya, S., Zhang, X.H., Vanharanta, S., Tavazoie, S.F., Morris, P.G., et al. Breast cancer cells produce tenascin C as a metastatic niche component to colonize the lungs. Nature medicine 17, 867-874 (2011). 13. Kudo-Saito, C., Shirako, H., Takeuchi, T. & Kawakami, Y. Cancer metastasis is accelerated through immunosuppression during Snail-induced EMT of cancer cells. Cancer cell 15, 195-206 (2009). 14. Marchetti, A., Tinari, N., Buttitta, F., Chella, A., Angeletti, C.A., Sacco, R., et al. Expression of 90K (Mac-2 BP) correlates with distant metastasis and predicts survival in stage I non-small cell lung cancer patients. Cancer Res 62, 2535-2539 (2002). 15. Lobb, R.J., Hastie, M.L., Norris, E.L., van Amerongen, R., Gorman, J.J. & Moller, A. Oncogenic transformation of lung cells results in distinct exosome protein profile similar to the cell of origin. Proteomics (2017). 16. Thiery, J.P., Acloque, H., Huang, R.Y. & Nieto, M.A. Epithelial-mesenchymal transitions in development and disease. Cell 139, 871-890 (2009). 17. Lobb, R.J., van Amerongen, R., Wiegmans, A., Ham, S., Larsen, J.E. & Moller, A. Exosomes derived from mesenchymal non-small cell lung cancer cells promote chemoresistance. International journal of cancer (2017). 18. Fischer, K.R., Durrans, A., Lee, S., Sheng, J., Li, F., Wong, S.T., et al. Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature 527, 472-476 (2015). 19. Larsen, J.E., Nathan, V., Osborne, J.K., Farrow, R.K., Deb, D., Sullivan, J.P., et al. ZEB1 drives epithelial-to-mesenchymal transition in lung cancer. The Journal of clinical investigation 126, 3219-3235 (2016). 20. Zheng, X., Carstens, J.L., Kim, J., Scheible, M., Kaye, J., Sugimoto, H., et al. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature 527, 525-530 (2015). 21. Sceneay, J., Chow, M.T., Chen, A., Halse, H.M., Wong, C.S.F., Andrews, D.M., et al. Primary Tumor Hypoxia Recruits CD11b+ / Ly6Cmed / Ly6G+ Immune Suppressor Cells and Compromises NK Cell Cytotoxicity in the Premetastatic Niche. Cancer Research 72, 3906 (2012). 22. Kalluri, R. & Weinberg, R.A. The basics of epithelial-mesenchymal transition. The Journal of clinical investigation 119, 1420-1428 (2009). 23. Sato, M., Vaughan, M.B., Girard, L., Peyton, M., Lee, W., Shames, D.S., et al. Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells. Cancer Res 66, 2116-2128 (2006). 24. Lobb, R.J., van Amerongen, R., Wiegmans, A., Ham, S., Larsen, J.E. & Moller, A. Exosomes derived from mesenchymal non-small cell lung cancer cells promote chemoresistance. International journal of cancer 141, 614-620 (2017). 25. Everitt, S.J., Ball, D.L., Hicks, R.J., Callahan, J., Plumridge, N., Collins, M., et al. Differential (18)F-FDG and (18)F-FLT Uptake on Serial PET / CT Imaging Before and During Definitive Chemoradiation for Non-Small Cell Lung Cancer. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 55, 1069-1074 (2014). 26. Wen, S.W., Sceneay, J., Lima, L.G., Wong, C.S., Becker, M., Krumeich, S., et al. The biodistribution and immune suppressive effects of breast cancer-derived exosomes. Cancer Research, canres. 0868.2016 (2016). 27. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature biotechnology 26, 1367-1372 (2008). 28. 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Claims

1. 1. A method for determining the aggressiveness of cancer in a subject, comprising determining the expression level of one or more markers in an exosome sample of the subject, wherein said markers comprise one or more of those proteins listed in Table 1 and / or Table 2, and wherein the expression level of the one or more markers is indicative of or correlates with the level of aggressiveness of the cancer.

2. 1. A method for determining the prognosis of cancer in a subject, comprising the step of determining the expression level of one or more markers in an exosome sample of the subject, wherein said markers comprise one or more of those proteins listed in Table 1 and / or Table 2, and wherein the expression level of the one or more markers is indicative of or correlates with a less favorable or more favorable prognosis for the cancer.

3. The method of claim 1 or 2, wherein a relatively reduced expression level of one or more markers indicates or correlates with a more favorable prognosis and / or a less aggressive cancer; and / or a relatively increased expression level of one or more markers indicates or correlates with a less favorable prognosis and / or a more aggressive cancer.

4. 4. The method of any one of claims 1 to 3, comprising the further step of diagnosing said subject as having (i) a highly aggressive cancer or a less aggressive cancer; and / or (ii) a less favorable prognosis or a more favorable prognosis.

5. The method of any one of claims 1 to 4, wherein the prognosis or aggressiveness of the cancer is used, at least in part, to determine the metastatic potential of the cancer in the subject.

6. 6. The method of claim 5, wherein a relatively reduced expression level of the one or more markers indicates or correlates with a reduced likelihood of metastasis of the cancer; and / or a relatively increased expression level of the one or more markers indicates or correlates with an increased likelihood of metastasis of the cancer.

7. 1. A method of predicting responsiveness of a cancer to an anti-cancer treatment in a subject, comprising determining the expression level of one or more markers in an exosome sample from the subject, wherein said markers comprise one or more of those proteins listed in Table 1 and / or Table 2, and wherein altered or regulated expression levels of the one or more markers are indicative of or correlate with a relatively increased or decreased responsiveness of the cancer to the anti-cancer treatment.

8. The method of any one of claims 1 to 7, comprising the further step of treating cancer in the subject.

9. 1. A method of treating cancer in a subject, comprising the step of determining the expression level of one or more markers in an exosome sample of the subject, wherein said markers comprise one or more of those proteins listed in Table 1 and / or Table 2, and based on the determination made, the method comprising initiating, continuing, modifying or discontinuing anti-cancer treatment.

10. 10. The method of any one of claims 7 to 9, wherein the anti-cancer treatment comprises administering to the subject a therapeutically effective amount of an anti-cancer agent that reduces the expression and / or activity of one or more markers.

11. The method of any one of claims 7 to 10, wherein the anti-cancer treatment comprises administering to the subject a therapeutically effective amount of an anti-cancer agent that prevents or inhibits metastasis of the cancer.

12. 12. The method of claim 10 or 11, wherein the anti-cancer agent is an antibody or a small molecule.

13. The method of any one of claims 1 to 12, further comprising the step of obtaining an exosome sample from the subject.

14. 14. The method of any one of claims 1 to 13, further comprising comparing the expression level of one or more markers in the exosome sample with a reference exosomal expression level of each one or more markers.

15. The method of any one of claims 1 to 14, wherein the cancer is or comprises lung cancer.

16. 16. The method of claim 15, wherein the lung cancer is or comprises non-small cell lung cancer.

17. 1. A method for identifying or producing an agent for use in treating cancer in a subject, comprising: (a) contacting a cell expressing a marker listed in Table 1 and / or Table 2 with a candidate agent; and (b) determining whether said candidate agent modulates the expression and / or activity of said marker.

18. 18. The method of claim 17, wherein the candidate agent at least partially reduces, eliminates, suppresses or inhibits the expression and / or activity of the marker.

19. A medicament produced by the method of claim 17 or 18 for use in accordance with the method of any one of claims 10 to 16.

20. The one or more markers are selected from the group consisting of galectin-3-binding protein, transitional endoplasmic reticulum ATPase, neutral α-glucosidase AB, 60 kDa heat shock protein, lysyl oxidase homolog 2, tenascin C, fatty acid synthase, agrin, aspartyl aminopeptidase, proteasome subunit α1, proteasome subunit α2, proteasome subunit α3, proteasome subunit α4, proteasome subunit α5, proteasome subunit α6, proteasome subunit α7, proteasome subunit α8, proteasome subunit α9, proteasome subunit α10, proteasome subunit α11, proteasome subunit α12, proteasome subunit α13, proteasome subunit α14, proteasome subunit α15, proteasome subunit α16, proteasome subunit α17, proteasome subunit α18, proteasome subunit α19 ...9, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α19, proteasome subunit α 20. The method of any one of claims 1 to 18, or the agent of claim 19, wherein the proteasome subunit is selected from the group consisting of proteasome subunit β1, proteasome subunit β2, proteasome subunit β3, proteasome subunit β4, proteasome subunit β5, proteasome subunit β6, proteasome subunit β7, proteasome subunit β8, thrombospondin-1, latent transforming growth factor β binding protein 3, and any combination thereof.

21. 21. The method or agent of claim 20, wherein the one or more markers are selected from the group consisting of galectin-3-binding protein, transitional endoplasmic reticulum ATPase, tenascin-C, proteasome subunit alpha type 2, thrombospondin-1, and any combination thereof.

22. 21. The method or agent of claim 20, wherein the one or more markers are selected from the group consisting of galectin-3-binding protein, transitional endoplasmic reticulum ATPase, tenascin-C, proteasome subunit alpha type 2, neutral alpha-glucosidase AB, and any combination thereof.

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