Tumor cell-derived microvesicles

Abstract

The present invention relates to a method for diagnosis of cancer and for monitoring the progression of cancer and/or the therapeutic efficacy of an anti-cancer treatment in a sample of a subject by detecting oncogenic and cancer related proteins in microvesicles, and to the use of an agent blocking exchange of microvesicles for treating cancer.

1 . A method of detecting the presence of an oncogenic protein or an MV-associated protein in a subject, comprising:
 collecting a sample from the subject;   isolating microvesicles from the sample; and   detecting the presence of the oncogenic protein or the MV-associated protein in the microvesicles.   
     
     
         2 . A method for diagnosing or determining prognosis of a cancer in a subject, comprising:
 detecting the presence of an oncogenic protein or an MV-associated protein in microvesicles in accordance with the method of  claim 1 , wherein the presence of the oncogenic protein or the MV-associated protein in the sample is indicative that the subject may have cancer.   
     
     
         3 . A method for monitoring progression of a cancer or therapeutic efficacy of an anti-cancer treatment in a subject, comprising:
 a) collecting a first sample from a subject having cancer at a first timepoint, isolating microvesicles from the first sample, and measuring an oncogenic protein or an MV-associated protein in the microvesicles obtained from the first sample; and   b) collecting a second sample from the subject having cancer at a second timepoint, the second timepoint occurring after the first timepoint, isolating microvesicles from the second sample, and measuring the oncogenic protein or the MV-associated protein in the microvesicles obtained from the second sample;   
       wherein a change in the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the second sample compared to the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the first sample is indicative of progression of the cancer, or a reduction or no change in the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the second sample compared to the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the first sample indicates therapeutic efficacy of the anti-cancer treatment when the first timepoint occurs before the subject has received an anti-cancer treatment, and the second timepoint occurs after the subject has received the anti-cancer treatment; and 
       wherein said sample is a bodily fluid selected from the group consisting of blood, lymph, urine, cerebrospinal fluid, ascites, saliva, lavage, semen, glandular secretions, exudate, contents of cysts and feces. 
     
     
         4 . The method according to any one of  claims 1 - 3 , wherein said oncogenic or MV-associated protein is selected from the group consisting of EGFRvIII, EGFR, HER-2, HER-3, HER-4, MET, cKit, PDGFR, Wnt, beta-catenin, K-ras, H-ras, N-ras, Raf, N-myc, c-myc, IGFR, Pl3K, Akt, BRCA1, BRCA2, PTEN, FGFR3, EphB2, ROR1, EphA2, EphA4, receptors of cancer associated cells selected from the group consisting of VEGFR-2, VEGFR-1, Tie-2, TEM-1 and CD276, and proteins listed in Tables 1 to 4. 
     
     
         5 . The method according to  claim 1 , further comprising the step of measuring the phosphorylation state of the oncogenic protein. 
     
     
         6 . The method according to  claim 3 , wherein:
 an increase in the amount of the oncogenic protein in the microvesicles obtained from the second sample compared to the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the first sample indicates that the cancer has progressed or continued to proliferate;   a reduction in the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the second sample, or a change in the composition of the microvesicles obtained from the second sample, compared to the amount of the oncogenic or MV-associated protein in the microvesicles, or the composition of the microvesicles, obtained from the first sample indicates that the cancer has regressed; and   no change in the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the second sample compared to the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the first sample indicates the cancer has not progressed.   
     
     
         7 . The method according to  claim 1 , wherein at least two oncogenic or MV-associated proteins are detected in the microvesicles. 
     
     
         8 . The method according to  claim 1 , wherein the microvesicles are isolated by ultracentrifugation, immunoprecipitation, affinity purification or microfiltration. 
     
     
         9 . The method according to  claim 1 , wherein the presence of the oncogenic or MV-associated protein in the microvesicles is detected or measured by immunoblot, immunoprecipitation, ELISA, RIA, flow cytometry, electron microscopy or mass spectrometry. 
     
     
         10 . The method according to  claim 1 , wherein the oncogenic or MV-associated protein in the microvesicles is detected or measured by ELISA with wells coated with Annexin V. 
     
     
         11 . The method according to  claim 3 , wherein the anti-cancer treatment is surgery, radiology, chemotherapy, a targeted cancer treatment, or a combination thereof. 
     
     
         12 . The method according to  claim 2 ,  3  or  11 , wherein said cancer is selected from the group consisting of breast cancer, glioma, brain cancer, lung cancer, pancreatic cancer, skin cancer, prostate cancer and colorectal cancer. 
     
     
         13 . A kit for detecting a cancer in a sample from a subject comprising:
 at least one antibody against an oncogenic or MV-associated protein, and instructions for using said at least one antibody to detect the oncogenic or MV-associated protein in microvesicles in the sample.   
     
     
         14 . The kit of  claim 13 , wherein said cancer is selected from the group consisting of breast cancer, glioma, brain cancer, lung cancer, pancreatic cancer, skin cancer, prostate cancer and colorectal cancer. 
     
     
         15 . The kit according to  claim 13 , wherein said oncogenic or MV-associated protein is selected from the group consisting of EGFRvIII, EGFR, HER-2, HER-3, HER-4, MET, cKit, PDGFR, Wnt, beta-catenin, K-ras, H-ras, N-ras, Raf, N-myc, c-myc, IGFR, Pl3K, Akt, BRCA1, BRCA2, PTEN, FGFR3, EphB2, ROR1, EphA2, EphA4, and receptors of cancer associated cells selected from the group consisting of VEGFR-2, VEGFR-1, Tie-2, TEM-1 and CD276. 
     
     
         16 . The kit according to  claim 13 , wherein said sample is a bodily fluid selected from the group consisting of blood, lymph, urine, cerebrospinal fluid, ascites, saliva, lavage, semen, glandular secretions, exudate and feces. 
     
     
         17 . The kit according to  claim 13 , further comprising a phosphospecific antibody. 
     
     
         18 . The method according to  claim 2 , wherein more than one oncogenic or MV-associated protein is detected in the microvesicles, and the combination of oncogenic or MV-associated proteins detected in the microvesicles is diagnostic of the cancer type. 
     
     
         19 . The method according to  claim 18 , wherein the phosphorylation state of the oncogenic or MV-associated proteins is determined. 
     
     
         20 . A method for monitoring progression of a cancer or therapeutic efficacy of an anti-cancer treatment in a subject, comprising:
 a) collecting a first blood sample from a subject having cancer at a first timepoint, isolating microvesicles from the first blood sample, and measuring the phosphorylation state of an oncogenic or MV-associated protein in the microvesicles obtained from the first blood sample; and   b) collecting a second blood sample from the subject having cancer at a second timepoint, the second timepoint occurring after the first timepoint, isolating microvesicles from the second blood sample, and measuring the phosphorylation state of the oncogenic or MV-associated protein in the microvesicles obtained from the second blood sample;   
       wherein a change in the phosphorylation state of the oncogenic or MV-associated protein, or in the amount of the oncogenic or MV-associated protein which is phosphorylated or unphosphorylated, in the microvesicles obtained from the second blood sample compared to the microvesicles obtained from the first blood sample is indicative of progression or regression of the cancer, or indicates therapeutic efficacy or ineffectiveness of the anti-cancer treatment when the first timepoint occurs before the subject has received an anti-cancer treatment, and the second timepoint occurs after the subject has received the anti-cancer treatment; and 
       wherein regression of the cancer or therapeutic efficacy of the anti-cancer treatment is indicated by an increase in the non-active form of the oncogenic or MV-associated protein, and progression of the cancer or ineffectiveness of the anti-cancer treatment is indicated by an increase in the activated form of the oncogenic or MV-associated protein, as determined by the phosphorylation state or the amount of phosphorylated protein detected in the MVs. 
     
     
         21 . The method of  claim 20 , wherein the oncogenic or MV-associated protein is a receptor tyrosine kinase. 
     
     
         22 . A method for monitoring the activation of an oncogenic receptor tyrosine kinase in a tumour, comprising collecting a blood sample from a subject having the tumour, isolating microvesicles from the blood sample, and measuring the phosphorylation state of the oncogenic receptor tyrosine kinase in the microvesicles, wherein the phosphorylation state of the oncogenic receptor tyrosine kinase indicates activation or non-activation of the receptor tyrosine kinase. 
     
     
         23 . An isolated microvesicle comprising an oncogenic or MV-associated protein.