Use of tenascin-c as an extracellular marker of tumor-derived microparticles

Abstract

The present disclosure provides methods for isolating tumor-derived microparticles from a subject for analysis, specifically by isolating Tenascin-C positive microparticles from a sample from the subject to obtain tumor-derived microparticles. Methods for determining the expression status of biomarkers in the tumor-derived microparticles and methods for determining additional characteristics of the tumor-derived microparticles are also provided.

What is claimed is: 
     
         1 . A method of isolating tumor-derived microparticles from a subject for analysis, the method comprising:
 providing a sample comprising bodily fluid from a subject;   isolating one or more microparticles from the sample; and   isolating one or more Tenascin-C positive microparticles from the one or more microparticles in the sample to obtain tumor-derived microparticles.   
     
     
         2 . A method of identifying tumor-derived microparticles from a subject for analysis, the method comprising:
 providing a sample comprising bodily fluid from a subject;   isolating one or more microparticles from the sample; and   identifying one or more Tenascin-C positive microparticles from the one or more microparticles in the sample to identify tumor-derived microparticles.   
     
     
         3 . The method of any one of  claim 1  or  2 , further comprising analyzing the tumor-derived microparticles to determine the expression status of one or more biomarkers. 
     
     
         4 . The method of any one of  claims 1  to  3 , further comprising analyzing the tumor-derived microparticles to determine the status of one or more tumor-derived microparticles. 
     
     
         5 . A method of isolating brain tumor-derived microparticles from a subject for analysis, the method comprising:
 providing a sample comprising bodily fluid from a subject;   isolating one or more microparticles from the sample; and   isolating one or more Tenascin-C positive microparticles from the one or more microparticles in the sample via immunopurification to obtain brain tumor-derived microparticles.   
     
     
         6 . The method of  claim 5 , further comprising analyzing the brain tumor-derived microparticles to determine the expression status of one or more biomarkers selected from the group consisting of EGFRvIII, 14-3-3, H2AX, PPIX, and glycoporin A, wherein the presence of EGFRvIII, the presence of 14-3-3, the presence of H2AX, the presence of PPIX, and the absence of glycoporin A are indicative of brain-tumor derived microparticles. 
     
     
         7 . A method of diagnosing brain cancer in a subject, the method comprising:
 providing a sample comprising bodily fluid from a subject;   isolating one or more microparticles from the sample;   isolating one or more Tenascin-C positive microparticles from the one or more microparticles in the sample to obtain tumor-derived microparticles;   determining the expression status of one or more biomarkers in the tumor-derived microparticles; and   diagnosing brain cancer in the subject based on the expression status of the one or more biomarkers.   
     
     
         8 . The method of  claim 7 , wherein the expression status of the one or more biomarkers is selected from the group consisting of the EGFRvIII, 14-3-3, H2AX, PPIX, and glycoporin A, wherein the presence of EGFRvIII, the presence of 14-3-3, the presence of H2AX, the presence of PPIX, and the absence of glycoporin A are indicative of brain tumor-derived microparticles. 
     
     
         9 . The method of any one of  claims 1  to  4 , wherein one or more of the tumor-derived microparticles are derived from a tumor arising from a cancer selected from the group consisting of brain cancer, breast cancer, ovarian cancer, lung cancer, and gastrointestinal cancer. 
     
     
         10 . The method of any one of  claims 1  to  9 , wherein the bodily fluid is selected from the group consisting of blood, serum, plasma, urine, saliva, and cerebrospinal fluid. 
     
     
         11 . The method of any one of  claims 1  to  10 , wherein the microparticles are isolated using a solid support. 
     
     
         12 . The method of  claim 11 , wherein the solid support is a column. 
     
     
         13 . The method of  claim 12 , wherein the column is a Sepharose 2B column. 
     
     
         14 . The method of any one of  claims 11  to  13 , wherein a mobile phase is added to the solid support. 
     
     
         15 . The method of  claim 14 , wherein the mobile phase is water. 
     
     
         16 . The method of  claim 14 , wherein the mobile phase comprises an amino acid. 
     
     
         17 . The method of  claim 16 , wherein the mobile phase comprises arginine. 
     
     
         18 . The method of  claim 11 , wherein the solid support comprises a HDN peptide. 
     
     
         19 . The method of  claim 11 , wherein the solid support comprises a Vn96 peptide. 
     
     
         20 . The method of any one of  claims 1  to  19 , wherein the Tenascin-C positive microparticles are isolated using a solid support. 
     
     
         21 . The method of  claim 20 , wherein the solid support comprises an anti-Tenascin-C antibody. 
     
     
         22 . The method of any one of  claims 3  and  9  to  21 , wherein the biomarkers are selected from the group consisting of EGFRvIII, HER-2, MET, K-ras, c-myc, PI3K, Akt, BRCA, PTEN, and VEGFR. 
     
     
         23 . The method of  claim 22 , wherein the expression status of the biomarkers is determined by Western blot or mass spectrometry. 
     
     
         24 . The method of any one of  claims 4  and  9  to  21 , wherein the microparticle characteristics are the number of Tenascin-C positive microparticles relative to the total microparticles isolated or the concentration of Ten-C positive microparticles. 
     
     
         25 . The method of any one of  claims 3 ,  4 ,  6 , and  9  to  24  wherein analyzing the tumor-derived microparticles is used to diagnose cancer, prognose cancer, and/or monitor the progression or recurrence of cancer. 
     
     
         26 . The method of any one of  claims 1  to  19 , wherein the Tenascin-C positive microparticles are isolated by direct affinity pulldown. 
     
     
         27 . The method of  claim 26 , wherein the Tenascin-C positive microparticles are contacted with an anti-Tenascin-C antibody. 
     
     
         28 . The method of  claim 27 , wherein the anti-Tenascin-C antibody is 81C6. 
     
     
         29 . The method of any one of  claims 1  to  28 , wherein the one or more microparticles isolated from the sample are incubated with a solution comprising LaurA. 
     
     
         30 . The method of  claim 29 , wherein the solution comprises about 5% LaurA. 
     
     
         31 . The method of any one of  claims 1 - 30 , wherein one or more microparticles isolated from the sample are contacted with a solution comprising arginine and glutamine. 
     
     
         32 . The method of  claim 31 , wherein the arginine and glutamine are present in the solution at substantially equal concentrations. 
     
     
         33 . The method of  claim 32 , wherein the concentration of arginine and glutamine is 1 M. 
     
     
         34 . The method of any one of  claims 1 - 30 , wherein one or more microparticles isolated from the sample are contacted with a solution comprising arginine and ethanol. 
     
     
         35 . The method of any one of  claims 1 - 30 , wherein one or more microparticles isolated from the sample are contacted with a solution comprising 1 M arginine buffer.