USE OF TENASCIN-C AS AN EXTRACELLULAR MARKER OF TUMOR-DERIVED MICROPARTICLES
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.
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.