Tissue-derived extracellular vesicles and their use as diagnostics
The present disclosure relates to a method of isolating extracellular vesicles directly from human tissues. The invention further relates to a method of identifying disease and tissue specific membrane proteins on extracellular vesicles by membrane isolation and proteomic analysis. The invention further relates to methods of diagnosing diseases by capturing extracellular vesicles by the use of disease specific membrane proteins from body fluids, and detecting or analyzing molecular signatures (proteome, DNA, and RNA) on captured extracellular vesicles. Moreover, the present invention relates to kits, apparatus and software required for implementing aforementioned methods.
1 . A method comprising the steps of:
(a) fragmenting a human tissue into a plurality of pieces;
(b) incubating the plurality of pieces with one or more enzymes, wherein the one or more enzymes are selected from a group of proteases including a matrix metalloproteinase, collagenases, and papain and nucleases including DNase, RNase, and Benzonase, wherein the incubating releases extracellular vesicles from the plurality of pieces, thereby generating a mixture of the extracellular vesicles, the plurality of pieces, and the one or more enzymes;
(c) isolating the extracellular vesicles from the mixture;
(d) treating the extracellular vesicles with an aqueous solution, wherein the aqueous solution has a pH in a range of 9 to 14 to generate membranes;
(e) isolating the membranes; and
(f) identifying proteins on the membranes by employing mass spectrometry to identify tissue specific membrane proteins.
2 . The method of claim 1 , wherein the human tissue comprises diseased tissue.
3 . The method of claim 2 , wherein the diseased tissue is a tumor.
4 . The method of claim 3 , wherein the tumor is a lung cancer, larynx cancer, stomach cancer, large intestine/rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, uterine cervical cancer, prostate cancer, or kidney cancer.
5 . The method of claim 3 , wherein the tumor is a melanoma, bone cancer, muscle cancer, adipose tissue sarcoma, or mesothelioma.
6 . The method of claim 3 , wherein the tumor is a neuroma, neuroblastoma, or medulloblastoma.
7 . The method of claim 1 , wherein the human tissue is liver, brain, kidney, heart, lung, skin, stomach, intestines, lymph nodes, bone marrow, peripheral or central nervous tissue, endocrine glands, adipose tissue or muscles.
8 . The method of claim 1 , wherein isolating the membranes at the step (e) comprises ultracentrifugation.
9 . The method of claim 2 , further comprising isolating extracellular vesicles from a diseased human tissue using a specific binder for a diseased tissue-specific membrane proteins identified in step (f) and characterizing at least one of:
i. their disease-specific proteome;
ii. their disease-related RNA cargo; and
iii. their disease-related DNA cargo.
10 . The method of claim 1 , wherein the enzymes comprise collagenase D and DNase I.
11 . The method of claim 1 , wherein isolating the extracellular vesicles at the step (c) comprises filtration of the mixture to generate a flow-through and centrifugation of the flow-through from the filtration to remove cells and tissue debris and provide a supernatant comprising the extracellular vesicles.