Method for detecting invasive microvesicles derived from tumor cells
The present application relates to the isolation and analysis of populations of microvesicles, such as populations of microvesicles that are shed by tumor cells and contain the protein ARF6. Invasive microvesicles from tumor cells contain a variety of specific proteins, including ARF6.
1. A method of identifying invasive microvesicles in a biological sample, the method comprising the steps of:
providing a biological sample;
centrifuging the biological sample to collect a population of microvesicles, and
assaying the population of microvesicles for the presence of the proteins ARF6 and Vamp3, wherein the presence of ARF6 and Vamp3 identifies the population of microvesicles as comprising invasive microvesicles.
2. The method of claim 1 , wherein the biological sample is a biological fluid obtained from a subject.
3. The method of claim 2 , wherein the biological fluid comprises one or more of blood, plasma, serum, urine, saliva, or ascites.
4. The method of claim 2 , wherein the biological fluid is obtained by washing an anatomical structure of the subject and collecting the wash fluid after it has come in contact with the anatomical structure.
5. The method of claim 1 , wherein assaying the prepared population of microvesicles is accomplished by contacting the microvesicles with an antibody that binds to ARF6.
6. The method of claim 1 , additionally comprising the step of:
assaying the prepared population of microvesicles for the presence of one or more proteins selected from the group consisting of MHC class I, β1-integrin and β1-integrin receptor.
7. The method of claim 6 , wherein the population of microvesicles is assayed by western blot.
8. The method of claim 1 , wherein the sample is centrifuged at about 10,000 g.
9. The method of claim 8 , wherein the sample is centrifuged at about 2,500 g prior to centrifugation at about 10,000 g.
10. A method of analyzing a population of microvesicles for the presence of invasive microvesicles comprising:
determining whether the protein ARF6 and a processed form of the protein MT1-MMP are present in the population of microvesicles, wherein the presence of ARF6and the processed form of MT1-MMP indicates that invasive microvesicles are present in the sample.
11. The method of claim 10 , additionally comprising determining whether one or more additional proteins selected from the group consisting of Vamp3, MHC class I, β1-integrin and β1-integrin receptor are present in the sample.
12. The method of claim 1 , wherein the biological sample is centrifuged at from 5,000 g to 15,000 g.
13. The method of claim 1 , additionally comprising assaying the population of microvesicles for the presence of MT1-MMP.
14. The method of claim 12 , wherein the sample is centrifuged two or more times at from 5,000 g to 15,000 g.
15. The method of claim 12 , additionally comprising centrifuging the sample at from about 1,200 g to about 3,000 g prior to centrifuging the sample at from 5,000 g to 15,000 g.
16. The method of claim 1 , wherein the population of microvesicles is assayed for the presence of ARF6 by western blot.
17. The method of claim 1 , wherein the biological sample is obtained from a patient suffering from a tumor.