Dynamic BH3 profiling
The present invention provides methods of predicting cell sensitivity or resistance to a therapeutic agent.
1. A method of determining whether cancer cells will be responsive to a therapeutic agent in vivo, the method comprising:
a) contacting mitochondria in vitro with a BH3 domain peptide, wherein the mitochondria are isolated from a test cancer cell that has been contacted with the therapeutic agent, wherein the test cancer cell is a primary cancer cell of a patient;
b) measuring an amount of mitochondrial outer membrane permeabilization (MOMP) induced by the BH3 domain peptide in the mitochondria of the test cancer cell; and
c) comparing the amount of MOMP in the mitochondria of the test cancer cell to an amount of MOMP induced by the BH3 domain peptide in mitochondria of a control cancer cell that has not been contacted with the therapeutic agent, wherein: (i) an increase in the amount of MOMP in the mitochondria of the test cancer cell compared to the amount of MOMP in the mitochondria of the control cancer cell indicates cancer cells of the patient are responsive to the therapeutic agent in vivo, or (ii) a decrease or no change in the amount of MOMP in the mitochondria of the test cancer cell compared to the amount of MOMP in the mitochondria of the control cancer cell indicates cancer cells of the patient are not responsive to the therapeutic agent in vivo.
2. The method of claim 1 , wherein measuring the amount of MOMP comprises detecting staining for cytochrome c.
3. The method of claim 2 , further comprising contacting the mitochondria of the test cancer cell with an antibody for cytochrome c.
4. The method of claim 1 , wherein the therapeutic agent is a chemotherapeutic agent.
5. The method of claim 1 , wherein the cancer cells of the patient comprise hematological cancer cells or solid tumor cells.
6. A method of determining whether cancer cells will be responsive to a therapeutic agent in vivo, the method comprising:
a) contacting a test cancer cell in vitro with a BH3 domain peptide, wherein the test cancer cell has been contacted with the therapeutic agent, wherein the test cancer cell is a primary cancer cell of a patient;
b) measuring an amount of MOMP induced by the BH3 domain peptide in the test cancer cell; and
c) comparing the amount of MOMP in the test cancer cell to an amount of MOMP induced by the BH3 domain peptide in a control cancer cell that has not been contacted with the therapeutic agent, wherein: (i) an increase in the amount of MOMP in the test cancer cell compared to the amount of MOMP in the control cancer cell indicates cancer cells of the patient are responsive to the therapeutic agent in vivo, or (ii) a decrease or no change in the amount of MOMP in the test cancer cell compared to the amount of MOMP in the control cancer cell indicates cancer cells of the patient are not responsive to the therapeutic agent in vivo.
7. The method of claim 6 , wherein the BH3 domain peptide comprises a transduction domain that translocates the BH3 domain peptide across a plasma membrane of the test cancer cell.
8. The method of claim 6 , wherein the test cancer cell is permeabilized to permit the BH3 domain peptide access to the mitochondria.
9. The method of claim 8 , further comprising permeabilizing the test cancer cell prior to contacting with the BH3 domain peptide.
10. The method of claim 6 , further comprising fixing the test cancer cell prior to measuring the amount of MOMP.
11. The method of claim 6 , wherein the test cancer cell is immobilized on a solid surface.
12. The method of claim 6 , wherein measuring the amount of MOMP comprises:
detecting emission from a potentiometric dye; or
detecting staining for cytochrome c.
13. The method of claim 6 , wherein the BH3 domain peptide is derived from a BH3 domain of a BH3 interacting domain death agonist (BID), a Bcl-2 interacting mediator of cell death (BIM), a Bcl-2-associated death promoter (BAD), a Noxa, a p53 up-regulated modulator of apoptosis (PUMA), a Bcl-2-modifying factor (BMF), or a harakiri (HRK) polypeptide.
14. The method of claim 6 , wherein the therapeutic agent is a chemotherapeutic agent.
15. The method of claim 14 , wherein the chemotherapeutic agent is a targeted chemotherapeutic agent.
16. The method of claim 14 , wherein the chemotherapeutic agent is a kinase inhibitor.
17. The method of claim 6 , wherein the cancer cells of the patient comprise hematological cancer cells.
18. The method of claim 17 , wherein the hematological cancer cells are selected from the group consisting of acute myeloid leukemia cells, chronic myelogenous leukemia cells, diffuse large B-cell lymphoma cells, and multiple myeloma cells.
19. The method of claim 6 , wherein the cancer cells of the patient comprise solid tumor cells.
20. The method of claim 19 , wherein the solid tumor cells are selected from the group consisting of breast cancer cells, non-small cell lung cancer cells, melanoma cells, and colon carcinoma cells.