Live cell imaging dynamic BH3 profiling
AssigneeDana-Farber Cancer Institute, Inc.
InventorsAnthony Letai, Patrick Bhola, Rebecca German
Life of this patent
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Abstract
Aspects of the application provide methods and compositions for identifying and evaluating putative therapeutic agents for cancer by live cell imaging. Cell samples comprising cancerous cells that have been pre-treated with a test agent are contacted with a BH3 peptide, and samples are imaged by live cell imaging over a time interval. Methods of the application can be used to determine whether a patient is likely to benefit from treatment with a particular test agent.
Claims20
A method of identifying a putative therapeutic agent for treating cancer, the method comprising:
- a. providing a test cell portion of a cell population that comprises primary cancerous cells, wherein the test cell portion has been contacted with a test agent;
- b. contacting the test cell portion with a BCL-2 homology domain 3 (BH3) peptide;
- c. capturing a series of images of the test cell portion over a time interval by live cell imaging;
- d. measuring BH3 peptide-induced mitochondrial outer membrane permeabilization (MOMP) in the test cell portion at different time points in the time interval by detecting emission of a potentiometric dye;
- and
- e. comparing BH3 peptide-induced MOMP measured in the test cell portion to BH3 peptide-induced MOMP in a control cell portion of the cell population, wherein the control cell portion has not been contacted with the test agent;
- wherein an increase in BH3 peptide-induced MOMP in the test cell portion compared to that in the control cell portion indicates that the test agent is a putative therapeutic agent for treating cancerous cells of the cell population, and no increase in BH3 peptide-induced MOMP in the test cell portion compared to that in the control cell portion indicates that the test agent is not a putative therapeutic agent for treating cancerous cells of the cell population.
The method of any one of the preceding claims, further comprising:
- i) providing the control cell portion of the cell population, wherein the control cell portion has not been contacted with the test agent;
- ii) contacting the control cell portion with the BH3 peptide;
- iii) capturing a series of images of the control cell portion over the time interval by live cell imaging;
- and
- iv) measuring BH3 peptide-induced MOMP in the control cell portion at the different time points based on the captured images.
The method of any one of the preceding claims, wherein the test cell portion is contacted with the BH3 peptide at a first peptide concentration, and the method further comprises contacting the test cell portion with the BH3 peptide at a second increased peptide concentration after measuring BH3 peptide-induced MOMP at the first peptide concentration.
A method of identifying a putative therapeutic agent for treating cancer, the method comprising:
- a. providing a test cell portion of a cell population that comprises primary cancerous cells, wherein the test cell portion has been contacted with a test agent;
- b. contacting the test cell portion with a BCL-2 homology domain 3 (BH3 ) peptide;
- c. capturing a series of images of the test cell portion over a time interval by live cell imaging;
- d. measuring BH3 peptide-induced mitochondrial outer membrane permeabilization (MOMP) in the test cell portion at different time points in the time interval by detecting emission of a potentiometric dye;
- and
- e. comparing BH3 peptide-induced MOMP measured in the test cell portion to BH3 peptide-induced MOMP in a control cell portion of the cell population, wherein the control cell portion has not been contacted with the test agent;
- wherein comparing comprises determining a value for delta priming, wherein delta priming is the difference between BH3 peptide-induced MOMP in the test cell portion and BH3 peptide-induced MOMP in the control cell portion;
- and
- wherein an increase in BH3 peptide-induced MOMP in the test cell portion compared to that in the control cell portion indicates that the test agent is a putative therapeutic agent for treating cancerous cells of the cell population, and no increase in BH3 peptide-induced MOMP in the test cell portion compared to that in the control cell portion indicates that the test agent is not a putative therapeutic agent for treating cancerous cells of the cell population.
A method of identifying a putative therapeutic agent for treating cancer, the method comprising:
- a. providing a test cell portion of a cell population that comprises primary cancerous cells, wherein the test cell portion has been contacted with a test agent;
- b. contacting the test cell portion with a BCL-2 homology domain 3 (BH3) peptide and treating it with a reagent for measuring BH3 peptide-induced mitochondrial outer membrane perrneabilization (MOMP);
- c. capturing a series of images of the test cell portion over a time interval by live cell imaging;
- d. analyzing the captured images to measure BH3 peptide-induced MOMP in the test cell portion at different time points in the time interval, wherein the analysis comprises measuring an increase or decrease in emission over time by the reagent for measuring BH3 peptide-induced MOMP;
- and
- e. comparing BH3 peptide-induced MOMP measured in the test cell portion to BH3 peptide-induced MOMP in a control cell portion of the cell population, wherein the control cell portion has not been contacted with the test agent;
- wherein an increase in BH3 peptide-induced MOMP in the test cell portion compared to that in the control cell portion indicates that the test agent is a putative therapeutic agent for treating cancerous cells of the cell population and no increase in BH3 peptide-induced MOMP in the test cell portion compared to that in the control cell portion indicates that the test agent is not a putative therapeutic agent for treating cancerous cells of the cell population.
Dependencies are inferred from the claim text.