E2F reporter melanoma cells
A method of using a modified melanoma cell capable of quantification of the effects of MEK inhibitors and CDK4/6 inhibitors in a quantitative, temporal and non-invasive manner both in vitro and in vivo.
1. A method for determining the efficacy of a compound of interest to inhibit E2 Factor (E2F) activity comprising:
a. applying a compound of interest to a tumor cell, wherein said tumor cell comprises a modified E2F reporter system to monitor the efficacy of CDK4/6 inhibitor-based therapies, wherein said tumor cells are transduced with tdTomato fluorescent protein and an EGFP-firefly luciferase fusion gene under the control of E2F transcriptional response elements; and, wherein the tumor cell is a melanoma cell harboring a serine/threonine protein kinase B-RAF (BRAF), a melanoma cell harboring a neuroblastoma RAS viral oncogene homolog (NRAS) mutation, or a melanoma cell wild type for both BRAF and NRAS;
b. determining the amount of tdTomato activity and determining the amount of firefly luciferase activity in the tumor cell, and
c. comparing the amount of tdTomato activity to the amount of firefly luciferase activity wherein the tdTomato activity measures tumor size and firefly luciferase activity measures pathway activity in the tumor; and quantitate the inhibition of the E2F reporter activity through visualizing the activity of the tdTomato and firefly luciferase and comparing the firefly luciferase activity normalized to tdTomato activity.
2. The method of claim 1 , wherein the method is performed in vitro.
3. The method of claim 1 , wherein the method is performed in vivo, further comprising the steps of:
d. injecting the tumor cell line expressing the reporter system subcutaneously or intradermally into a mouse model, wherein tumors are allowed to form; and
e. injecting said mouse intraperitoneally with luciferin wherein said firefly luciferase activity can be imaged and quantitated using an in vivo imaging system (IVIS) machine.
4. The method of claim 3 wherein tdTomato activity can be imaged and quantitated using an IVIS machine; and wherein firefly luciferase activity is normalized to tdTomato activity to quantify existing pathway activity.
5. The method of claim 1 comprising administering a second compound of interest to the tumor cell.
6. The method of claim 5 comprising administering a third compound of interest to the tumor cell.