A novel genetic- and cell-based tool for assessing the efficacy and XXXX of anticancer drugs in vitro.

@entity5 @entity137 @entity1265 @entity50001
AIMS: To develop an in vitro tool for assessing the efficacy and @entity137 of anticancer drugs using mixed culture containing both @entity5 and non- @entity5 cells. Such in vitro tool should have high application potential in drug-screening and personalized @entity5 care. METHODS: Fibroblasts were spiked as non- @entity5 cells into @entity5 cells of an established line. The mixed culture was treated with a test drug at various concentrations. After the treatment, DNA was prepared directly from the survived adhesive cells in the wells of the 96-well plates using a simple and inexpensive method, and subjected to digital PCR for measuring relative copy numbers of a target gene @entity1265 to that of a reference gene @entity50001 . The @entity1265 gene is known to be heterozygously deleted in these @entity5 cells while the @entity50001 gene has two @entity5 and non- @entity5 cells. Using the @entity1265 / @entity50001 ratios resulting from the dual digital PCR assay, the proportions of @entity5 cells were calculated for each drug concentration. RESULTS: Digital PCR confirmed that the @entity5 cells have only one copy of the @entity1265 gene while the non- @entity5 fibroblasts have two copies. By contrast, both types of cells have two copies of the reference gene @entity50001 . Using the ratio of the two genes, we successfully calculated the proportion of @entity5 cells which decreased as the dose of the test drug increased up to a certain concentration, indicating that the drug is more effective for the @entity5 cells than for the non- @entity5 cells in this dose-range. At the highest dose, we observed a slight increase in the proportion of @entity5 cells, likely reflecting the toxic effect of the @entity5 and non- @entity5 cells. CONCLUSION: This pilot study demonstrated the feasibility of a genetic- and cell-based tool for testing efficacy and @entity137 of anticancer drugs in vitro. The promising results suggest that additional efforts are merited, for further development since such a tool will likely have high application potential (1) in drug discovery where it enables simultaneously assessing therapeutic effect on target cells and toxic effect on non-target cells, and (2) in personalized adjuvant chemotherapy where multiple drugs can be tested in primary cultures derived from surgically removed @entity5 .
