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

Abstract

Aims.To develop an in vitro tool for assessing the efficacy and toxicity of anticancer drugs using mixed culture containing both tumor and non-tumor cells. Such in vitro tool should have high application potential in drug-screening and personalized cancer care. Methods. Fibroblasts were spiked as non-tumor cells into tumor 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 NF1 to that of a reference gene RPP30. The NF1 gene is known to be heterozygously deleted in these tumor cells while the RPP30 gene has two copies in both tumor and non-tumor cells. Using the NF1/ RPP30 ratios resulting from the dual digital PCR assay, the proportions of tumor cells were calculated for each drug concentration. Results. Digital PCR confirmed that the tumor cells have only one copy of the NF1 gene while the non-tumor fibroblasts have two copies. By contrast, both types of cells have two copies of the reference gene RPP30. Using the ratio of the two genes, we successfully calculated the proportion of tumor 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 tumor cells than for the non-tumor cells in this dose-range. At the highest dose, we observed a slight increase in the proportion of tumor cells, likely reflecting the toxic effect of the drug on both tumor and non-tumor cells.Conclusion. This pilot study demonstrated the feasibility of a genetic-and cell-based tool for testing efficacy and toxicity 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 tumor.

Combined Targeting of AKT and mTOR Inhibits Proliferation of Human NF1-Associated Malignant Peripheral Nerve Sheath Tumour Cells In Vitro but not in a Xenograft Mouse Model In Vivo

“…Therefore, novel experimental therapies have to be tested and validated in preclinical models of NF1-associated neoplasms. MPNST-derived cell lines, which recapitulate the genetic landscape of their corresponding primary tumour, have been demonstrated in the past to be eligible model systems for the evaluation of novel therapeutic agents or combinations of established anticancer drugs in vitro [26][27][28][29] and, when implanted into immunodeficient mice, also in vivo [15,30,31]. MPNST cell lines have also been shown to contain a subpopulation of cells with stem-like properties (cancer stem-like cells, CSLC), thereby representing the intratumoural heterogeneity of MPNST in cell culture [32].…”

New insights into the role of ribonuclease P protein subunit p30 from tumor to internal reference

“…Because RPP30 is stably expressed in the vast majority of tumor cells and non-tumor cells, while the neurofibromatosis type 1 (NF1) gene loses heterozygosity in tumor cells, the number of tumor cells may be evaluated by the quantitative RT-PCR ratio of NF1 to RPP30, which may be used to evaluate the efficacy and side effects of tumor drugs, and may also be used in personalized adjuvant chemotherapy. Due to the different behavior of cells in vivo and in vitro, this method has some limitations (76)(77)(78). As an internal reference gene, RPP30 may also accurately and effectively evaluate the concentration of antiretroviral drugs in cells (79).…”

Separating Response of Tumor and non-Tumor Cells to Drug In Vitro by Quantifying a Mutation

“…Previously, we demonstrated the feasibility of using a tumor-specific allele loss for determining the proportion of tumor cells in a mixed culture (5,6). The present study aims to further explore the feasibility of using the BRAF mutation c.1799T>A to determine the proportion of tumor over nontumor cells in a mixed culture.…”

“…A strategy for determining the proportion of tumor cells in a mixed culture is therefore highly desirable. One possible strategy is to quantify a tumor-specific genetic alteration such as a mutation or an allele loss (5,6). For melanomas with the BRAF mutation c.1799T>A, the ratio of the mutant 1799A allele to the wild-type 1799T allele should, at least in theory, enable the calculation of the proportion of the tumor cells in a mixed culture containing both tumor and non-tumor cells.…”