title: Assessing Specificity of Anticancer Drugs In Vitro.
abstract: A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.
authors.affiliation: Department of Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf; Department of Neurology, University Medical Center Hamburg-Eppendorf; Laboratory for Tumor Biology and Regenerative Medicine, University Medical Center Hamburg-Eppendorf; kluwe@uke.de.
authors.forename: Lan
authors.initials: L
authors.lastname: Kluwe
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D055808:Drug Discovery / Q000379:methods*; D006801:Humans; D009369:Neoplasms; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D000970:Antineoplastic Agents