Establishing in vitro systems addressing the clonality of drug tolerant persister cells

Among women, breast cancer is the type of cancer with the highest mortality and incidence.Despite treatment with a combination of therapies and surgery patients often show signs ofincomplete response and eventual relapse. Lately a temporary state of drug resistance knownas drug tolerant persistence was found. Those so-called drug-tolerant persister (DTP) cellsand their mechanisms require further research.Currently, it is not known whether DTP cells are randomly selected for survival or are derivedfrom a pre-determined sub-population of drug-naive cells, and whether such “pre-DTP” cellswould be clonally determined. To address these possibilities in in vitro experiments, this thesisassessed an experimental setup using lentiviral tagging of cells and so-called repopulationassays where modeling acute drug response induces the DTP state. It was shown that 250 nMfor MCF-7 cells and 125 nM of doxorubicin for CSTr cells was the best concentration to inducethe DTP state. CSTr cells were shown to repopulate after about two weeks while MCF-7 cellstook in average approximately five weeks.Several genes are believed to be involved in the phenotypic transition. Their functionalrelevance can be assessed in knockdown experiments requiring two cell lines with differentfluorescent tags to distinguish cells with and without gene expression. This thesis tested if theintroduction of red and green fluorescent proteins drastically influences drug sensitivity ofMCF-7 cells, time of dormancy of DTP-cells and whether there is a selection for either one ofthe cell types when running repopulation assays. It was found that MCF-7 cells with twodifferent fluorescent proteins show similar drug sensitivity and that the time until repopulationis not significantly different. Moreover, it was shown that the ratio of RFP+ and GFP+ cellsdoes not drastically change over time, thus allowing planned knockdown experiments.