Development of Methods to Validate Synthetic Lethalitty In vitro and In vivo

ANDRES CASTELLARO; AYELEN NIGRA; , GERMÁN A. GIL; GASTON SORIA

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de BioCiencias; 2017

Reunión Conjunta de Sociedades de BioCiencias

One of the current challenge in the development of anticancer therapies lies in the identification of drugs that selectively kill cancer cells without destroying normal cells. A strategy of selective cytotoxicity that has yielded promising results is the synthetic lethality (SL) induction. The SL induction involves previous tumor-mutations and some other partner mutation which becomes deadly when are in combination. We focused in the study of drugs that cause SL in BRCA1/2 deficient cancer cells (Bd-cells). Herein we show the trials for the validation of potential drugs which have previously been found as possible SL-inducers using cell-based screening high-throughput flow cytometry platform. All experiments are based on the study of the performance of different drugs tested on a Bd-cell model and comparing it with the same wild type cell line (Bw-cells). First, it was analyzed the capacity of drugs to cause DNA damage by In-Cell Western through the quantification of H2AX phosphorylation. Furthermore, Bd-cells and Bw-cells drugs treated were evaluated by clonogenic assays to produce progeny cells. In addition, by using 3D spheroids it was validated the SL induction and cytotoxicity before been test in vivo model. To accomplish this, the chimeric spheroids formed by an equal proportion of Bd-cells and Bw-cells, both tagged with a fluorescent protein, were drug treated and then SL was measured by flow cytometry. The last step of the validation was performed in a double tumor xenograft model in Nu/Nu mice. This model consists of generating two xenograft tumors in a single mouse, one from Bd-cells and the other from Bw-cells. Mice were treated with a possible SL-inducers compound or vehicle and then was to assess the tumor progression of each tumor-type.