Gene expression analysis of tumors of TN and R clonal pairs in vivo

We previously had generated clonal mouse melanoma cell lines, where each clone was isolated in a RAFi/MEKi-resistant ( R ) or treatment-naïve ( TN ) state using the method CaTCH (clones: 2646TN, 2646R, 14559TN, 14559R, 13TN). We assessed the response to targeted therapy of these clones in in vivo experiments using RAFi/MEKi-treatments. Herey, we saw that, compared to the other treatment-naïve clones, 2646TN exhibited a strongly reduced response to targeted therapy. 2646TN displayed fast growth on drug after only five days, indicating an increased persistence and fast adaption to the treatment, while all other TN clones responded to the treatments and showed tumor regression for weeks before relapsing. To uncover intrinsic programs that might underlie th minimal response of 2646TN, we examined the transcriptome of untreated and short-term treated tumors comprising 2646TN, 2646R, 14559TN, 14559R, or 13TN clones . These clonal cell lines were injected subcutaneously into C57BL/6 mice and tumors were harvested either at tumor engraftment (untreated) or after 3 days of RAFi/MEKi treatment. Lviving cancer cells were sorted from the tumors by FACS and the markers CD45- and the cell line specific marker GFP+. Libraries were prepared using the Lexogen Quantseq 3' mRNA seq kit and sqeuencing was performed on an Illumina HiSeqV4 sequencer (single-read 50 read mode). 5 different mouse melanoma cell lines FACS sorted from subcutaneously injectd tumors, each untreated and RAFi/MEKi treated, 3 tumors per cell line and condition (= 30 samples total)