Fig. 1

Platform for the identification of drug response biomarker genes with single cell lineage tracing. A An overview of strategies of lineage tracing. Once cells are marked (i.e. barcoded), they can be exposed to a selected drug concentration to drive the selection of resistant clones (yellow cells). Two types of analyses can then be performed. Prospective analyses where resistant clones (i.e. lineages) are followed over time during treatment to reconstruct driving mechanisms of drug adaptation. Retrospective analyses where resistant lineages are retrospectively mapped on the untreated condition to reconstruct pre-existing mechanisms of drug resistance. B Average log IC50 distribution of MDAMB468 cells and of all the other available TNBC cell lines to anticancer drugs targeting EGFR. C The Cellecta’s lentiviral construct contain a pool of random 48 base pairs (bp) barcodes located 70 bp far from ploy-A tail of the puromycin‐resistance gene cloned downstream of a Venus fluorescent protein. D To enable lineage tracing in MDAMB468 cells, we seeded 50,000 cells and infected them with Cellecta's lentiviral libraries at a multiplicity of infection (MOI) of 0.05. After five days of antibiotic selection, surviving cells were assessed by flow cytometry and subsequently expanded. E Scheme of the time series experiment performed on the barcoded MDAMB468 cell line. D0 are untreated parental MDAMB468 cells (MDAMB468-P) a few hours before afatinib addition, and D3 through D40 is the duration of the experiment where cells where subjected to incremental doses of afatinib ranging from 250 to 2000 nM. Cells at D40 were considered as afatinib tolerant persisted cells (ATPC). 10 × Chromium sequencing was performed at Day 0 (D0), Day 3 (D3), Day 6 (D6) and Day 9 (D9) of 250 nM afatinib treatment. Quantseq-Flex to retrieve afatinib-tolerant lineages in bulk was performed at Day 40 with 2000 nM afatinib treatment. F Dose–response curve showing cell viability of MDAMB468-P and MDAMB468-ATPC cells following treatment with the indicated concentrations of afatinib. G Estimated IC50 for the dose response curve in (G). H Number of unique lineages present in the MDAMB468-P and MDAMB468-ATPC cell lines