Transcriptomic analysis of DOXO-Resistant cells compared to parental DOXO-Sensitive mda-mb-436 cell line

The major obstacle in successfully treating triple negative breast cancer (TNBC) is resistance to cytotoxic chemotherapy, the mainstay of treatment in this disease. Previous pre-clinical models of chemoresistance in TNBC have suffered from a lack of clinical relevance. Using a single high dose chemotherapy treatment, we developed a novel MDA-MB-436 cell-based model of chemoresistance characterized by a unique and complex morphological phenotype, which consists of polyploid giant cancer cells (PGCCs) giving rise to neuron-like mononuclear daughter cells filled with smaller but functional mitochondria and numerous lipid droplets. This resistant phenotype is associated with metabolic reprogramming with a shift to a greater dependence on fatty acids and oxidative phosphorylation. We validated both the molecular and histologic features of this model in a clinical cohort of primary chemoresistant TNBCs and identified several metabolic vulnerabilities including a dependence on PLIN4, a perilipin coating the observed lipid droplets, expressed both in the TNBC resistant cells and clinical chemoresistant tumors treated with neoadjuvant doxorubicin-based chemotherapy. These findings thus reveal a novel mechanism of chemotherapy resistance that has therapeutic implications in the treatment of drug resistant cancer. The MDA-MB-436 cell line (referred to as parental cells), representative of a mesenchymal TNBC subtype (16), was exposed to a single high dose (IC75) of DOXO or PTX followed by drug removal after 48h. Parental cells treated with either DOXO or PTX exhibited the same phenotypic response to these treatments. First, after massive cell death, a few small, round mononucleated cells appeared one-week post-treatment. At 3-weeks post-treatment, polyploid giant cancer cells (PGCCs) exhibiting a massive cytoplasm and growing extrusions appeared (Fig. 1B) and, after 5 weeks, colonies of smaller mononuclear cells with thin cellular projections had appeared around these PGCCs. Biological replicates of colonies selected with DOXO (named C1 and C8) and PTX (CA, CD and CF) were isolated and grown individually without drug and then re-tested for chemosensitivity at different passages. Colonies C1 and C8 were resistant to DOXO (henceforth called DOXO-R). Colonies selected with PTX (CA, CD, CF) were also resistant to DOXO on re- testing even though they shared the cellular phenotypic response.