Primary triple negative breast cancers (TNBC) are prone to dissemination but sub-clonal relationships between tumors and resulting metastases are poorly understood. Here we use cellular barcoding of two treatment-naïve TNBC patient-derived xenografts (PDXs) to track the spatio-temporal fate of thousands of barcoded clones in primary tumors, and their metastases. Tumor resection had a major impact on reducing clonal diversity in secondary sites, indicating that most disseminated tumor cells lacked the capacity to ‘seed’, hence originated from ‘shedders’ that did not persist. The few clones that continued to grow after resection i.e. ‘seeders’, did not correlate in frequency with their parental clones in primary tumors. Cisplatin treatment of one BRCA1-mutated PDX model to non-palpable levels had a surprisingly minor impact on clonal diversity in the relapsed tumor yet purged 50% of distal clones. Therefore, clonal features of shedding, seeding and drug resistance are important factors to consider for the design of therapeutic strategies.

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Androgens have anabolic actions in skeletal muscle and could potentially act to: (a) increase proliferation of myoblasts; (b) delay differentiation to myotubes; and (c) induce protein accretion in post-proliferative myofibers. To identify the site of androgens action, we investigated the proliferative response of the C2C12 mouse myoblast cell line to testosterone and dihydrotestosterone (DHT) treatment. Neither androgens affected cell proliferation after up to 7 days treatment, nor was there a synergistic effect of androgens on the proliferative response of C2C12 cells to IGF-I treatment. However, proliferating C2C12 cells expressed 0.1% of the level of androgen receptor (AR) mRNA found in adult mouse gastrocnemius muscle (p<0.01). Therefore, we generated mouse C2C12 myoblast cell lines stably transfected with the mouse AR cDNA driven by the SV40 promoter (C2C12-AR). C2C12-AR cell proliferation, differentiation, and protein content were analyzed in response to androgen treatment. Our data demonstrated that androgen treatment does not alter either proliferation rate or differentiation rate of C2C12-AR cells. However, treatment of differentiated C2C12-AR myotubes with 100 nM DHT for 3 days caused a 20% increase in total protein content vs vehicle treatment (p<0.05). This effect was not observed in control C2C12 cells transfected with empty vector. These data suggest that androgens act via the AR to upregulate myotube protein content. This model cell line will be useful to further investigate the molecular mechanisms via which androgens regulate protein accretion.