Supplementary Components1. (HER2), (HER3), compared to parental cells (Supplementary Physique 2). Open in a separate window Physique 1: Trastuzumab resistance is usually reversible.(A) Trastuzumab-resistant pools were generated by exposing parental BT474 cells to increasing doses of trastuzumab over the course of 3+ months. (B) Schematic of resistance reversal experiment for BT-TR cells. (C-D) Pools of BT474 cells made resistant to trastuzumab were cultured in trastuzumab (+T; triangle) or without drugs (washout; square) for 20 doublings (9 passages) and their proliferation after ten days of trastuzumab treatment was measured by WST-1 assays. BT474 cells (circle) were included as a control. Proliferation is usually shown as a percentage of no treatment control growth. (E) Schematic of resistance reversal experiment for BT-TR2-derived clones. (F-G) Clones of BT-TR2 cells were cultured in trastuzumab (+T; down triangle) or without drugs (washout; square) for 23 doublings. Proliferation after ten days of trastuzumab treatment was measured by WST-1 assays. BT474 cells (circle) and BT-TR2 cells cultured constantly in trastuzumab (up triangle) were included as controls. Proliferation is usually shown as a percentage of no treatment control growth. Data points in C, D, F and G represent means of three replicate wells SEM. We Necrostatin 2 racemate passaged BT474-derived resistant pools side by side in trastuzumab or drug-free media (referred to as washout) and Necrostatin 2 racemate examined their sensitivity to trastuzumab periodically. After twenty doublings (nine passages) in drug-free media, all pools became more sensitive to the drug, and three out of four pools of resistant cells tested regained sensitivity to trastuzumab (Physique 1BCD, Supplementary Physique 3ACB). To decipher if the pools regained sensitivity due to clonal selection or flexibility of individual clones, we generated single cell clones from the resistant pool BT-TR2 and repeated the assay (Physique 1E). After 23 doublings, two of three resistant clones tested regained sensitivity to trastuzumab (Physique 1FCG). The third clone demonstrated increased sensitivity after 34 doublings (Supplementary Physique 3C). Taken together, these results suggested that non-genetic changes may mediate resistance to trastuzumab. The oxidative phosphorylation gene signature is usually enriched in resistant cells. We hypothesized that alterations in gene expression programs could be the major contributors to resistance. Thus, RNA-sequencing was performed for sensitive BT474 cells, two pools of BT-TR cells and two pools of BT-TPR cells cultured in the absence of drug(s) for seven days in order to exclude gene expression changes induced by the drug(s) (Supplementary Furniture 2C5). We utilized GSEA to identify differences between resistant pools and BT474 parental cells (Supplementary Furniture 6C13). Several hallmark pathways were positively enriched with nominal p-value <0.05 and FDR q-value <0.1 in each resistant pool compared to BT474 cells. Only one hallmark pathway, protein secretion, was common to both BT-TR pools, but not BT-TPR pools (Physique 2A). Surprisingly, no pathways were common to both BT-TPR pools without also being enriched in BT-TR pools, highlighting similarities in pools resistant to single and combination therapies. Three GSEA hallmark pathways were positively enriched in all four resistant pools compared to BT474 cells: oxidative phosphorylation, fatty acid metabolism, and MYC targets V1 (Physique 2A). Oxidative phosphorylation (OXPHOS) was the top positively enriched pathway in BT-TR2, BT-TPR1, and BT-TPR2 cells, and third for BT-TR1 (Physique 2BCC, Supplementary Furniture 6C9). Open in a separate window Physique 2: The oxidative phosphorylation gene program is usually elevated in resistant cells.(A) GSEA hallmark pathways positively enriched with nominal p-value<0.05 and FDR q-value<0.1 in resistant pools versus BT474 parental cells (left). NES scores of each resistant pool for pathways enriched in all pools compared to BT474 cells (right). (B-C) GSEA enrichment plots of the hallmark oxidative phosphorylation pathway for BT-TR2 (B) and BT-TPR1 (C) versus BT474 parental cells. (D) GSEA hallmark pathways negatively enriched with nominal p-value<0.05 and FDR q-value<0.1 in resistant pools versus BT474 parental cells (left). NES scores of each resistant pool for pathways enriched in all pools compared to BT474 cells (right, top) or in BT-TPR pools only (right, bottom). (E) GSEA Necrostatin 2 racemate enrichment plots of the hallmark estrogen response early pathway for BT-TR2 versus BT474 parental cells. (F) GSEA enrichment plots of the hallmark epithelial mesenchymal transition pathway for BT-TPR1 Rabbit Polyclonal to KALRN versus BT474 parental cells. (G) GSEA enrichment Necrostatin 2 racemate plots of the hallmark IL6 JAK STAT3 signaling pathway.