Additional file 1: of A novel anti-HER2 anthracycline-based antibody-drug conjugate induces adaptive anti-tumor immunity and potentiates PD-1 blockade in breast cancer

Figure S1 Generation of a murine EMT6-hHER2 breast cancer cell line. A, Human-HER2 expression in EMT6-hHER2 or EMT6-WT tumor cell line determined by FACS. B, H&E (top panel) and 〈-hHER2 (bottom panel) immunohistochemical staining of orthotopic EMT6-WT and EMT6-hHER2 tumors. C, Cell proliferation assay showing EMT6-WT and EMT6-hHER2 growth inhibition after T-PNU or T-DM1 treatment in vitro. Figure S2 Trastuzumab, free PNU or mitoxantrone treatment is ineffective in promoting EMT6-hHER2 tumor growth inhibition. A, Therapeutic response of EMT6-hHER2 tumors exposed to T-PNU (1 mg/kg, 2x), free PNU (6 and 8 μg/kg, 2x), trastuzumab (20 mg/kg, 4x, once per week) and mitoxantrone (4 mg/kg 1x). B, Survival curves of A. Figure S3 T-PNU samples cluster apart from untreated, trastuzumab and T-DM1 treated samples based on gene expression. A, Sample similarity in a 2D projection by multi-dimensional scaling. Only the top 1000 differentially expressed genes (DEGs) are taken into account. B, Heatmap of the gene expression vs sample matrix. Displayed are custom selected genes plus the top 1000 DEGs (rows) of all samples (columns). Figure S4 Heatmap PID CD8 TCR downstream pathway. Figure S5 Gene signatures of innate immune and cytokine pathways. A, Network cluster of gene sets with overlapping genes with shared function of inflammatory pathway. The network includes 36 gene sets and 7551 genes (see Table S2). B-D, Heatmaps of BIOCARTA inflammatory, dendritic cell (DC) and cytokine pathway, respectively. Asterisks denote low-responding T-PNU samples. Figure S6 Characterization of intratumoral T cells upon treatment. *p ≤ 0.05, **p ≤ 0.01. A, MvA plot depicting expression and effect size of selected key immune genes. B, Validation by FACS of selected CD8 T cell markers of functional activation and proliferation identified in A in between the comparisons. Table S1 Gene sets of network TCR pathway. Table S2 Gene sets of network activated TLR pathway. (PDF 10859 kb