Tolinapant promotes activation of the innate immune system by inducing necroptosis and immunogenic cell death in tumor cells. (A) Myeloid pathway score for the BW5147 model (top) and the HH model (bottom) calculated using NanoString Advanced Analysis pathway scores module. (B) Heatmap of the expression of selected genes that are differentially expressed between vehicle and tolinapant-treated BW5147 tumors (adjusted P < .05), displayed as gene-wise z-scores (log2 normalized expression). Selected genes were obtained from gene expression signatures associated with different pathways scored with NanoString Advanced Analysis GSA module. (C) Expression of CD25, CD40, and HLA-DR on M0 macrophages at day 6 of treatment with vehicle (DMSO) or 1 µM tolinapant analyzed by flow cytometry (3 donors). *P < .05, **P < .01 (ratio paired t test). (D) Expression of CD86 (top) and CD40 (bottom) on polarized M1 and M2 macrophages at day 8 of treatment with vehicle (DMSO) or 1 µM tolinapant analyzed by flow cytometry (3 donors). *P < .05, **P < .001 (ratio paired t test). (E) Effect of tolinapant or tolinapant + 10 ng/mL mouse TNF-α on BW5147 cell viability by CTG assay after 72 hours. (F) As for panel E, except addition of 0.5 µM emricasan to inhibit caspase-8. (G) As for panel E, except addition of 20 µM Necrostatin-1 (Nec-1) to inhibit RIPK1. (H) Viability of BW5147 cells by cytometry (PI and CC3) after 48 hours’ treatment with tolinapant alone or with 0.5 µM emricasan or with 0. 5 µM emricasan plus 20 µM Nec-1. (I) Western blots of BW5147.1.4 cell lysates prepared after 48-hour treatment with tolinapant alone or with 0.5 µM emricasan or with 0. 5 µM emricasan plus 20 µM Nec-1. (J) BW5147 cell surface calreticulin or HSP90 staining analyzed by cytometry after 24-hour treatment with tolinapant or 0.2 µg/mL mitoxantrone. Data shown as flow cytometry histograms gated on total cells. (K) HMGB1 measured by enzyme-linked immunosorbent assay in BW5147 cell supernatants after 24-hour treatment with tolinapant.