Cholangiocarcinoma (CCA), a malignant tumor originating in the biliary tract, is well known to be associated with adverse clinical outcomes and high mortality rates due to the lack of effective therapy. Evasion of apoptosis is considered a key contributor to therapeutic success and chemotherapy resistance in CCA, highlighting the need for novel therapeutic strategies. In this study, we demonstrated that the induction of necroptosis, a novel regulated form of necrosis, could potentially serve as a novel therapeutic approach for CCA patients. The RNA sequencing data in The Cancer Genome Atlas (TCGA) database were analyzed and revealed that both receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), two essential mediators of necroptosis, were upregulated in CCA tissues when compared with the levels in normal bile ducts. We demonstrated in a panel of CCA cell lines that RIPK3 was differentially expressed in CCA cell lines, while MLKL was more highly expressed in CCA cell lines than in nontumor cholangiocytes. We therefore showed that treatment with both tumor necrosis factor-α (TNF-α) and Smac mimetic, an inhibitor of apoptosis protein (IAP) antagonist, induced RIPK1/RIPK3/MLKLdependent necroptosis in CCA cells when caspases were blocked. The necroptotic induction in a panel of CCA cells was correlated with RIPK3 expression. Intriguingly, we demonstrated that Smac mimetic sensitized CCA cells to a low dose of standard chemotherapy, gemcitabine, and induced necroptosis in an RIPK1/RIPK3/MLKL-dependent manner upon caspase inhibition but not in nontumor cholangiocytes. We further demonstrated that Smac mimetic and gemcitabine synergistically induced an increase in TNF-α mRNA levels and that Smac mimetic reversed gemcitabine-induced cell cycle arrest, leading to cell killing. Collectively, our present study demonstrated that TNF-α and gemcitabine induced RIPK1/ RIPK3/MLKL-dependent necroptosis upon IAP depletion and caspase inhibition; therefore,

Necroptosis, a regulated form of necrosis, has emerged as a novel therapeutic strategy that could enhance cancer immunotherapy. However, its role in tumorigenesis is still debated because recent studies have reported both anti- and pro-tumoral effects. Here, we aimed to systematically evaluate the associations between tumor necroptosis (mixed lineage kinase domain-like protein, MLKL; phosphorylated MLKL, pMLKL; and receptor-interacting protein kinase 1–receptor-interacting protein kinase 3, RIPK1–RIPK3 interaction) and tumor-infiltrating immune cells (CD8+ and FOXp3+ T cells and CD163+ M2 macrophages) and tumor PD-L1 by immunohistochemistry in 88 cholangiocarcinoma (CCA) patients who had undergone surgical resection. Their associations with clinicopathological characteristics, survival data, and prognosis were evaluated. MLKL was found to be an unfavorable prognostic factor (p-value = 0.023, HR = 2.070) and was inversely correlated with a clinically favorable immune cell signature (high CD8+/high FOXp3+/low CD163+). Both pMLKL and RIPK1–RIPK3 interaction were detected in CCA primary tissues. In contrast to MLKL, pMLKL status was significantly positively correlated with a favorable immune signature (high CD8+/high FOXp3+/low CD163+) and PD-L1 expression. Patients with high pMLKL-positive staining were significantly associated with an increased abundance of CD8+ T cell intratumoral infiltration (p-value = 0.006). Patients with high pMLKL and PD-L1 expressions had a longer overall survival (OS). The results from in vitro experiments showed that necroptosis activation in an RMCCA-1 human CCA cell line selectively promoted proinflammatory cytokine and chemokine expression. Jurkat T cells stimulated with necroptotic RMCCA-1-derived conditioned medium promoted PD-L1 expression in RMCCA-1. Our findings demonstrated the differential associations of necroptosis activation (pMLKL) and MLKL with a clinically favorable immune signature and survival rates and highlighted a novel therapeutic possibility for combining a necroptosis-based therapeutic approach with immune checkpoint inhibitors for more efficient treatment of CCA patients.