Tumor necroptosis is correlated with a favorable immune cell signature and programmed death-ligand 1 expression in cholangiocarcinoma

Abstract

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.

Necroptosis-driving genesRIPK1, RIPK3andMLKL-pare associated with intratumoral CD3+and CD8+T cell density and predict prognosis in hepatocellular carcinoma

“…In both cohorts, an increasing level of immunoreaction score of RIPK1, RIPK3 and MLKL-p resulted in an increasing level of intratumoral density of CD3 + and CD8 + T-lymphocytes (figure 5A and figure S4 in online supplemental file 1), enforcing the immunogenic role of NCP in cancer, 28 as also previously shown in breast cancer 29 and, recently, in cholangiocarcinoma. 30 NCS, as well as tumor-infiltrating CD8 + T cells, showed also prognostic relevance in terms of OS, while TILs were not found associated with a worse prognosis in both Italian and Japanese cohorts (figure 5B with table 3 and figure S6 with table S5 in online supplemental file 1 for NCS; figure S7 with table S7 in online supplemental file 1 for CD8 + T cells and TILs). These results were also confirmed in TCGA-LIHC data considering NCS as a combined mRNA expression score of the three receptors and the inferred CD8 + T cells infiltration from RNAseq data through deconvolution approaches (figure S9 and table S8 in online supplemental file 1).…”

A Risk Model Developed Based on Necroptosis Predicts Overall Survival for Hepatocellular Carcinoma and Identification of Possible Therapeutic Drugs

“…Studies by Lomphithak et al. have shown that in cholangiocarcinoma, necroptosis is associated with a favorable immune cell signature ( 32 ). In addition, inflammation can also regulate necroptosis through cytokines such as IFN, increasing the expression of MLKL.…”

Necroptosis contributes to chronic inflammation and fibrosis in aging liver

“…necroptosis can either promote or reduce tumor growth depending on the type of cancer. For example, reduced expression of necroptotic markers promote tumor growth in breast cancer (Stoll et al, 2017) and acute myeloid leukemia (Höckendorf et al, 2016), whereas in pancreatic ductal adenocarcinoma (Seifert et al, 2016) and cholangiocarcinoma (Lomphithak et al, 2021) increased expression of necroptosis markers are associated with tumor growth. Because aging is a slow and gradual process, further studies with long-term Nec-1s treatment will be needed to address the feasibility of using necroptosis inhibitor(s) for inflammaging and aging.…”