Pancreatic ductal adenocarcinoma (PDAC) is the worst prognoses among all the malignancies. Now, gemcitabine (Gem) is the first line chemotherapeutic drug for advanced pancreatic cancer. However, Gem is usually ineffective to the PDAC because of high degree of drug resistance. Hypoxia and immune suppressive milieu are the best-described hallmarks of PDAC; therefore, we investigated the impact of hypoxia inducible factor-1 (HIF-1) inhibitor, PX-478, in combination with Gem on the induction of immunogenic cell death (ICD). We verified that combined treatment with Gem/PX-478 significantly enhanced the anti-tumor effect and increased proportion of tumor infiltrating T-lymphocytes in Panc02-bearing immune-competent but not in immune-deficient mice. Vaccination using Panc02 cell line treated with single agent or in combination showed significant anti-tumor effects. Pancreatic cell lines treated with Gem and PX-478 can induce an increase in eIF2α phosphorylation was correlated with down-regulation of HIF-1α and elicited exposure of CRT and release of HMGB1 and ATP. Only co-treated cells induced DC maturation/phagocytosis and IFN-γ secretion by cytotoxic T lymphocytes. Altogether, combined treatment with Gem/PX-478 showed significantly inhibition on tumor growth and anti-tumor immunization. We propose that inhibition HIF-1α elicits Gem-induced immune response and eliminates PDAC cells by inducing ICD.

High expression of PD-L1 marks the poor prognosis of pancreatic ductal adenocarcinomas (PDAC). However, the regulatory mechanism of PD-L1 remains elusive. We recently reported that cancer Forkhead box protein 3 (Cancer-FOXP3 or C-FOXP3) promoted immune evasion of PDAC by recruiting Treg cells into PDAC via upregulation of CCL5. In this study, we confirmed that PD-L1 was overexpressed in PDAC samples from two independent cohorts of patients with radical resection. Moreover, C-FOXP3 was colocalized and correlated with the expression of PD-L1 in tumor cells at the mRNA and protein levels, and this finding was confirmed by the The Cancer Genome Atlas (TCGA) database. Chromatin immunoprecipitation (ChIP) revealed that C-FOXP3 directly bound to the promoter region of PD-L1 in pancreatic cancer cells. Furthermore, overexpression of C-FOXP3 activated the luciferase reporter gene under the control of the PD-L1 promoter. However, mutation of the binding motif-a completely reversed the luciferase activity. In addition, C-FOXP3-induced upregulation of PD-L1 effectively inhibited the activity of CD8⁺ T cells. Based on our recent finding that the CCL-5 antibody achieved a better response to PDAC models with high C-FOXP3 levels, we further demonstrated that the PD-L1 antibody strengthened the antitumor effect of CCL-5 blockade in xenograft and orthotopic mouse models with high C-FOXP3 levels. In conclusion, C-FOXP3 directly activates PD-L1 and represents a core transcription factor that mediates the immune escape of PDAC. Combined blockade of PD-L1 and CCL-5 may provide an effective therapy for patients with PDAC that have high C-FOXP3 levels.

Interleukin 35 (IL-35) is a novel member of the IL-12 family, consisting of an EBV-induced gene 3 (EBI3) subunit and a P35 subunit. IL-35 is an immune-suppressive cytokine mainly produced by regulatory T cells. However, the role of IL-35 in cancer metastasis and progression is not well understood. Here we demonstrate that IL-35 is overexpressed in human pancreatic ductal adenocarcinoma (PDAC) tissues, and that IL-35 overexpression is associated with poor prognosis in PDAC patients. IL-35 has critical roles in PDAC cell extravasation and metastasis by facilitating the adhesion to endothelial cells and transendothelial extravasation. Mechanistically, IL-35 promotesICAM1overexpression through a GP130-STAT1 signalling pathway, which facilitates endothelial adhesion and transendothelial migration via an ICAM1–fibrinogen–ICAM1 bridge. In an orthotopic xenograft model, IL-35 promotes spontaneous pancreatic cancer metastasis in anICAM1-dependent manner. Together, our results indicate additional functions of IL-35 in promoting PDAC metastasis through mediatingICAM1expression.