The current pathological and molecular classification of pancreatic ductal adenocarcinoma (PDAC) provides limited guidance for treatment options, especially for immunotherapy. Cancer-associated fibroblasts (CAFs) are major players of desmoplastic stroma in PDAC, modulating tumor progression and therapeutic response. Using single-cell RNA sequencing, we explored the intertumoral heterogeneity among PDAC patients with different degrees of desmoplasia. We found substantial intertumoral heterogeneity in CAFs, ductal cancer cells, and immune cells between the extremely dense and loose types of PDACs (dense-type, high desmoplasia; loose-type, low desmoplasia). Notably, no difference in CAF abundance was detected, but a novel subtype of CAFs with a highly activated metabolic state (meCAFs) was found in loose-type PDAC compared to dense-type PDAC. MeCAFs had highly active glycolysis, whereas the corresponding cancer cells used oxidative phosphorylation as a major metabolic mode rather than glycolysis. We found that the proportion and activity of immune cells were much higher in loose-type PDAC than in dense-type PDAC. Then, the clinical significance of the CAF subtypes was further validated in our PDAC cohort and a public database. PDAC patients with abundant meCAFs had a higher risk of metastasis and a poor prognosis but showed a dramatically better response to immunotherapy (64.71% objective response rate, one complete response). We characterized the intertumoral heterogeneity of cellular components, immune activity, and metabolic status between dense- and loose-type PDACs and identified meCAFs as a novel CAF subtype critical for PDAC progression and the susceptibility to immunotherapy.

Perineural invasion is a common feature of pancreatic ductal adenocarcinoma (PDAC). Here, we investigated the effect of perineural invasion on the microenvironment and how this affects PDAC progression. Transcriptome expression profiles of PDAC tissues with different perineural invasion status were compared, and the intratumoral T-cell density and levels of neurotransmitters in these tissues were assessed. Perineural invasion was associated with impaired immune responses characterized by decreased CD8+ T and Th1 cells, and increased Th2 cells. Acetylcholine levels were elevated in severe perineural invasion. Acetylcholine impaired the ability of PDAC cells to recruit CD8+ T cells via HDAC1-mediated suppression of CCL5. Moreover, acetylcholine directly inhibited IFNγ production by CD8+ T cells in a dose-dependent manner and favored Th2 over Th1 differentiation. Furthermore, hyperactivation of cholinergic signaling enhanced tumor growth by suppressing the intratumoral T-cell response in an orthotopic PDAC model. Conversely, blocking perineural invasion with bilateral subdiaphragmatic vagotomy in tumor-bearing mice was associated with an increase in CD8+ T cells, an elevated Th1/Th2 ratio, and improved survival. In conclusion, perineural invasion–triggered cholinergic signaling favors tumor growth by promoting an immune-suppressive microenvironment characterized by impaired CD8+ T-cell infiltration and a reduced Th1/Th2 ratio. Significance: These findings provide a promising therapeutic strategy to modulate the immunosuppressive microenvironment of pancreatic ductal adenocarcinoma with severe perineural invasion.

Background and aims: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death worldwide. Neurotransmitter-initiated signalling pathway is profoundly implicated in tumour initiation and progression. Here, we investigated whether dysregulated neurotransmitter receptors play a role during pancreatic tumourigenesis. Methods: The Cancer Genome Atlas and Gene Expression Omnibus datasets were used to identify differentially expressed neurotransmitter receptors. The expression pattern of gamma-aminobutyric acid type A receptor pi subunit (GABRP) in human and mouse PDAC tissues and cells was studied by immunohistochemistry and western blot analysis. The in vivo implications of GABRP in PDAC were tested by subcutaneous xenograft model and lung metastasis model. Bioinformatics analysis, transwell experiment and orthotopic xenograft model were used to identify the in vitro and in vivo effects of GABRP on macrophages in PDAC. ELISA, co-immunoprecipitation, proximity ligation assay, electrophysiology, promoter luciferase activity and quantitative real-time PCR analyses were used to identify molecular mechanism. Results: GABRP expression was remarkably increased in PDAC tissues and associated with poor prognosis, contributed to tumour growth and metastasis. GABRP was correlated with macrophage infiltration in PDAC and pharmacological deletion of macrophages largely abrogated the oncogenic functions of GABRP in PDAC. Mechanistically, GABRP interacted with KCNN4 to induce Ca ²⁺ entry, which leads to activation of nuclear factor κB signalling and ultimately facilitates macrophage infiltration by inducing CXCL5 and CCL20 expression. Conclusions: Overexpressed GABRP exhibits an immunomodulatory role in PDAC in a neurotransmitter-independent manner. Targeting GABRP or its interaction partner KCNN4 may be an effective therapeutic strategy for PDAC.

Purpose: Extensive research has reported that the tumor microenvironment components play crucial roles in tumor progression. Thus, blocking the supports of tumor microenvironment is a promising approach to prevent cancer progression. We aimed to determine whether blocking extracellular ATP–P2RY2 axis could be a potential therapeutic approach for PDAC treatment. Experimental Design: Expression of P2RY2 was determined in 264 human PDAC samples and correlated to patient survival. P2RY2 was inhibited in human PDAC cell lines by antagonist and shRNA, respectively, and cell viability, clonogenicity, and glycolysis were determined. RNA sequencing of PDAC cell line was applied to reveal underlying molecular mechanisms. Multiple PDAC mouse models were used to assess the effects of the P2RY2 inhibition on PDAC progression. Results: P2RY2 was upregulated and associated with poor prognosis in PDAC. Activated P2RY2 by increased extracellular ATP in tumor microenvironment promoted PDAC growth and glycolysis. Further studies showed that the agonist-activated P2RY2 triggered PI3K/AKT–mTOR signaling by crosstalk with PDGFR mediated by Yes1, resulting in elevated expression of c-Myc and HIF1α, which subsequently enhanced cancer cell glycolysis. Genetic and pharmacologic inhibition of P2RY2 impaired tumor cell growth in subcutaneous and orthotopic xenograft model, as well as delayed tumor progression in inflammation-driven PDAC model. In addition, synergy was observed when AR-C118925XX, the selective antagonist of P2RY2 receptor, and gemcitabine were combined, resulting in prolonged survival of xenografted PDAC mice. Conclusions: These findings reveal the roles of the P2RY2 in PDAC metabolic reprogramming, suggesting that P2RY2 might be a potential metabolic therapeutic target for PDAC.

The immunosuppressive microenvironment that is shaped by hepatic metastatic pancreatic ductal adenocarcinoma (PDAC) is essential for tumor cell evasion of immune destruction. Neutrophils are important components of the metastatic tumor microenvironment and exhibit heterogeneity. However, the specific phenotypes, functions and regulatory mechanisms of neutrophils in PDAC liver metastases remain unknown. Here, we show that a subset of P2RX1-negative neutrophils accumulate in clinical and murine PDAC liver metastases. RNA sequencing of murine PDAC liver metastasis-infiltrated neutrophils show that P2RX1-deficient neutrophils express increased levels of immunosuppressive molecules, including PD-L1, and have enhanced mitochondrial metabolism. Mechanistically, the transcription factor Nrf2 is upregulated in P2RX1-deficient neutrophils and associated with PD-L1 expression and metabolic reprogramming. An anti-PD-1 neutralizing antibody is sufficient to compromise the immunosuppressive effects of P2RX1-deficient neutrophils on OVA-activated OT1 CD8+ T cells. Therefore, our study uncovers a mechanism by which metastatic PDAC tumors evade antitumor immunity by accumulating a subset of immunosuppressive P2RX1-negative neutrophils.