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Regulatory T cells (Treg) are abundant in human and mouse pancreatic cancer. To understand the contribution to the immunosuppressive microenvironment, we depleted Tregs in a mouse model of pancreatic cancer. Contrary to our expectations, Treg depletion failed to relieve immunosuppression and led to accelerated tumor progression. We show that Tregs are a key source of TGFβ ligands and, accordingly, their depletion reprogramed the fibroblast population, with loss of tumor-restraining, smooth muscle actin–expressing fibroblasts. Conversely, we observed an increase in chemokines Ccl3, Ccl6, and Ccl8 leading to increased myeloid cell recruitment, restoration of immune suppression, and promotion of carcinogenesis, an effect that was inhibited by blockade of the common CCL3/6/8 receptor CCR1. Further, Treg depletion unleashed pathologic CD4+ T-cell responses. Our data point to new mechanisms regulating fibroblast differentiation in pancreatic cancer and support the notion that fibroblasts are a heterogeneous population with different and opposing functions in pancreatic carcinogenesis.Significance:Here, we describe an unexpected cross-talk between Tregs and fibroblasts in pancreatic cancer. Treg depletion resulted in differentiation of inflammatory fibroblast subsets, in turn driving infiltration of myeloid cells through CCR1, thus uncovering a potentially new therapeutic approach to relieve immunosuppression in pancreatic cancer.See related commentary by Aykut et al., p. 345.This article is highlighted in the In This Issue feature, p. 327

SF1: ATM regulates type I interferon signaling in pancreatic cancer. SF2: ATM regulation of the type I interferon pathway is cGAS/STING independent but SRC dependent. SF3: ATM inhibition cooperates with radiation to activate type I interferon signaling. SF4: ATM regulates pancreatic tumor immunity and ICB efficacy in vivo.

Combinatorial strategies are needed to overcome the resistance of pancreatic cancer to immune checkpoint blockade (ICB). DNA damage activates the innate immune response and improves ICB efficacy. Because ATM is an apical kinase in the radiation-induced DNA damage response, we investigated the effects of ATM inhibition and radiation on pancreatic tumor immunogenicity. ATM was inhibited through pharmacologic and genetic strategies in human and murine pancreatic cancer models both in vitro and in vivo. Tumor immunogenicity was evaluated after ATM inhibition alone and in combination with radiation by assessing TBK1 and Type I interferon (T1IFN) signaling as well as tumor growth following PD-L1/PD-1 checkpoint inhibition. Inhibition of ATM increased tumoral T1IFN expression in a cGAS/STING-independent, but TBK1- and SRC-dependent, manner. The combination of ATM inhibition with radiation further enhanced TBK1 activity, T1IFN production, and antigen presentation. Furthermore, ATM silencing increased PD-L1 expression and increased the sensitivity of pancreatic tumors to PD-L1–blocking antibody in association with increased tumoral CD8+ T cells and established immune memory. In patient pancreatic tumors, low ATM expression inversely correlated with PD-L1 expression. Taken together, these results demonstrate that the efficacy of ICB in pancreatic cancer is enhanced by ATM inhibition and further potentiated by radiation as a function of increased tumoral immunogenicity, underscoring the potential of ATM inhibition in combination with ICB and radiation as an efficacious treatment strategy for pancreatic cancer.Significance:This study demonstrates that ATM inhibition induces a T1IFN-mediated innate immune response in pancreatic cancer that is further enhanced by radiation and leads to increased sensitivity to anti–PD-L1 therapy.See related commentary by Gutiontov and Weichselbaum, p. 3815

SF1 Legend: ATM regulates type I interferon signaling in pancreatic cancer. SF2 Legend: ATM regulation of the type I interferon pathway is cGAS/STING independent but SRC dependent. SF3 Legend: ATM inhibition cooperates with radiation to activate type I interferon signaling. SF4 Legend: ATM regulates pancreatic tumor immunity and ICB efficacy in vivo.

The aim of this retrospective study was to compare the outcomes of trauma patients directly transported to a level I trauma center (SCENE) versus those who were stabilized at a critical access hospital (CAH) and subsequently transferred.Patients were grouped based on their transfer status, interventions performed at CAH and outcomes. Google Maps was used to calculate the distances from the location of injury (LOI). Each transfer group data was analyzed separately to examine associations of different factors on the outcomes. Outcomes were compared using univariate and multivariate analyses and propensity score matching analysis.There were 262 patients in SCENE and 684 in CAH. Compared to SCENE, CAH had higher rates of blunt injury and a greater distance from LOI, whereas lower ISS score and length of stay (LOS) (p 0.05). The majority of CAH group survived compared to SCENE (p = 0.007). For both groups, baseline factors (e.g., age) were associated with outcomes (p 0.05). Interestingly, longer LOS in the CAH was associated with an increase in survival (p = 0.009), whereas an increased number of CT/MRI performed was associated with increased LOS (p 0.05)., and an increased number of procedures was associated with longer LOS and ICU stay (p 0.05). After matching, the two groups had no significant differences in survival, LOS, or ICU stay (p 0.05).Equivalent overall clinical outcomes were seen in both groups, suggesting that existing trauma system protocols in the West Texas region are functioning well to select appropriate patients for each transfer modality.Retrospective Analysis.

Pancreatic Ductal Adenocarcinoma (PDA) has an exceedingly poor prognosis with only 10% 5-year survival rate. Kras mutations are found in over 90% of cases of pancreatic cancer and drive the formation of pancreatic intraepithelial neoplasia (PanIN), precursor lesions to PDA. Both PanIN and PDA are characterized by dense stroma, containing fibroblasts and immune cells. The infiltrating immune cells have a suppressive phenotype and prevent anti-tumor immunity by cytotoxic T cells. The mechanisms underlying the immunosuppression in pancreatic cancer are only partially understood. Our goal was to unravel the non-cell autonomous role of oncogenic Kras (Kras*) expressing epithelial cells in driving the formation of a complex, tumor promoting microenvironment during the onset of pancreatic cancer. Our laboratory has described a mouse model (iKras*) of inducible and reversible expression of Kras* in the pancreas. Taking advantage of the reversibility of Kras* expression in this model, we conducted a thorough characterization of the immune infiltration and function upon modulation of Kras* at different stages of pancreatic carcinogenesis. iKras* mice and wild type littermates were enrolled in experiments at the age of 8-12 weeks. Kras* expression was activated, then we induced acute pancreatitis to promote the formation of pre-neoplastic lesions. After 3 weeks, a time when widespread low-grade lesions and fibrosis are observed, mice were either harvested or Kras* expression was inactivated and 3 days or 1 week later the pancreas was harvested. We performed flow cytometry, immunohistochemistry and cytometry time of flight (CyTOF) in the pancreas to analyze myeloid cell populations, as well as functional markers (Arg1, iNOS, IFN). Myeloid cells and T cells infiltrated the pancreas in presence of active Kras*. Inactivation of Kras* resulted in a relatively modest decrease in infiltrating myeloid cells. However, analysis of the functional marker Arg1, a putative immune suppressive molecule expressed in myeloid cells, indicated that its expression depends on Kras*-expressing cells. When we further studied the interactions among cell types using single cell RNA sequencing, we discovered that non-cell autonomous (extrinsic) oncogenic KRAS signaling reprograms pancreatic fibroblasts, activating an inflammatory gene expression program. As a result, fibroblasts become a hub of extracellular signaling, mediating the polarization and function of pro-tumorigenic myeloid cells while also preventing tissue repair. Citation Format: Ashley Velez-Delgado, Katelyn L. Donahue, Kristee L. Brown, Wenting Du, Valerie Irizarry-Negron, Rosa E. Menjivar, Emily L. Lasse-Opsahl, Nina G. Steele, Stephanie The, Jenny Lazarus, Veerin R. Sirihorachai, Filip Bednar, Timothy L. Frankel, Yaqing Zhang, Marina Pasca di Magliano. Extrinsic KRAS signaling shapes the pancreatic microenvironment through fibroblast reprogramming [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-016.