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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.

Pancreatic ductal adenocarcinoma (PDA) is characterized by an immune-suppressive tumor microenvironment that renders it largely refractory to immunotherapy. We implemented a multimodal analysis approach to elucidate the immune landscape in PDA. Using a combination of CyTOF, single-cell RNA sequencing and multiplex immunohistochemistry on patient tumors, matched blood and non-malignant samples, we uncovered a complex network of immune-suppressive cellular interactions. These experiments revealed heterogeneous expression of immune checkpoint receptors in individual patients’ T cells and increased markers of CD8+ T cell dysfunction in the advanced disease stage. Tumor-infiltrating CD8+ T cells had an increased proportion of cells expressing an exhausted expression profile that included upregulation of the immune checkpoint TIGIT, a finding that we validated at the protein level. Our findings point to a profound alteration of the immune landscape of tumors, and to patient-specific immune changes that should be taken into account as combination immunotherapy becomes available for pancreatic cancer. Using single-cell RNA sequencing, CyTOF and multiplex immunohistochemistry, Steele et al. survey the immune landscape in pancreatic cancers, adjacent tissue and blood, observing heterogeneous immune checkpoint receptor expression within patients.

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

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.

Oncogenic KRAS is the hallmark mutation of human pancreatic cancer and a driver of tumorigenesis in genetically engineered mouse models of the disease. While the tumor cell-intrinsic effects of oncogenic Kras expression have been widely studied, its role in regulating the extensive pancreatic tumor microenvironment is less understood. Using a genetically engineered mouse model of inducible and reversible oncogenic Kras expression and a combination of approaches that include mass cytometry and single cell RNA sequencing, we have discovered that non-cell autonomous (i.e., 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. Our study provides fundamental new knowledge on the mechanisms underlying the formation of the fibroinflammatory stroma in pancreatic cancer and highlights stromal pathways with the potential to be exploited therapeutically.

Background The tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) contains abundant immunosuppressive tumor-associated macrophages. High level of infiltration is associated with poor outcome and is thought to represent a major roadblock to lymphocyte-based immunotherapy. Efforts to block macrophage infiltration have been met with some success, but noninvasive means to track tumor-associated macrophagess in PDAC are lacking. Translocator protein (TSPO) is a mitochondrial membrane receptor which is upregulated in activated macrophages. We sought to identify if a radiotracer-labeled cognate ligand could track macrophages in PDAC. Materials and Methods A murine PDAC cell line was established from a transgenic mouse with pancreas-specific mutations in KRAS and p53. After confirming lack of endogenous TSPO expression, tumors were established in syngeneic mice. A radiolabeled TSPO-specific ligand ([11C] peripheral benzodiazepine receptor [PBR]28) was delivered intravenously, and tumor uptake was assessed by autoradiography, ex vivo, or micro-positron emission tomography imaging. Results Resected tumors contained abundant macrophages as determined by immunohistochemistry and flow cytometry. Immunoblotting revealed murine macrophages expressed TSPO with increasing concentration on activation and polarization. Autoradiography of resected tumors confirmed [11C]PBR28 uptake, and whole mount sections demonstrated the ability to localize tumors. To confirm the findings were macrophage specific, experiments were repeated in CD11b-deficient mice, and the radiotracer uptake was diminished. Micro-positron emission tomography imaging validated radiotracer uptake and tumor localization in a clinically applicable manner. Conclusions As new immunotherapeutics reshape the PDAC microenvironment, tools are needed to better measure and track immune cell subsets. We have demonstrated the potential to measure changes in macrophage infiltration in PDAC using [11C]PBR28.

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.

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of disease processes. The principle techniques used in the past include immunohistochemistry (IHC) and immunofluorescence (IF) which enable visualization of cells as a snapshot in time using between 1 and 4 markers. Both techniques have shortcomings including difficulty staining poorly antigenic targets and limitations related to cross-species reactivity. IHC is reliable and reproducible, but the nature of the chemistry and reliance on the visible light spectrum allows for only a few markers to be used and makes co-localization challenging. Use of IF broadens potential markers but typically relies on frozen tissue due to the extensive tissue autofluorescence following formalin fixation. Flow cytometry, a technique that enables simultaneous labeling of multiple epitopes, abrogates many of the deficiencies of IF and IHC, however, the need to examine cells as a single cell suspension loses the spatial context of cells discarding important biologic relationships. Multiplex fluorescent immunohistochemistry (mfIHC) bridges these technologies allowing for multi-epitope cellular phenotyping in formalin fixed paraffin embedded (FFPE) tissue while preserving the overall microenvironment architecture and spatial relationship of cells within intact undisrupted tissue. High fluorescent intensity fluorophores that covalently bond to the tissue epitope enables multiple applications of primary antibodies without worry of species specific cross-reactivity by secondary antibodies. Although this technology has been proven to produce reliable and accurate images for the study of disease, the process of creating a useful mfIHC staining strategy can be time consuming and exacting due to extensive optimization and design. In order to make robust images that represent accurate cellular interactions in-situ and to mitigate the optimization period for manual analysis, presented here are methods for slide preparation, optimizing antibodies, multiplex design as well as errors commonly encountered during the staining process.

Although immune-based therapy has proven efficacious for some patients with microsatellite instability (MSI) colon cancers, a majority of patients receive limited benefit. Conversely, select patients with microsatellite stable (MSS) tumors respond to checkpoint blockade, necessitating novel ways to study the immune tumor microenvironment (TME). We used phenotypic and spatial data from infiltrating immune and tumor cells to model cellular mixing to predict disease specific outcomes in patients with colorectal liver metastases. Formalin fixed paraffin embedded metastatic colon cancer tissue from 195 patients were subjected to multiplex immunohistochemistry (mfIHC). After phenotyping, the G-function was calculated for each patient and cell type. Data was correlated with clinical outcomes and survival. High tumor cell to cytotoxic T lymphocyte (TC-CTL) mixing was associated with both a pro-inflammatory and immunosuppressive TME characterized by increased CTL infiltration and PD-L1+ expression, respectively. Presence and engagement of antigen presenting cells (APC) and helper T cells (Th) were associated with greater TC-CTL mixing and improved 5-year disease specific survival compared to patients with a low degree of mixing (42% vs. 16%, p = 0.0275). Comparison of measured mixing to a calculated theoretical random mixing revealed that PD-L1 expression on APCs resulted in an environment where CTLs were non-randomly less associated with TCs, highlighting their biologic significance. Evaluation of immune interactions within the TME of metastatic colon cancer using mfIHC in combination with mathematical modeling characterized cellular mixing of TCs and CTLs, providing a novel strategy to better predict clinical outcomes while identifying potential candidates for immune based therapies.

Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant infiltration of tumor-associated macrophages (TAMs). TAMs have been reported to drive resistance to gemcitabine, a frontline chemotherapy in PDA, though the mechanism of this resistance remains unclear. Profiling metabolite exchange, we demonstrate that macrophages programmed by PDA cells release a spectrum of pyrimidine species. These include deoxycytidine, which inhibits gemcitabine through molecular competition at the level of drug uptake and metabolism. Accordingly, genetic or pharmacological depletion of TAMs in murine models of PDA sensitizes these tumors to gemcitabine. Consistent with this, patients with low macrophage burden demonstrate superior response to gemcitabine treatment. Together, these findings provide insights into the role of macrophages in pancreatic cancer therapy and have potential to inform the design of future treatments. Additionally, we report that pyrimidine release is a general function of alternatively activated macrophage cells, suggesting an unknown physiological role of pyrimidine exchange by immune cells.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the US. Unfortunately, recent clinical trials using immunotherapy targeting the highly immunosuppressive tumor microenvironment have showed disappointing results, as there is no method to predict which patients will respond to therapy. More recently, the development of single cell technology has allowed for in-depth profiling at the cellular level using small amounts of tissue, raising the potential to develop precision medicine tools at time of endoscopic fine needle biopsy. Results: We performed single-cell RNA sequencing (scRNAseq) on endoscopic fine needle biopsies from 10 PDAC tumors at time of diagnostic endoscopic biopsy or 6 surgically resected tissues. We also sequenced 3 adjacent or normal pancreas tissues. We captured 8,521 cells from 3 surgical normal adjacent samples and 46,244 cells from PDAC tumors. Mapping of putative interactions between ligands and receptors demonstrated upregulation of key signaling pathways, including Hedgehog, NOTCH, and chemokine signaling within myeloid, epithelial, T, and NK cells. Differential expression analysis in cytotoxic CD8 T cells of PDA patients revealed increased expression of genes involved in T cell activation (GZMB, GZMA), exhaustion (GZMK, EOMES) as well as immune checkpoint pathway upregulation when compared to cytotoxic T cells in adjacent normal pancreatic tissue. Among the most significantly increased genes in CD8 T cells of PDAC tumor was the immune checkpoint TIGIT. Upon further analysis of the CD8 T cells, we found TIGIT was almost exclusively expressed in exhausted CD8 T cells, while other checkpoints such as PD-1 and LAG3 were equally distributed across effector and exhausted T cell populations. Interestingly, we were able to capture patient-specific heterogeneity of gene expression in T cells, suggesting the possibility of individualized T cell gene signatures present in PDAC tumors. We used mass cytometry and immunostaining to validate our transcript-based findings. Conclusion: Overall, we have successfully performed robust in-depth profiling using single-cell sequencing of PDAC tumors from fine needle biopsies. TIGIT, but not other immune checkpoints, correlates with T cells exhaustion in tumors, revealing an important biological function of this relatively understudied checkpoint. Analysis of our results identified patient-specific heterogeneity of key signaling pathways in different cell compartments of PDAC tumors that have to potential to be leveraged for precision medicine. Citation Format: Nina Steele, Eileen Carpenter, Samantha Kemp, Veerin Sirihorachai, Stephanie The, Lawrence Delrosario, Jenny Lazarus, El-ad Amir, Valerie Gunchick, Carlos Espinoza, Samantha Bell, Lindsey Harris, Valerie Irizarry-Negron, Dan Paglia, Justin Macchia, Fatima Lima, Angel Ka Yan Chu, Heather Schofield, Erik Jan Wamsteker, Richard Kwon, Allison Schulman, Anoop Prabhu, Ryan law, Arjun Sondhi, Katelyn Donahue, Hari Nathan, Clifford Cho, Michelle Anderson, Vaibhav Sahai, Costas Lyssiotis, Benjamin Allen, Arvind Rao, Weiping Zou, Filip Bednar, Timothy Frankel, Marina Pasca di Magliano. Multimodal mapping of the immune landscape in human pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3442.

Background & Aims Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy that invades surrounding structures and metastasizes rapidly. Although inflammation is associated with tumor formation and progression, little is known about the mechanisms of this connection. We investigate the effects of interleukin (IL) 22 in the development of pancreatic tumors in mice. Methods We performed studies with Pdx1-Cre;LSL-KrasG12D;Trp53+/–;Rosa26EYFP/+ (PKCY) mice, which develop pancreatic tumors, and PKCY mice with disruption of IL22 (PKCY Il22–/–mice). Pancreata were collected at different stages of tumor development and analyzed by immunohistochemistry, immunoblotting, real-time polymerase chain reaction, and flow cytometry. Some mice were given cerulean to induce pancreatitis. Pancreatic cancer cell lines (PD2560) were orthotopically injected into C57BL/6 mice or Il22–/–mice, and tumor development was monitored. Pancreatic cells were injected into the tail veins of mice, and lung metastases were quantified. Acini were collected from C57BL/6 mice and resected human pancreata and were cultured. Cell lines and acini cultures were incubated with IL22 and pharmacologic inhibitors, and protein levels were knocked down with small hairpin RNAs. We performed immunohistochemical analyses of 26 PDACs and 5 nonneoplastic pancreas specimens. Results We observed increased expression of IL22 and the IL22 receptor (IL22R) in the pancreas compared with other tissues in mice; IL22 increased with pancreatitis and tumorigenesis. Flow cytometry indicated that the IL22 was produced primarily by T-helper 22 cells. PKCY Il22–/–mice did not develop precancerous lesions or pancreatic tumors. The addition of IL22 to cultured acinar cells increased their expression of markers of ductal metaplasia; these effects of IL22 were prevented with inhibitors of Janus kinase signaling to signal transducer and activator of transcription (STAT) (ruxolitinib) or mitogen-activated protein kinase kinase (MEK) (trametinib) and with STAT3 knockdown. Pancreatic cells injected into Il22–/– mice formed smaller tumors than those injected into C57BL/6. Incubation of IL22R-expressing PDAC cells with IL22 promoted spheroid formation and invasive activity, resulting in increased expression of stem-associated transcription factors (GATA4, SOX2, SOX17, and NANOG), and increased markers of the epithelial–mesenchymal transition (CDH1, SNAI2, TWIST1, and beta catenin); ruxolitinib blocked these effects. Human PDAC tissues had higher levels of IL22, phosphorylated STAT3, and markers of the epithelial–mesenchymal transition than nonneoplastic tissues. An increased level of STAT3 in IL22R-positive cells was associated with shorter survival times of patients. Conclusions We found levels of IL22 to be increased during pancreatitis and pancreatic tumor development and to be required for tumor development and progression in mice. IL22 promotes acinar to ductal metaplasia, stem cell features, and increased expression of markers of the epithelial–mesenchymal transition; inhibitors of STAT3 block these effects. Increased expression of IL22 by PDACs is associated with reduced survival times.

Objective: Pancreatic cancer is a deadly malignancy, and little progress has been made in the survival of patients during the past 40 years. Harnessing the immune system to elicit anticancer response is an attractive therapeutic option, alone or in combination with antitumor agents. Targeting regulatory T cell (Treg), which acts to suppress immune responses, holds promise as one potential approach to relieve the immune suppression of pancreatic cancer, but functional studies investigating whether Treg depletion at different stages of pancreatic cancer has therapeutic benefit are needed. Methods: We are using KC; Foxp3DTR mice generated by crossing KC (p48-Cre; LSL-KrasG12D) with Foxp3DTR (B6.129(Cg)-Foxp3tm3(DTR/GFP)Ayr/J, Jackson Laboratory) to determine the requirement of Tregs during oncogenic Kras-induced pancreatic cancer precursor lesion formation and progression. We depleted Foxp3-expressing Tregs by Diphtheria Toxin (DT) injection at different time points—either before or after PanIN formation. Multiplex immunohistochemistry and Cytometry by Time-of-Flight (CyTOF) were performed for tumor immunophenotyping; RNA sequencing was used for transcriptomic analysis of the tumor epithelial cells, fibroblasts, and myeloid cells. Results: Surprisingly, instead of blocking tumorigenesis, depletion of Tregs during these early stages led to accelerated tumor progression. We show that Tregs are a key source of TGFb, and accordingly their depletion alters fibroblast population, exemplified by loss of Smooth Muscle Actin expression. Treg depletion further resulted in a compensatory influx of suppressive myeloid cells, thereby preventing an antitumor immune response. Mechanistically, we show that this myeloid recruitment was driven through the CCR1 axis, with the receptor expressed on tumor-associated myeloid cells and several ligands upregulated in epithelial cells and fibroblasts upon Treg depletion. Finally, blockade of CCR1 signaling successfully inhibits tumor progression upon Treg-depletion. Conclusion: Our study revealed an immunosuppressive network of interactions between different components within the pancreatic tumor microenvironment, including Tregs, fibroblasts, tumor cells, and macrophages. Depleting one component (Tregs) can lead to the compensation of immunosuppression from the rest of components within the network. Thus, a thorough understanding of the immunosuppressive network in pancreatic neoplasia is necessary to optimize our approaches to treatment and identify opportunities for combination therapy and prevent potential feedback mechanisms resulting in tumor resistance and relapse. Citation Format: Yaqing Zhang, Jenny Lazarus, Nina Steele, Wei Yan, Ho-Joon Lee, Christopher J. Halbrook, Rosa Menjivar, Samantha B. Kemp, Veerin Sirihorachai, Eileen S. Carpenter, Anna C. Nevison, Alekya Vinta, Michelle A. Anderson, Howard C. Crawford, Costas A. Lyssiotis, Timothy L. Frankel, Filip Bednar, Marina Pasca di Magliano. Regulatory T-cell depletion promotes oncogenic Kras-driven pancreatic carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A62.

Objective: Type 2 diabetes mellitus (DM) is a known risk factor for pancreatic ductal adenocarcinoma (PDAC), but the mechanisms underlying this association remain poorly understood. Multiple reports implicate adipocytes and preadipocytes as putative promoters of PDAC in humans and mice. To test whether preadipocytes regulate PDAC tumorigenic functions in the context of DM, we explored the effects of conditioned media (CM) prepared from preadipocytes from nondiabetic (NDM) and DM patients on PDAC cells. Methods: Preadipocytes were isolated from omental adipose tissue obtained from NDM and DM patients undergoing bariatric surgery. Serum-free conditioned media was prepared from these preadipocytes cultured in vitro for 72 hours. Sphere assays using T3M4 and Capan1 PDAC lines were performed in the presence or absence of conditioned media, and sphere surface area was measured over 2-3 weeks. Modifications of the sphere assays, including boiling of conditioned media as well as addition of neutralizing antibodies or recombinant proteins, were implemented. A proteome profiler array was used to identify differences in cytokine composition between CM from DM and NDM. A phospho-kinome array was used to identify differentially activated signaling pathways when PDAC cells were treated with NDM and DM conditioned media. Results: Conditioned media from human DM preadipocytes, compared to media from NDM preadipocytes, increased PDAC cell sphere surface area in both T3M4 and Capan1 cell lines. Sphere surface area correlated directly with hemoglobin A1c (HbA1c) but not sex or body mass index of the human subjects from which preadipocytes were derived. Boiling of preadipocyte supernatants abrogated their pro-growth effects. Proteome profiler array analysis identified different proteins that were differentially expressed by DM and NDM preadipocytes, among which chitinase-3-like protein 1 (CHI3L1) and interleukin-6 (IL-6) were increased over 2-fold in DM relative to NDM preadipocyte supernatants. Neutralizing antibody to human CHI3L1 and IL-6 attenuated the increase in PDAC sphere surface area in response to DM preadipocyte conditioned media in a dose-dependent manner. Recombinant human CHI3L1 and IL-6 enhanced the growth of PDAC cells when they were exposed to NDM conditioned media. Phospho-kinome analysis demonstrated that treatment of PDAC cells with DM-derived conditioned media increased activation of inflammation-associated signal transduction pathways. Conclusions: Preadipocyte-PDAC cell crosstalk regulates PDAC cell growth in a DM-specific and CHI3L1 and IL-6-dependent manner. These results suggest that the positive clinical correlation between DM and PDAC may be mediated by preadipocytes expressing higher levels of CHI3L1 and IL-6. Preadipocytes CHI3L1 and IL-6 represent targets for further research directed towards developing novel therapies for PDAC based on manipulation of the tumor microenvironment. Citation Format: Lawrence Delrosario, Carmen Flesher, Nicki Baker, Jenny Lazarus, Mirna Perusina-Lanfranca, Robert O'Rourke, Timothy Frankel. Human preadipocytes promote pancreatic cancer cellular growth in a diabetes-specific manner [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A08.

In human patients and in mouse models, immunosuppressive regulatory T cells (Tregs) accumulate in pancreatic cancer. Tregs are considered a potential therapeutic target with the goal to reverse immunosuppression in a number of malignancies, including pancreatic cancer. Using a genetically engineered mouse model of pancreatic cancer, we determined that—unlike in other malignancies—Treg depletion failed to relieve immunosuppression. Contradicting the current paradigm, depletion of Tregs led to accelerated tumor progression. We show that Tregs are a key source of TGFb ligands both in human samples and in mouse models. Accordingly, their depletion reprograms the fibroblast population, exemplified by loss of smooth muscle actin expression and an increase in the myeloid-recruiting chemokines Ccl3, Ccl6, and Ccl8. Our findings point to a dual role of Tregs in pancreatic cancer, with their immune-suppressive function being balanced by their induction of a SMA-high (tumor-restraining) fibroblast phenotype. Further, our data point to a compensatory immunosuppression mechanism that confers resistance to Treg depletion in pancreatic cancer. Finally, blockade of the common CCL3/6/8 receptor CCR1 combined with Treg-depletion successfully inhibited tumorigenesis, a finding with potential therapeutic implications. Citation Format: Yaqing Zhang, Jenny Lazarus, Nina Steele, Ho-Joon Lee, Wei Yan, Cristopher J. Halbrook, Rosa Menjivar, Samantha B. Kemp, Veerin Sirihoracai, Eileen S. Carpenter, Anna C. Nevison, Alekya Vinta, Michelle A. Anderson, Howard C. Crawford, Costas A. Lyssiotis, Timothy L. Frankel, Filip Bednar, Marina Pasca di Magliano. Regulatory T-cell depletion causes compensatory immune suppression and accelerates pancreatic carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I04.

Pancreatic ductal adenocarcinoma (PDA) has an exceedingly poor prognosis, with only 9% 5-year survival rate. Kras mutations are found in 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 antitumor immunity by cytotoxic T cells. The mechanisms underlying the immunosuppression in pancreatic cancer are only partially understood. Our goal is to investigate the interactions between tumor cells and the immune cells, with the long-term objective to identify new therapeutic targets. Our laboratory has described a mouse model (iKras*) of inducible and reversible expression of oncogenic Kras (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 preneoplastic 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 CyTOF in the pancreas to analyze T cell and myeloid cell populations, as well as functional markers (Arg1, iNOS, IFNγ). 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 and a modest increase in CD4+ T cells, particularly regulatory T 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. Kras* in the neoplastic cells initiates the infiltration of immune cells into the pancreas and induces them to express immune-suppressive factors. Going forward, we are investigating the signals downstream of oncogenic Kras that mediate the crosstalk between neoplastic cells and infiltrating immune cells. Citation Format: Ashley Velez-Delgado, Valerie Irizarry-Negron, Rosa Menjivar, Jenny Lazarus, Murali Bollampally, Nina Steele, Timothy Frankel, Fillip Bednar, Yaqing Zhang, Marina Pasca di Magliano. Oncogenic Kras modulates pancreas plasticity and the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A54.

Paramount to the efficacy of immune checkpoint inhibitors is proper selection of patients with adequate tumor immunogenicity and a robust but suppressed immune infiltrate. In colon cancer, immune-based therapies are approved for patients with DNA mismatch repair (MMR) deficiencies, in whom accumulation of genetic mutations results in increased neoantigen expression, triggering an immune response that is suppressed by the PD-L1/PD-1 pathway. Here, we report that characterization of the microenvironment of MMR-deficient metastatic colorectal cancer using multiplex fluorescent immunohistochemistry (mfIHC) identified increased infiltration of cytotoxic T lymphocytes (CTLs), which were more often engaged with epithelial cells (ECs) and improved overall survival. A subset of patients with intact MMR but a similar immune microenvironment to MMR-deficient patients was identified and found to universally express high levels of PD-L1, suggesting that they may represent a currently untreated, checkpoint inhibitor-responsive population. Further, PD-L1 expression on antigen-presenting cells (APCs) in the tumor microenvironment (TME) resulted in impaired CTL/EC engagement and enhanced infiltration and engagement of Tregs. Characterization of the TME by mfIHC highlights the interconnection between immunity and immunosuppression in metastatic colon cancer and may better stratify patients for receipt of immunotherapies.

Elevated levels of Interleukin 22 (IL-22) and its receptor (IL-22R) are associated with poor prognosis in pancreatic ductal adenocarcinoma (PDAC) and the underlying mechanism is currently unknown. Our aim is to investigate the role of the IL-22 axis in PDAC initiation and progression. Impact of IL-22 on tumor initiation was assayed by subcutaneous and intravenous inoculation of PDAC cells into wild-type and IL-22−/− mice. The PKCY (Pdx1-Cre; KrasG12D; p53fl/+; RosaYFP) model of pancreas cancer was used to study the in-vivo presence and significance of IL-22 in spontaneous tumors. Results were confirmed in human specimens of surgically resected pancreas cancer. IL-22R was present in all tested PDAC lines and IL-22 treatment led to STAT3 phosphorylation and subsequent increased expression of EMT (Epithelial to Mesenchymal Transition) transcription factors. Cells transitioned to a mesenchymal phenotype and robust tumor sphere formation was observed. While tumors readily formed in wild-type mice, initiation, establishment and growth were impaired in IL-22−/− mice and PKCY-IL-22−/− mice. Increased levels of IL-22 were found in both spontaneous murine tumors and surgical specimens compared to control tissue. IHC of tumors showed diffuse IL-22R staining with increasing intensity of pSTAT3 and EMT markers as tumors progressed to invasive cancer. FACS analysis identified type 3 innate lymphoid cells and TH22 cells as the source of IL-22 in both human and murine PDAC. Our data suggests that IL-22 is integral in the initiation, progression and establishment of pancreatic cancer, positioning it as an attractive target for cancer therapy.