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 fi broblast population, with loss of tumor-restraining, smooth muscle actin-expressing fi broblasts. 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 fi broblast differentiation in pancreatic cancer and support the notion that fi broblasts are a heterogeneous population with different and opposing functions in pancreatic carcinogenesis. SIGNIFICANCE:Here, we describe an unexpected cross-talk between Tregs and fi broblasts in pancreatic cancer. Treg depletion resulted in differentiation of infl ammatory fi broblast subsets, in turn driving infi ltration of myeloid cells through CCR1, thus uncovering a potentially new therapeutic approach to relieve immunosuppression in pancreatic cancer.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of Hedgehog signaling in PDAC have been contradictory, with Hedgehog signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how Hedgehog pathway inhibition reprograms the PDAC microenvironment.Experimental Design: We used a combination of pharmacologic inhibition, gain-and loss-of-function genetic experiments, cyto-metry by time-of-flight, and single-cell RNA sequencing to study the roles of Hedgehog signaling in PDAC.Results: We found that Hedgehog signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAF) compared with inflammatory CAFs (iCAF). Sonic Hedgehog overexpression promotes tumor growth, while Hedgehog pathway inhibition with the smoothened antagonist, LDE225, impairs tumor growth. Furthermore, Hedgehog pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in regulatory T cells, consistent with increased immunosuppression.Conclusions: Hedgehog pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with few effective therapeutic options. PDA is characterized by an extensive fibroinflammatory stroma that includes abundant

Pancreatic ductal adenocarcinoma (PDA) is accompanied by reprogramming of the local microenvironment, but changes at distal sites are poorly understood. We implanted biomaterial scaffolds, which act as an artificial premetastatic niche, into immunocompetent tumor-bearing and control mice, and identified a unique tumor-specific gene expression signature that includes high expression of C1qa, C1qb, Trem2, and Chil3. Single-cell RNA sequencing mapped these genes to two distinct macrophage populations in the scaffolds, one marked by elevated C1qa, C1qb, and Trem2, the other with high Chil3, Ly6c2 and Plac8. In mice, expression of these genes in the corresponding populations was elevated in tumor-associated macrophages compared with macrophages in the normal pancreas. We then analyzed single-cell RNA sequencing from patient samples, and determined expression of C1QA, C1QB, and TREM2 is elevated in human macrophages in primary tumors and liver metastases. Single-cell sequencing analysis of patient blood revealed a substantial enrichment of the same gene signature in monocytes. Taken together, our study identifies two distinct tumor-associated macrophage and monocyte populations that reflects systemic immune changes in pancreatic ductal adenocarcinoma patients.

Pancreatic ductal adenocarcinoma (PDA) is associated with activation of WNT signaling. Whether this signaling pathway regulates the tumor microenvironment has remained unexplored. Through single-cell RNA sequencing of human pancreatic cancer, we discovered that tumor-infiltrating CD4+ T cells express TCF7, encoding for the transcription factor TCF1. We conditionally inactivated Tcf7 in CD4 expressing T cells in a mouse model of pancreatic cancer and observed changes in the tumor immune microenvironment, including more CD8+ T cells and fewer regulatory T cells, but also compensatory upregulation of PD-L1. We then used a clinically available inhibitor of Porcupine, a key component of WNT signaling, and observed similar reprogramming of the immune response. WNT signaling inhibition has limited therapeutic window due to toxicity, and PD-L1 blockade has been ineffective in PDA. Here, we show that combination targeting reduces pancreatic cancer growth in an experimental model and might benefit the treatment of pancreatic cancer.

An extensive fibroinflammatory stroma rich in macrophages is a hallmark of pancreatic cancer. In this disease, it is well appreciated that macrophages are immunosuppressive and contribute to the poor response to immunotherapy; however, the mechanisms of immune suppression are complex and not fully understood. Immunosuppressive macrophages are classically defined by expression of the enzyme Arginase 1 (Arg1), which we demonstrated is potently expressed in pancreatic tumor associated macrophages from both human patients and mouse models. While routinely used as a polarization marker, Arg1 also catabolizes arginine, an amino acid required for T cell activation and proliferation. To investigate this metabolic function, we used a genetic and a pharmacologic approach to target Arg1 in pancreatic cancer. Genetic inactivation of Arg1 in macrophages, using a dual recombinase genetically engineered mouse model of pancreatic cancer, delayed formation of invasive disease, while increasing CD8+ T cell infiltration. Additionally, Arg1 deletion induced compensatory mechanisms, including Arg1 overexpression in epithelial cells, namely Tuft cells, and Arg2 overexpression in a subset of macrophages. To overcome these compensatory mechanisms, we used a pharmacological approach to inhibit arginase. Treatment of established tumors with the arginase inhibitor CB-1158 exhibited further increased CD8+ T cell infiltration, beyond that seen with the macrophage-specific knockout, and sensitized the tumors to anti-PD1 immune checkpoint blockade. Our data demonstrate that Arg1 drives immune suppression in pancreatic cancer by depleting Arginine and inhibiting T cell activation.

<div>Abstract<p>Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with few effective therapeutic options. PDAC is characterized by an extensive fibroinflammatory stroma that includes abundant infiltrating immune cells. Tumor-associated macrophages (TAM) are prevalent within the stroma and are key drivers of immunosuppression. TAMs in human and murine PDAC are characterized by elevated expression of apolipoprotein E (ApoE), an apolipoprotein that mediates cholesterol metabolism and has known roles in cardiovascular and Alzheimer's disease but no known role in PDAC. We report here that ApoE is also elevated in peripheral blood monocytes in PDAC patients, and plasma ApoE protein levels stratify patient survival. Orthotopic implantation of mouse PDAC cells into syngeneic wild-type or in <i>ApoE^−/−</i> mice showed reduced tumor growth in <i>ApoE^−/−</i> mice. Histologic and mass cytometric (CyTOF) analysis of these tumors showed an increase in CD8⁺ T cells in tumors in <i>ApoE^−/−</i> mice. Mechanistically, ApoE induced pancreatic tumor cell expression of <i>Cxcl1</i> and <i>Cxcl5</i>, known immunosuppressive factors, through LDL receptor and NF-κB signaling. Taken together, this study reveals a novel immunosuppressive role of ApoE in the PDAC microenvironment.</p>Significance:<p>This study shows that elevated apolipoprotein E in PDAC mediates immune suppression and high serum apolipoprotein E levels correlate with poor patient survival.</p><p><i><a href="https://aacrjournals.org/cancerres/article/doi/10.1158/0008-5472.CAN-21-1930" target="_blank">See related commentary by Sherman, p. 4186</a></i></p></div>

<p>SFigure5</p>