Highlights d Macrophages polarized by pancreatic cancer cells release pyrimidine nucleosides d Pyrimidine release is a property of alternatively activated macrophage metabolism d Deoxycytidine from macrophages inhibits gemcitabine treatment of cancer cells d Targeting macrophages enhances gemcitabine treatment of pancreatic cancer

Pancreatic Ductal Adenocarcinoma (PDA) is characterized by abundant infiltration of tumor associated macrophages (TAMs). TAMs have been reported to drive resistance to gemcitabine, the front-line chemotherapy in PDA, though the mechanism of this resistance remains unclear. Profiling metabolite exchange, we demonstrate 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. Additionally, we report pyrimidine release is a general function of anti-inflammatory myeloid cells, suggesting an unknown physiological role of pyrimidine exchange by immune cells.

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Pancreatic ductal adenocarcinoma (PDA) remains a leading cause of cancer related death, contrasting a relatively low incidence rate. A principle barrier in PDA treatment is the physiology of the tumors, characterized by a densely fibrotic stroma, rich with immune cell infiltration including macrophages. Macrophages are polarized by environmental cues which dictate their function. In PDA, macrophages within the tumor (tumor associated macrophages, or TAMs) are strongly immunosuppressive, inhibiting both infiltration and activation of cytotoxic T-cells. Additionally, TAMs have been shown to drive resistance to chemotherapy, though the mechanism for which remains unclear. Several pathways have been described by which PDA cells recruit and polarize macrophages into TAMs, and the effects that TAMs have on the tumor microenvironment. These pathways have largely focused on signaling proteins, however, metabolic byproducts also influence the behavior of immune cells within the tumor microenvironment. To explore potential metabolic crosstalk, we have profiled the metabolic factors exchanged between PDA cells and TAMs. Among these, we have found that TAMs release metabolites which can regulate the response of PDA cells to chemotherapy. Importantly, this response is consistent across several murine and patient-derived pancreatic cancer cell lines, and this metabolite release appears to be a general property of anti-inflammatory macrophage metabolism. We further validated these findings in vivo, using a combination of pharmacological and genetic models to modulate myeloid cells within the tumor microenvironment. Taken together, these data suggest that further development of interventions which target either PDA-mediated polarization of TAMs or TAM-mediated inhibition of chemotherapy represent opportunities to improve the efficacy of currently available treatment options. Citation Format: Christopher J. Halbrook, Corbin Pontious, Ilya Kovalenko, Laura Lapienyte, Stephan Dreyer, Yaqing Zhang, Barbara Nelson, Hanna Hong, Daivid Chang, Jennifer P. Morton, Marina Pasca di Magliano, Costas A. Lyssiotis. Macrophage-epithelial metabolic crosstalk impairs chemotherapy in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4549.

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Background: Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate of 10% due to a lack of early detection and effective treatments. Aspartic protease Cathepsin E (CTSE) expression is increased early in PDAC, but its role in tumorigenesis is unknown. We hypothesize that high CTSE expression promotes proliferation, invasion, inhibition of apoptosis, and the secretion of cancer-associated cytokines in PDAC. Methods: Human PDAC cell lines (Mpanc96, Panc-1, and Miapaca2) were engineered to express high levels of CTSE. Cell proliferation, invasion, and apoptosis were analyzed and compared to control cell lines expressing low levels of CTSE. Cells were treated with aspartic protease inhibitors (Pepstatin A and Ritonavir) and monitored for proliferation (24 - 72 hours) and invasion (48 hours). Apoptosis was measured using propidium iodide and analyzed via flow cytometry. Conditioned media from cells was collected (72 hours) after treatment to assess differential secretion of cancer-associated cytokines using a cytokine array. Results: Proliferation was not affected by the overexpression of CTSE; however, inhibition of CTSE slowed proliferation in the Panc-1 and MiaPaca2, but not Mpanc96 cells. CTSE inhibitors promoted invasion of Mpanc96 and Panc-1, but not Miapaca2 cells, regardless of CTSE levels. Apoptosis was not significantly different between the cell lines treated with the aspartic protease inhibitors compared to controls. Increased expression of CTSE induced the secretion of various cytokines such as epithelial neutrophil-activating protein 78 (ENA-78), a chemokine expressed in inflammatory pancreatic disorders. Treatment with the inhibitor Pepstatin A decreased ENA-78 secretion. Conclusions: CTSE levels does not appear to affect proliferation or apoptosis; however, inhibition of CTSE increased cell invasion and resulted in altered cytokine secretion. Further studies examining how CTSE influences invasiveness, and cytokine secretion may create new opportunities for developing effective therapies for PDAC. Citation Format: Ericka Velez-Bonet, Sabrina Kaul, Corbin Pontious, Marcus Hong, Kelly Dubai, Zobeida Cruz-Monserrate. The role of Cathepsin E expression in pancreatic ductal adenocarcinoma tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-061.