Interruption of cross-communication pathways alters the immune cell signature of pancreatic cancer and decreases tumor growth

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with limited therapeutic options in part due to a dense stroma and the ability of the tumor cells to defy therapy by creating an immunosuppressive milieu. A histologic feature of PDAC is the abundance of immunosuppressive M2-polarized tumor-associated macrophages (TAMs) that favor cancer growth and metastases. Cancer cells communicate with immune cells of the tumor microenvironment (TME) by several mechanisms including exosomes to induce plasticity of the immune cells favoring pro-cancer instead of anti-cancer phenotypes. We examined a novel strategy to interrupt the cross-communication between cancer and immune cells by reprogramming the TME with a cholecystokinin-B receptor (CCK-BR) antagonist, proglumide.

METHODS: Cross-communication was examined using 3-dimensional human PDAC spheroids that were either co-cultured with macrophages or treated with supernatant spent media from macrophages. RNA was extracted from the spheroids and subjected to RNA sequencing and Reverse Phase Protein Array (RPPA). RNA sequencing revealed that the most upregulated gene in the cancer cells after co-culture was ELANE, which codes for neutrophil elastase. Next, ELANE expression was down regulated by shRNA transfection in PDAC cells and the effect of the decreased expression was examined on the polarization of immune cells in vitro and in vivo in mice. Exosomes were also collected from PDAC spheroids for evaluation by RPPA.

RESULTS: Co-culture of spheroids with macrophages increased cancer cell number and induced polarization of M0 macrophages to the M2-phenotype. Co-culture increased oncogenic pathways and genes by RPPA and RNA sequencing, respectively, and these changes were reversed with proglumide. ELANE knockdown in PDAC cells prevented the M2-polarization of macrophages in vitro and decreased tumor growth in vivo. Immunohistochemistry of ELANE knockdown tumors showed a shift in plasticity from M2-polarized TAMs to M1-polarized cells compared to wild-type tumors. Neutrophil elastase in cancer exosomes increased after co-culture and was abolished by treatment with proglumide.

CONCLUSION: ELANE expression in PDAC cells regulates polarization of macrophages through the release of neutrophil-elastase rich exosomes. Proglumide therapy with CCK-B receptor antagonism is a novel method to interrupt cross-communication pathways between PDAC and immune cells in the TME and decrease cancer growth.