Downregulation of<i>ELANE</i>in pancreatic cancer cells regulates macrophage polarization

Background: Macrophages are phenotypically called classical (M1) or pro-inflammatory and alternative (M2) or anti-inflammatory, pro-repair and pro-cancer types. Pancreatic cancer is associated with an immunosuppressive microenvironment with a predominance of M2-polarized tumor-associated macrophages (TAMs). Pancreatic tumor progression is driven by the communication between the cancer epithelial cells and the surrounding cells in the microenvironment. Macrophages exhibit remarkable phenotypic plasticity, enabling them to respond dynamically to a spectrum of immune-metabolic stimuli. Recent research has focused on strategies to target the immunosuppressive TAMs in order to improve efficacy of therapies for pancreatic cancer. Hypothesis: Cancer epithelial cells cross-communicate with TAMs and interruption of the communication may induce plasticity of the macrophages, reverting them to the pro-inflammatory and anti-cancer state. Methods: In this study, we used human AsPC-1 pancreatic cancer cells and human U937 monocytes. The U937 cells exhibit plasticity and can polarize to M1 or M2 TAMs. Human pancreatic cancer AsPC-1 3-dimensional spheroids were developed in vitro. AsPC-1 spheroids were grown in culture alone or co-cultured with U937 cells. After 72h, the number of spheroids were counted and the U937 cells were examined for M2-polarization with arginase-1 staining. RNA sequencing was done to compare differentially expressed genes between AsPC-1 spheroids alone and the AsPC-1 spheroids that were co-cultured with U937 cells. Since ELANE mRNA or neutrophil elastase was the most upregulated gene by RNAseq, AsPC-1 cells were transfected with ELANE shRNA to establish clones with ELANE downregulation. The AsPC-1 ELANE -knockdown (KD) clones were studied in vitro in co-culture trans-wells with U937 monocytes and as tumors grown in female athymic nude mice compared to wild-type AsPC-1 controls. Tumor growth was measured weekly and tumor cell immune cells were examined by flow cytometry and immunohistochemistry. Results: When AsPC-1 spheroids were co-cultured with U937 cells for 72h, the number of spheroids significantly increased in number (p&lt;0.0001) and the U937 cells polarized to M2-phenotype. ELANE mRNA expression was low in wild-type AsPC-1 cells, but increased more than 20-fold by PCR when co-cultured with U937 cells suggesting that U937 cells communicated with AsPC-1 pancreatic cancer cells to promote a pro-cancer phenotype. In vitro, co-culture of AsPC-1 wild-type cells caused U937 monocytes to polarize to the M2 phenotype. ELANE mRNA expression by PCR was downregulated after shRNA transfection of AsPC-1 cells by 30% in clone B. Co-culture of ELANE -knockdown (KD) AsPC-1 cells with U937 cells in vitro failed to induce M2-polaization. Tumor growth of ELANE -KD tumors was slower than that of wild type tumors. Flow cytometry revealed increased M1-polarized macrophages in ELANE -KD AsPC-1 tumors compared to wild-type tumors. Immunohistochemistry showed fewer M2-polarized TAMs in AsPC-1 tumors. Conclusion: ELANE expression in pancreatic cancer cells regulates polarization of macrophages and is a potential new target for therapy. Targeting ELANE may represent a novel strategy to induce plasticity in macrophages and decrease the immunosuppressive signature in pancreatic cancer tumors. Support of this research was provided by NIH/NCI grant R01 CA277680 to JPS. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.