Abstract A044: Pancreatic cancer cell-derived EVs facilitate monocyte recruitment to the tumor site

Abstract Introduction: Tumor-associated macrophages (TAMs) are the most abundant immune cell population within the pancreatic tumor microenvironment (TME) and majorly contribute to its immunosuppressive nature. Circulating monocytes are deemed to be the primary source of TAMs instead of organ-specific resident macrophages. Extracellular vesicles (EVs) are membrane-bound nanoparticles containing bioactive molecules released by all cell types. Recently, the role of EVs in immune modulation has gained increasing credence. Our present study aims to examine the role of pancreatic cancer cell-derived EVs (cEV) in monocyte recruitment to TME and their differentiation into the TAM phenotype. Material and methods: EVs were isolated from non-tumorigenic pancreatic epithelial cell line, (hTERT-HPNE), one pancreatic cancer cell line (Mia PaCa-2), and pancreatic PDX cell line, (PDX188), using size exclusion chromatography (SEC). The EVs were characterized per ISEV guidelines using cryo-electron microscopy, nanoparticle tracking analysis (NTA), and immunoblot analysis. The effect of cEVs on monocyte phenotype, differentiation, and chemotaxis was investigated using multi-omics analysis, flow cytometry, western blot, and cytokine profiling in co-culture experiments with the human monocyte cell line THP-1, as well as primary monocytes derived from PBMCs. The in vivo effect of normal and cancer derived EVs on monocyte recruitment was investigated by injecting PDAC tumor-bearing mice. Results: Proteomics data revealed that cargo of cEVs but not of HPNE-derived EVs, was enriched with proteins associated with myeloid cell activation. Additionally, flow cytometry data established that cEV-treated THP1 monocytes expressed significantly high levels of surface CD14 compared to HPNE-EV-treated monocytes, suggesting that cEVs likely activate monocyte differentiation. Chemotaxis results showed that cEV-treated THP1 monocytes moved more rapidly towards monocyte chemoattractant protein-1 (CCL2), aligning with our in vivo data, where tumor-bearing mice injected with cEVs had a higher number of macrophages in their tumors. Western blot and cytokine profile analysis confirmed expression of anti-inflammatory proteins such as STAT3 and showed that monocytes treated with cEVs secreted an elevated level of anti-inflammatory cytokines such as IL-10 and IL-4. Conclusion: Our results provide credence to a novel finding that pancreatic cancer-derived EVs carry specific cargo that, upon uptake, enhance monocyte recruitment to TME and their subsequent differentiation to TAMs. Further studies examining the underlying mechanisms is ongoing. Citation Format: Amrita K. Cheema, Yanjun R. Zhang, Baldev Singh, Pritha Bose, Jeyalakshmi Kandhavelu, Sunain Deol, Meth Jayatilake, Shu Wang. Pancreatic cancer cell-derived EVs facilitate monocyte recruitment to the tumor site [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A044.