Targeted Effects of FPRL1 Signaling on Tumor Microenvironment and Immune Response in Cholangiocarcinoma

Objective: Urokinase plasminogen activator (uPAR) plays critical roles in inflammation, extracellular matrix remodeling and immune responses. uPAR and soluble uPAR (suPAR) are increased and are prognostic indicators of poor outcome in cholangiocarcinoma (CCA), a metastatic liver cancer. The receptor for suPAR (FPRL1) is expressed by tumor cells, T cells and macrophages; when bound on the tumor, can lead to T cell exhaustion and immune evasion. Studies from the lab have shown that FPRL1 antagonist, WRW4, reduced tumor burden and altered immune response in CCA tumor-bearing mice. We investigated the effects of WRW4 on the immune response and tumor microenvironment. Hypothesis: We hypothesized that targeting of FPRL1 significantly impacts the immune response and tumor microenvironment. Methods: CCA orthotopic mouse models were established and treated with vehicle or WRW4. Liver, liver lymph node, bone marrow, and tumor tissues were collected after 4 weeks. Bone marrow derived macrophages (BMDM) from these mice were treated with CSF-1, from which the conditioned media (CM) was collected. Splenocytes were isolated from a wildtype C57Bl/6 mouse and co-cultured with BMDM CM or SB-1 cells, a murine CCA cell line. Splenocytes and SB-1 cells were either non-treated or pre-treated with WRW4 prior to co-culture. Data: SB-1 pre-treatment with WRW4 drove expression of uPAR, MMP2, VEGFR3, and fibronectin on splenocytes. However, splenocyte pre-treatment with WRW4 altered expression of TIM3 and LAG3, (T cell exhaustion markers). When splenocytes were co-cultured with BMDM CM from CCA mice, both vehicle and WRW4 treated reduced cytotoxic CD8+ T cells. SB-1 CM decreased T regulatory cells, which was opposed in SB-1+WRW4 CM. Macrophages isolated from CCA mice exhibited an M2 phenotype, but WRW4 treatment re-polarized to an M1 phenotype. Summary of Results: SB-1 pre-treatment with WRW4 impacted splenic cell Non-B Non-T populations. Both splenocyte and SB-1 pre-treatment impacted SB-1 cell markers, but pre-treated SB-1 had a greater impact on splenocyte mRNA expression than SB-1. Induced BMDM CM from mock mice altered immune profiles of cultured splenocytes, with a greater difference in CCA groups, and WRW4 altered mRNA expression of immune regulatory and invasion genes. Conclusions: Data suggest macrophages and their signaling play a key role in crosstalk with the peripheral immune system and polarization of macrophages induces specific immune responses, which is further impacted by suPAR-FPRL1. T cell responses resulting from BMDM CM co-culture indicates SB-1 suppresses T regulatory cell responses, which is reversed with WRW4 treatment. CPRIT RP210213 and RP230204, Texas A&M ASCEND Research Leadership Fellowship and TAMU Health Seedling Grant to SC. NIH T32 LYMPH Training Program Fellowship awarded to SB. 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.