Role of cell-intrinsic C5AR1 in colorectal cancer survival and chemoresistance.

Abstract

225 Background: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. Although immune checkpoint blockade benefits ~15% of patients with high mutational burden and microsatellite instability, the majority of CRC remains refractory, with cytotoxic chemotherapy as the standard of care. Identifying mechanisms that sustain cancer cell survival and proliferation is therefore critical to uncovering vulnerabilities that may enhance therapeutic efficacy. The complement peptide C5a, a potent effector generated by proteolytic activation of the complement cascade, signals through its receptor C5AR1 to drive cell survival, cytokine production, and chemotaxis. While historically regarded as a systemic pathway, recent evidence suggests that complement activation can also occur locally. We hypothesized that CRC tumors locally generate C5a, with cancer cell-intrinsic C5AR1 driving survival and chemoresistance. Methods: CRC tissue microarrays (TMAs) were generated from de-identified specimens (Hackensack Meridian Health , N = 28; commercial TMA, N = 17). Preliminary spatial transcriptomics was performed on the commercial TMA using the Nanostring CosMx Spatial Molecular Imager. Publicly available single-cell transcriptomes from two independent CRC cohorts ( N = 63 and N = 62) were reanalyzed. Multiplexed immunofluorescence (24-plex Cyclic IF) was applied to detect complement fragments (C3, C3b/iC3b, C5a) and C5AR1, and to map their cell type-specific expression in situ. Cancer cell-intrinsic C5AR1 expression was validated in two human CRC cell lines and four patient-derived organoids. Functional relevance of C5AR1 was assessed using in vitro cytotoxicity assays combining chemotherapy with pharmacologic C5AR1 inhibition. Results: Spatial transcriptomics revealed induction of complement pathway transcripts in CRC tumors, further confirmed by reanalysis of two independent CRC single-cell datasets. Multiplexed CycIF revealed an enriched localization of complement ligands (C3, C3b/iC3b, C5a) to tumor-adjacent regions, whereas C5AR1 was enriched in the tumor core. Furthermore, CycIF demonstrated robust C5AR1 protein expression in cytokeratin-positive cancer cells and infiltrating myeloid phagocytes; cancer cell-specific C5AR1 expression was further validated in two human CRC cell-lines and patient-derived organoids. Functionally, cancer cell-intrinsic C5AR1 proved essential for survival, and its pharmacologic inhibition synergistically enhanced 5-fluorouracil cytotoxicity. Conclusions: Complement activation is spatially organized in CRC, with cancer cell-intrinsic C5AR1 supporting survival. Pharmacologic C5AR1 inhibition synergizes with chemotherapy, positioning the C5a-C5AR1 axis as a tractable therapeutic vulnerability. Ongoing studies aim to define how C5AR1 supports cancer cell survival and mediates chemoresistance.