Colorectal cancer cell-derived arginine drives SMS-mediated spermine synthesis and PI3K-AKT-mTOR-dependent M2 polarization of tumor-associated macrophages

Abstract Colorectal cancer (CRC) often exhibits severe tolerance to immunotherapy due to the highly immunosuppressive nature of the tumor microenvironment (TME), with the polarization of tumor-associated macrophages (TAMs) toward the pro-tumor M2 phenotype playing a key role. Arginine (Arg), as a core precursor for the synthesis of the immune-microenvironment-reshaping metabolite spermine (Spm), is a crucial factor influencing the fate of TAMs. Unlike some tumors with Arg auxotrophy, CRC cells exhibited elevated argininosuccinate synthetase 1 (ASS1) expression, which was associated with increased extracellular Arg availability in our co-culture system. This study aimed to investigate the specific regulatory mechanisms of CRC cell metabolic reprogramming on immune function in the TME. Single-cell and clinical tissue analyses revealed elevated ASS1 expression in CRC epithelial cells and enrichment of SMS in M2-like TAMs, while higher spermine synthase (SMS) expression was associated with poorer survival in the analyzed PD-1-treated CRC cohort. The Transwell co-culture model demonstrated that CRC cells specifically upregulated ASS1 and released large amounts of Arg into the TME. Macrophages took up Arg and utilized their significantly enriched SMS to convert it into Spm. The intracellular accumulation of Spm further activated the PI3K-AKT-mTOR signaling pathway, driving macrophage polarization toward the M2 phenotype and upregulating immunosuppressive factors such as IL-10 and TGF-β, which in turn induced the exhaustion of local CD8 + T cells. Based on these findings, combined treatment with AMXT 1501 and Nivolumab attenuated M2-like polarization and the CD8⁺ T-cell exhaustion-associated phenotype, reduced CRC-cell proliferation, and increased apoptosis in vitro, with the major findings further validated in the MSS CRC cell line SW480. In conclusion, this study clarified the importance of the ASS1/Arg/SMS/Spm cross-cellular metabolic axis in CRC immune escape and demonstrated that targeting polyamine metabolism in combination with immune checkpoint blockade represents a promising strategy for overcoming immune tolerance across molecularly distinct CRC models.