Multi-omics integration reveals ATG2A-regulated Ammonia-Induced cell death as a key driver of the immunosuppressive niche in colorectal cancer liver metastases

Colorectal cancer liver metastasis (CRLM) is the leading cause of mortality among patients. Tumors exhibit highly active metabolic processes, resulting in significant ammonia production within the tumor microenvironment (TME). Increasing evidence indicates that ammonia accumulation impairs T-cell function, facilitating tumor immune escape and metastasis. However, the molecular mechanisms underlying ammonia’s role in T-cell dysfunction in CRLM remain unclear. We combined single-cell RNA sequencing (scRNA-seq) and spatial transcriptome data to map the immune landscape in primary colorectal cancer (CRC) and colorectal liver metastases (CRLM). By analyzing T-cell subpopulations, conducting pseudo-time trajectory analysis, and employing high-dimensional weighted gene co-expression network analysis (hdWGCNA) alongside various machine learning algorithms, we identified key regulatory factors responsible for ammonia-related immune dysfunction.We applied scTenifoldKnk to perform in-silico ATG2A knockout, and further characterised ATG2A function under ammonia stress using in-vitro tumour-cell experiments. The study found significant differences in ammonia death-related gene set scores between colorectal liver metastases (CRLM) and primary colorectal cancer. Notably, CD8+ Tem_GZMK+ cells exhibited the highest scores, indicating a strong association with severe immune dysfunction. Pseudo-temporal analysis revealed a distinct trajectory of ammonia death-related dysfunction, differing from the conventional T-cell depletion pathway. High-dimensional gene network analysis (hdWGCNA) and various machine learning techniques identified ATG2A as a potential core gene linking ammonia death to T-cell dysfunction. ScTenifoldKnk-based in-silico perturbation predicted that ATG2A alteration may modulate gene networks associated with antigen-processing and NK-cell-mediated cytotoxicity. In-vitro assays demonstrated that ammonia triggers dose-dependent cytostatic/cytotoxic stress in CRC cells, with ATG2A expression levels increasing alongside ammonia concentration. Silencing ATG2A enhanced cell sensitivity to ammonia and inhibited proliferation and invasion capabilities. Additionally, a six-gene signature constructed from ammonia-stress-related genes shows association with prognosis in CRC patients. The shift in metabolic weight in colorectal liver metastasis resulted in ammonia buildup, which increased ATG2A levels. This increase impaired CD8⁺ Tem_GZMK⁺ T cell function, aiding tumor immune evasion. This six-gene signature reflects systemic ammonia-stress burden in colorectal cancer and may serve as a prognostic indicator for general CRC; its direct applicability to CRLM patients remains a hypothesis requiring dedicated CRLM-specific validation. Targeting this axis may offer a potential strategy, pending further in-vivo validation.