Atlas of metastatic gastric cancer links ferroptosis to disease progression and immunotherapy response

Gastric adenocarcinoma (GAC) remains a significant cause of cancer-related deaths worldwide, with the majority of mortality resulting from metastatic spread. To elucidate the evolution of cancer cells and their interactions with the tumour microenvironment (TME), we conducted a comprehensive single-cell transcriptome and immune repertoire profiling of primary tumours (PRIs), matched liver metastases (LMs) or peritoneal carcinomatosis (PC), adjacent normal tissues, and blood samples from 20 treatment-naïve patients. We found that the TME within metastases varied substantially from PRIs and differed between LMs and PC samples. Indicative of immune evasion, we observed diminished T cell clonal expansion, a marked reduction in the fraction of tumour-reactive CD8 T cells, and an enrichment of naïve T cells, M2-like, and proliferative macrophages in PC samples compared to PRIs. LMs displayed significantly reduced B cells, with increased fractions of exhausted CD8 T cells and NK/NKT cells. Both LMs and PC showed nearly depleted plasma cells. Malignant cells largely exhibited diminished gastric lineage identity, predominantly adopting intestinal-like and mixed cell states, with the acquired expression of gastrointestinal stem-cell markers. LM cancer cells showed increased epithelial-to-mesenchymal signalling, while PC cancer cells had increased levels of aneuploidy. We observed differentially expressed cancer meta-programs and gene modules (GMs) between LMs and PC, with high GM2 expression in the PRI predicting an increased risk of distant metastases. Further analysis of GM signature genes revealed ferroptosis evasion through GPX4 upregulation during malignant progression. Functional validation via patient-derived xenografts and a novel autochthonous mouse model of GAC confirmed that genetic or pharmacological inhibition of ferroptosis resistance attenuates tumor growth and metastatic progression. Together, our mapping of malignant progression provides a rationale for targeting ferroptosis defense in combination with next-generation CAR T-cell immunotherapy as a potential avenue to overcome T cell dysfunction in advanced metastatic GAC therapy. A total of 20 patients with stage-IV gastric adenocarcinoma (GAC) treated at Zhejiang Cancer Hospital were enrolled in this study. All patients were at stage IV and treatment-naïve prior to sample collection. This cohort included nine patients with liver metastases (LM), six patients with peritoneal carcinomatoses (PC), one patient with both LM and PC, and four patients with both PC and ovarian metastases. Based on the Lauren classification, 13 primary GACs were defined as intestinal type, six as diffuse type, and one as mixed type. Three patients had signet-ring cell carcinoma and seven patients had Heliobacter pylori (H. Pylori) infection. The primary GAC diagnosis of all patients was confirmed through endoscopic biopsies. LMs were shown as hypoechoic nodules on B-scan ultrasonography. The diagnosis of PC was confirmed by cytologic examination or laparoscopic exploration, which was also used to verify whether the patient had ovarian metastases. The pathology results were confirmed by two experienced gastrointestinal pathologists independently. In addition, computed tomography (CT) images of all patients were verified by two experienced radiologists. Except for patient 17 (Pt-17) who underwent radical extremital partial gastrectomy in 2006, none of the patients had a past medical history of gastrointestinal tumours. **Raw data not provided due to patient privacy concerns**