Spatial proteomics reveals four-stage molecular evolution in cancer immunotherapy-related gastritis

Immune checkpoint inhibitors (ICIs) have transformed cancer treatment, yet immune-related adverse events (irAEs) including immunotherapy-related gastritis (IRAEG) pose significant clinical challenges-often necessitating treatment interruption that may compromise antitumor efficacy. IRAEG presents with atypical symptoms, lacks specific biomarkers, and shows histopathological overlap with other forms of gastritis, complicating diagnosis and management. Despite increasing clinical recognition, a systematic understanding of spatial molecular alterations across the full disease course remains limited. Here, we used spatial proteomics to map the molecular landscape of IRAEG during disease progression and to define stage-specific patterns of molecular evolution relevant to cancer immunotherapy management.

We analyzed tissue samples from seven patients, including four non-immunotherapy-related gastritis controls and three cancer patients who developed IRAEG following ICI therapy for solid tumors, sampled longitudinally across four disease stages: baseline (G1), acute severe inflammation (G2), early recovery (G3), and complete recovery (G4). Using laser capture microdissection coupled with data-independent acquisition mass spectrometry, we profiled 177 spatially resolved gastric tissue regions. Multiplex immunohistochemistry and immunofluorescence characterized features of the immune microenvironment, while Gene Ontology, KEGG pathway analysis, Gene Set Variation Analysis, and xCell inference enabled functional, metabolic, and immune profiling. Key immune and NET-related findings were further validated by multiplex immunofluorescence in an independent, expanded cohort of IRAEG and non-immunotherapy-related gastritis samples.

IRAEG was characterized by widespread HLA molecule activation and enhanced antigen processing, resembling the immune phenotype observed in organ transplant rejection. The acute G2 stage exhibited excessive neutrophil extracellular trap formation, profound metabolic suppression, and collapse of immune homeostasis-features that may inform early intervention strategies to preserve ICI treatment continuity. During early recovery (G3), inflammatory injury transitioned toward repair, marked by activation of fatty acid metabolism and PPAR signaling. Notably, even at complete clinical recovery (G4), more than 1,000 proteins remained differentially expressed, reflecting sustained enhancement of metabolic and immune functions and establishing a distinct molecular "memory" state with implications for ICI rechallenge decisions.

These findings define four molecularly distinct stages of IRAEG progression and recovery. The stage-specific signatures identified here serve as candidate biomarkers for diagnosis, disease staging, and therapeutic response assessment, and may guide clinical decisions regarding irAE management, treatment modification, and safe ICI rechallenge to support continued antitumor therapy.