Spatial profiling defines three immunophenotypes and a perineural invasion-associated immune-evasion niche in gallbladder cancer.

[BACKGROUND & AIMS] Gallbladder cancer (GBC) has a high risk of postoperative recurrence, and immunotherapy-based adjuvant approaches remain unstandardised. We aimed to define spatial immunophenotypes in GBC and evaluate their associations with clinical outcomes and immune-evasion mechanisms.

[METHODS] We used multiplex immunohistochemistry to quantify 16 immune-cell subsets across the tumour core, tumour margin and peritumour regions in 86 treatment-naïve GBCs, and integrated nerve bundle-centred spatial analysis with transcriptomic profiling (n = 9) to investigate perineural invasion (PNI)-associated immune-evasion mechanisms.

[RESULTS] Unsupervised clustering and a CD8/α-SMA dual-marker approach defined three spatial immunophenotypes: immune-inflamed, immune-excluded, and immune-desert. The immune-inflamed phenotype featured high intratumoural immune infiltration with elevated PD-L1 expression (median combined positive score: 13.85 vs. 0.41 vs. 0.25, p <0.001), was associated with better prognosis (overall survival [OS]: hazard ratio [HR] 0.25, 95% CI 0.12-0.54, p <0.001; recurrence-free survival [RFS]: HR 0.41, 95% CI 0.21-0.79, p = 0.008), and improved outcomes with adjuvant chemoimmunotherapy vs. chemotherapy alone (OS: HR 0.10, 95% CI 0.02-0.48, p <0.001; RFS: HR 0.29, 95% CI 0.12-0.70, p = 0.003). The immune-excluded phenotype was stroma-enriched (α-SMA, p <0.001), with immune cells largely restricted to the tumour margin or peritumour regions; the immune-desert phenotype showed sparse immune infiltration. PNI was associated with immunosuppressive chronic inflammation, characterised by enrichment of PD-L1 myeloid cells and exhausted CD8 T cells near invaded nerve bundles (cell density, p <0.001). Transcriptomic analyses suggested enhanced tumour neurotropism in PNI-positive tumours (gene-set enrichment, p <0.05), together with activation of IL-6/IGF-1 related chronic inflammatory signalling and lipid-metabolic rewiring involving LEP, CD36, and ADIPOQ, collectively shaping a local immune-evasion niche (FDR <0.05).

[CONCLUSIONS] We defined three spatial immunophenotypes in GBC with distinct immune features and clinical outcomes. PNI was associated with the formation of a local immune-evasion niche.

[IMPACT AND IMPLICATIONS] GBC lacks validated biomarkers to guide postoperative immunotherapy, whereas spatial immune architecture provides a clinically meaningful indicator of tumour immune heterogeneity. By defining three spatial immunophenotypes, we delineate their distinct clinical outcomes and immune-evasion features. Moreover, we demonstrate an association between PNI and the formation of a local immune-evasion niche. These findings may inform clinicians and clinical trialists seeking to optimise postoperative risk stratification and adjuvant treatment strategies, and researchers investigating PNI-related molecular mechanisms. Clinically, integrating spatial immunophenotyping with PNI assessment into routine pathology workflows may improve prognostic stratification and inform adjuvant treatment decisions; however, prospective validation is required before broad clinical adoption.