Tumor immune microenvironment features associated with response to neoadjuvant durvalumab plus gemcitabine, cisplatin, and nab-paclitaxel (DURGAP) in resectable biliary tract cancer

Background: Neoadjuvant DurGAP therapy has shown promising efficacy in resectable biliary tract cancer (BTC). In an updated cohort of 30 patients (2025 The Lancet Summit: Cancer Control in China, Abstract #TLSU2025_0087; NCT05640791), the objective response rate (ORR) was 60%, disease control rate (DCR) 86.7%, and R0 resection rate among resected patients 93.8%. However, mechanisms underlying variable response remain unclear. This study explored the tumor immune microenvironment (TIME) associated with differential tumor regression grades (TRG). Methods: Four TRG1 (major response) and four TRG3 (minimal response) patients from the DurGAP cohort were analyzed. Multiplex immunofluorescence quantified CD8⁺ T cells, CD4⁺ T cells, and regulatory T cells (Tregs). One representative case per group underwent spatial transcriptomic sequencing to assess cellular composition and spatial distribution. Immunofluorescence data were compared using Mann-Whitney U test. Spatial transcriptomic data were processed with the Seurat and sctransform pipelines, normalized and batch-corrected via Harmony, followed by PCA and UMAP-based clustering. Spatial domains were annotated using FindAllMarkers to identify domain-specific genes. Differentially expressed genes (p<0.05, |log₂FC|>log₂1.5) were subjected to GO and KEGG pathway enrichment analysis. Results: TRG1 tumors displayed significantly higher infiltration of CD8⁺ and CD4⁺ T cells compared with TRG3, while Tregs were enriched in TRG3. Spatial transcriptomics confirmed these findings, revealing greater T and B lymphocyte density and fewer malignant cells in TRG1, whereas TRG3 exhibited higher tumor cell proportions and reduced effector infiltration. Subset analysis showed that the proportion of Tregs among total T cells was markedly higher in TRG3, while the CD8⁺/Treg ratio was substantially greater in TRG1. Functional enrichment highlighted immune activation and antigen presentation pathways in TRG1, contrasting with metabolic and proliferative signaling enrichment in TRG3. These spatial and transcriptional patterns indicate that robust effector T-cell infiltration and coordinated immune architecture underlie effective tumor regression, whereas immunosuppressive microenvironments limit therapeutic benefit. Conclusions: Distinct TIME profiles distinguish responders from non-responders to neoadjuvant DurGAP in BTC. Higher effector T-cell infiltration, an increased CD8⁺/Treg ratio, and immune-enriched spatial domains are linked to major tumor regression, whereas Treg-dominant microenvironments correspond to poor response. These findings highlight potential immune biomarkers to refine patient selection and improve future therapeutic strategies.