Spatial transcriptomics reveal depth‐dependent nerve growth factor and M2 ‐like macrophage enrichment in the perineural invasion of distal cholangiocarcinoma

Abstract Perineural invasion (PNI) is a frequent pathological feature of distal cholangiocarcinoma. The spatial molecular programs associated with PNI are unclear. We aimed to characterize tumor–nerve–immune interactions linked to PNI using spatial transcriptomics and independent validation. Spatial transcriptomics was performed using GeoMx Digital Spatial Profiler on six cases of surgically resected distal cholangiocarcinoma (non‐PNI, n = 3; PNI, n = 3). Regions of interest included non‐PNI tumor regions, surface mucosal tumor regions without neural contact (PNI‐surface), and tumor regions adjacent to S100‐positive nerves (PNI‐perineural). Tumor compartments were enriched by CK19‐based segmentation. Differential expression and pathway analyses were performed using iDEP/DESeq2 and Reactome/Kyoto Encyclopedia of Genes and Genomes enrichment. Validation at the protein level was conducted using immunohistochemistry in an independent cohort (non‐PNI, n = 22 and PNI, n = 82) for nerve growth factor (NGF), CD68, and CD163. Three‐dimensional nerve architecture was assessed in nine additional cases using tissue clearing. PNI‐associated compartments and non‐PNI regions had distinct transcriptional profiles. Compared to that in non‐PNI, 109 genes were differentially expressed in PNI‐surface (34 up‐ and 75 downregulated) and 179 in PNI‐perineural (64 up‐ and 115 downregulated). Pathway analyses identified enrichment of immune‐, microenvironment‐, and neurotrophic‐related programs, including NGF‐associated signatures. Tumor‐cell NGF expression was significantly higher in PNI‐positive tumors (median H ‐score: 215 versus 119, p < 0.0001) and increased with depth in PNI cases ( p < 0.0001). Deep invasive regions of PNI‐positive tumors saw a significant increase in CD68‐positive (median 313 versus 210 cells per high‐power field, p < 0.001) and CD163‐positive (median 64 versus 40 cells per high‐power field, p < 0.01) macrophages. CD163/CD68 ratio increased in deep versus surface regions, specifically in PNI‐positive tumors ( p < 0.0001). PNI‐positive distal cholangiocarcinoma exhibited spatially distinct tumor programs and depth‐dependent remodeling involving NGF upregulation and M2‐like macrophage enrichment, identifying potential therapeutic targets. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.