Integrated single-cell and spatial transcriptomics uncover distinct cellular subtypes involved in neural invasion in...

Nerves are integral to tumor biology, yet the peri- and intra-neural microenvironment and their roles in cancer-neural invasion (NI) remain underexplored. Here, we perform single-cell/single-nucleus RNA sequencing (sc/snRNA-seq) and spatial transcriptomics on 62 samples from 25 pancreatic ductal adenocarcinoma (PDAC) patients, mapping cellular composition, lineage dynamics, and spatial organization across varying NI statuses. Tertiary lymphoid structures are abundant in low-NI tumor tissues and co-localize with non-invaded nerves, while NLRP3⁺ macrophages and cancer-associated myofibroblasts surround invaded nerves in high-NI tissues. We identify a unique endoneurial NRP2⁺ fibroblast population and characterize three distinct Schwann cell subsets. TGFBI⁺ Schwann cells locate at the leading edge of NI, can be induced by transforming growth factor β (TGF-β) signaling, promote tumor cell migration, and correlate with poor survival. We also identify basal-like and neural-reactive malignant subpopulations with distinct morphologies and heightened NI potential. This landscape depicting tumor-associated nerves highlights critical cancer-immune-neural interactions in situ and enlightens treatment development targeting NI.