Single-cell spatial analysis identifies ID1-high endothelial cells in tertiary lymphoid structures as predictors of durable response to immunotherapy in non-small cell lung cancer

BACKGROUND: Tertiary lymphoid structures (TLSs) maturity and cellular composition shape resistance or sensitivity to immune-checkpoint blockade (ICB) across cancers. Single-cell spatial resolution data for the endothelial compartment of TLSs are lacking; therefore, we investigated the cellular composition, endothelial dynamics, and cell-cell interactions within TLS regions. METHODS: We applied high-resolution Xenium In Situ spatial transcriptomics to eight samples collected from non-small-cell lung cancers (NSCLC) (four after chemoimmunotherapy, four untreated), extracted 90 TLS regions, generated approximately 320,000 single-cell profiles from those regions, and analyzed the endothelial compartment within each TLS. We independently assessed the exploratory findings using multiplex immunofluorescence and survival analysis in a separate cohort of 45 patients receiving immunotherapy. RESULTS: Spatial mapping revealed a reproducible, B-cell-dominant TLS architecture across all samples. Mature TLSs contained CXCL13-rich follicular cores and were associated with a favorable prognosis. Within the endothelial compartment, high endothelial cells (HECs) forming high-endothelial venules (HEVs) segregated from other endothelial cells; in mature TLSs these HECs exhibited high inhibitor of DNA binding 1 (ID1) expression and an adhesion molecular signature, promoting lymphocyte recruitment. Patients with tumors harboring ID1-high HEVs showed higher response rates and pronounced survival advantage; multivariable analysis confirmed ID1-high status as an independent prognostic factor. Conversely, ICB exposure eventually reduced ID1 expression and adhesion molecule levels in HECs, impaired lymphocyte trafficking, and promoted a shift toward a remodeled endothelial state. CONCLUSIONS: ID1expression, linked to HEC differentiation during TLS maturation or ICB exposure, marks a functionally mature, lymphocyte-recruiting HEV that predict durable response to immunotherapy. These findings provide a novel mechanistic framework and practical biomarkers for HEV-centered immuno-oncology strategies in NSCLC.