Targeting MAN1A1 to address immune checkpoint inhibitors resistance: A novel mechanism of vascular-endothelial reprogramming in NSCLC.

2566 Background: Despite the efficacy of immune checkpoint inhibitors (ICIs) in NSCLC, resistance remains a major hurdle. The role of non-immune components, particularly vascular endothelial cells (VECs), is poorly understood. We aimed to identify key drivers of ICI resistance by focusing on VEC-mediated remodeling of the tumor microenvironment (TME). Methods: We integrated transcriptomic data from public cohorts of ICI-treated and treatment-naïve NSCLC patients (GSE225620), proteomic profiling from in-house whole blood samples, and peripheral blood lymphocyte subset analysis from NSCLC patients and healthy controls. Differential expression analysis and machine learning (LASSO, SVM-RFE) were employed to identify key resistance-associated genes, including MAN1A1. Functional validation was performed using shRNA-mediated MAN1A1 knockdown in A549 and H1299 NSCLC cell lines, with apoptosis measured by flow cytometry. Single-cell RNA-seq data (GSE207422) from pre- and post-treatment NSCLC tumors were analyzed using Seurat for cell annotation. Cell-cell communication (CellChat) and pathway activity (AUCell) analyses were used to investigate VEC-immune cell interactions and their association with treatment response. Statistical analyses included Kaplan-Meier survival, Cox regression, and ROC curves to assess the prognostic and predictive value of MAN1A1. Results: Multi-omics analysis identified MAN1A1 as a key candidate associated with ICI resistance. Elevated MAN1A1 expression predicted inferior clinical outcomes, including shorter mPFS (3.2 vs. 8.5 months; P=0.019) in our cohort and reduced OS (47.4 vs. 65.1 months; HR=0.95, P=0.017) in the TCGA dataset. MAN1A1 expression effectively distinguished ICI non-responders (AUC=0.784). MAN1A1 knockdown significantly induced apoptosis in NSCLC cell lines (A549, P=0.00022; H1299, P=0.0004). Increased peripheral B-cell counts post-chemotherapy correlated with treatment response, and MAN1A1 protein levels inversely associated with B-cell levels (r=-0.38, P<0.05). Single-cell RNA-seq analysis revealed VECs as the primary expressors and communication hubs of MAN1A1. In non-responders, MAN1A1-high VECs were reprogrammed to drive a pro-inflammatory feedback loop with neutrophils via ANXA1-FPR1/NAMPT-integrin signaling axes. In contrast, VECs from responders exhibited a shift towards T-cell-recruiting LGALS9-CD45 signaling. Conclusions: Our study identifies MAN1A1 as a novel driver of ICI resistance in NSCLC, mediating vascular-endothelial reprogramming that fosters an immunosuppressive TME characterized by pro-tumor neutrophil recruitment and impaired T- and B-cell immunity via the ANXA1-NAMPT axes. Targeting MAN1A1 represents a promising therapeutic strategy to overcome ICI resistance, providing a strong rationale for future translational development.