DSG2+ Cancer Stem Cells Co‐Located With FAP+ Myofibroblasts in the Tumor Boundary That Determines the Efficacy of Immunotherapy in Non‐Small Cell Lung Cancer

ABSTRACT Lung cancer remains a leading cause of cancer‐related mortality owing to its aggressiveness and pronounced heterogeneity. Cancer stem cells (CSCs) have been implicated in tumor progression and therapeutic resistance; however, their transcriptional programs and spatial organization in non‐small cell lung cancer (NSCLC) are not fully characterized. Here, we integrated single‐cell RNA sequencing with spatial transcriptomics to systematically define CSC‐associated phenotype in NSCLC. Weighted gene co‐expression network analysis identified a 127‐gene CSC signature enriched for stemness‐related pathways, with DSG2 emerging as a dominant marker. Elevated DSG2 expression was associated with chemotherapy resistance and predicted response to EGFR tyrosine kinase inhibitors based on independent clinical proteomic datasets. Spatial mapping revealed preferential enrichment of DSG2+CSCs at tumor margins, where they co‐localized with FAP+myofibroblasts (myCAFs). Multiplex immunofluorescence demonstrated that FAP+myCAFs expressed MMP9 and MMP12, consistent with a microenvironment supportive of epithelial‐mesenchymal transition and CSC maintenance. Functional co‐culture assays showed that myCAFs enhanced CSC‐associated phenotypes in DSG2ʰigh tumor cells in an MMP‐dependent manner. Collectively, these findings delineate a spatially organized DSG2+CSC–myCAF niche that contributes to therapeutic resistance in NSCLC.