A Spatially Constrained Fibroblast-Myeloid Program Associates With Immune Exclusion and Poor Prognosis in Lung Adenocarcinoma

<h4>Background</h4>Although immune checkpoint blockade (ICB) therapy has improved clinical outcomes for some patients with lung adenocarcinoma (LUAD), only a subset of cases can achieve durable benefits, and primary resistance is often associated with an immune-excluded tumor microenvironment (TME). Existing studies indicate that cancer-associated fibroblasts (CAFs) are increasingly recognized as important participants in extracellular matrix (ECM) remodeling and stroma-immune crosstalk. However, in LUAD, it is still unclear which CAFs-related programs are involved in immune exclusion, particularly in relation to their spatial interactions with myeloid cell states.<h4>Methods</h4>We integrated eight publicly available LUAD single-cell RNA-seq cohorts (164 samples; 471,501 cells) using Harmony and annotated major lineages and stromal subsets. CAFs were reclustered to resolve subtype heterogeneity, followed by pathway activity scoring, weighted gene coexpression network analysis (WGCNA), pseudotime trajectory inference (Slingshot), and regulon analysis. Bulk TCGA-LUAD data were used for immune-exclusion correlation and survival analyses. Spatial transcriptomics was applied for in situ validation, and ligand-receptor analysis together with NicheNet was used to prioritize CAFs-derived signaling interactions and downstream targets.<h4>Results</h4>The integrated atlas identified 10 major cell lineages and revealed tumor-associated expansion of stromal and myeloid compartments. We further resolved six CAFs subtypes with distinct molecular and functional features. Tumor-enriched CAFs subsets showed stronger activation of ECM remodeling, focal adhesion, TGF-<i>β</i> signaling, hypoxia-related pathways, and inflammatory programs. Bulk-level analyses in TCGA-LUAD demonstrated that CAFs-related signatures were associated with a T-cell exclusion index and increased expression of the immune checkpoint-related molecule CD276 (B7-H3). Spatial transcriptomic mapping further showed that selected CAFs signatures were enriched in hypoxic, nonepithelial regions and colocalized with myeloid-associated signals, supporting the presence of a spatially constrained fibro-myeloid niche. Cell-cell communication analysis revealed extensive ligand-receptor interactions between CAFs subtypes and myeloid populations, whereas survival and immunotherapy cohort analyses showed that specific CAFs-related programs were associated with worse clinical outcomes and less favorable treatment responses.<h4>Conclusions</h4>Multicohort single-cell integration and spatial validation define a CAFs-centered, immune-excluded niche in LUAD characterized by coordinated stromal and myeloid programs. These findings improve current understanding of CAFs heterogeneity and fibroblast-myeloid coupling in LUAD and provide a framework for future strategies aimed at targeting stromal barriers and remodeling the immune microenvironment to enhance ICB responsiveness.