Multi-scale integrative analyses identify THBS2+ cancer-associated fibroblasts as a key orchestrator promoting aggressiveness in early-stage lung adenocarcinoma.

Rationale: Subsets of patients with early-stage lung adenocarcinoma (LUAD) have a poor post-surgical course after curative surgery. However, biomarkers stratifying this high-risk subset and molecular underpinnings underlying the aggressive phenotype remain unclear. Methods: We integrated bulk and single-cell transcriptomics, proteomics, secretome and spatial profiling of clinical early-stage LUAD samples to identify molecular underpinnings that promote the aggressive phenotype. Results: We identified and validated THBS2, at multi-omic levels, as a tumor size-independent biomarker that robustly predicted post-surgical survival in multiple independent clinical cohorts of early-stage LUAD. Furthermore, scRNA-seq data revealed that THBS2 is exclusively derived from a specific cancer-associated fibroblast (CAF) subset that is distinct from CAFs defined by classical markers. Interestingly, our data demonstrated that THBS2 was preferentially secreted via exosomes in early-stage LUAD tumors with high aggressiveness, and its levels in the peripheral plasma associated with short recurrence-free survival. Further characterization showed that THBS2-high early-stage LUAD was characterized by suppressed antitumor immunity. Specifically, beyond tumor cells, THBS2+ CAFs mainly interact with B and CD8+ T lymphocytes as well as macrophages within tumor microenvironment of early-stage LUAD, and THBS2-high LUAD was associated with decreased immune cell infiltrates but increased immune exhaustion marker. Clinically, high THBS2 expression predicted poor response to immunotherapies and short post-treatment survival of patients. Finally, THBS2 recombinant protein suppressed ex vivo T cells proliferation and promoted in vivo LUAD tumor growth and distant micro-metastasis. Conclusions: Our multi-level analyses uncovered tumor-specific THBS2+ CAFs as a key orchestrator promoting aggressiveness in early-stage LUAD.