Heterogeneous SPP1-expressing esophageal cancer cells license immunotherapy resistance by organizing an immunosuppressive niche

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) remains a major cause of cancer-related mortality worldwide, with immunotherapy resistance representing a critical therapeutic challenge. This resistance is driven by profound tumor heterogeneity that fosters diverse immune evasion mechanisms, limiting the efficacy of current treatments. Thus, an urgent need exists to identify the key drivers within heterogeneous tumors that potentiate immunotherapeutic resistance. METHODS: Single-cell, spatial, and bulk transcriptomic data from multiple ESCC cohorts were integrated to map the cellular ecosystems and decipher the heterogeneity of immunotherapy-resistant tumors. Multi-cohort analyses were conducted to assess clinical relevance, and key findings were validated through targeted functional assays in vitro and in vivo. RESULTS: We discovered a previously unrecognized secreted phosphoprotein 1 (SPP1)-expressing malignant epithelial subpopulation that is markedly enriched in non-responders, representing a critical component of tumor heterogeneity. Elevated SPP1 expression correlated with immunotherapy resistance and poorer survival across independent cohorts. Mechanistically, SPP1 promoted tumor proliferation and migration while upregulating PD-L1 via the mTORC1-STAT3 pathway, as revealed by our RNA-sequencing data. Furthermore, SPP1 induced CD8⁺ T cell exhaustion through SPP1-CD44 signaling, accompanied by CD44-dependent MAPK activation, and polarized macrophages toward an immunosuppressive phenotype via α5β1 integrin-dependent FAK-AKT-mTOR activation. Spatial transcriptomics validation demonstrated that SPP1⁺ cells shape immunosuppressive niches within the tumor microenvironment. Therapeutically, SPP1 blockade enhanced the antitumor effect of anti-PD-1 therapy to suppress tumor growth in vivo. To directly address tumor heterogeneity, we employed drug sensitivity profiling that nominated microtubule and kinase inhibitors as potent agents against SPP1⁺ cells, providing a strategy to target resistant subpopulations. CONCLUSION: This study establishes SPP1 as both a key driver of immunotherapy resistance in heterogeneous ESCC tumors and a promising therapeutic target for overcoming treatment failure.