Integrative single-cell and spatial transcriptomic analyses identify LMX1B as a tumor suppressor orchestrating the GDF15-ATP4B axis in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is characterized by profound inter- and intratumoral heterogeneity and extensive tumor microenvironment (TME) remodeling, yet the spatially resolved cellular programs and intercellular communication networks orchestrating malignant progression remain poorly defined. LMX1B, a LIM homeodomain transcription factor essential for kidney morphogenesis, has not been previously investigated in renal carcinogenesis. METHODS: We integrated single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) across three independent RCC cohorts, complemented by hierarchical differential weighted gene co-expression network analysis (hdWGCNA), CellChat-based ligand-receptor interaction analysis, RCTD spatial deconvolution, and mistyR colocalization analysis. Multi-platform validation included qRT-PCR, western blot, TCGA and CCLE database mining, chromatin immunoprecipitation, dual-luciferase reporter assays, and functional studies in vitro and in subcutaneous xenograft models. RESULTS: ScRNA-seq and ST analyses revealed that LMX1B is predominantly expressed in epithelial cells and is markedly downregulated in tumor-derived epithelial populations compared to normal counterparts, with reduced expression correlating with advanced tumor grade and lymphatic metastasis. LMX1B-associated epithelial cells (LMX1B-Epi) function as central signaling hubs within the TME, engaging in extensive bidirectional communication with fibroblasts, endothelial cells, myeloid cells, and T/NK cells through VEGFA-, SPP1-, and GDF15-mediated pathways. Spatial deconvolution demonstrated that LMX1B-Epi occupy specialized tissue niches distinct from LMX1B-negative epithelial cells. HdWGCNA identified five co-expression modules enriched for stress response, metabolism, and mitochondrial ATP synthesis, all exhibiting significant differential activation between normal and tumor LMX1B-Epi. Functionally, LMX1B overexpression suppressed RCC cell proliferation, migration, and invasion. Mechanistically, LMX1B directly binds to the GDF15 promoter to transcriptionally activate its expression. GDF15, in turn, positively regulates ATP4B and induces mitochondrial apoptosis through ROS-mediated mitochondrial membrane potential depolarization and Bax/Bcl-2-caspase-3 cascade activation. Rescue experiments confirmed that ATP4B overexpression reverses the oncogenic effects of GDF15 knockdown both in vitro and in vivo. CONCLUSIONS: Our integrative multi-omic and functional analyses establish the LMX1B-GDF15-ATP4B axis as a critical tumor-suppressive cascade in RCC. These findings illuminate how a developmentally essential transcription factor governs TME organization and mitochondrial homeostasis in renal malignancy, and identify LMX1B as a promising prognostic biomarker and therapeutic target for RCC.