Pan-cancer analysis identifies SLC9A9, a Floatptosis-associated gene, as a regulator of tumor progression and immune modulation in ccRCC

Solute carrier family 9 member A9 (SLC9A9) is a vesicle-associated Na⁺/H⁺ antiporter regulating endosomal pH homeostasis, recently implicated in floatptosis. Its pan-cancer expression landscape, prognostic relevance, and immunological roles remain largely unexplored. A multi-level pan-cancer integrative approach was employed, incorporating transcriptomic data from TCGA, GTEx, and GEO to assess differential expression, diagnostic performance, and prognostic value. Immune infiltration profiling, genomic characterization, single-cell and spatial transcriptomic analyses were conducted. In vitro and in vivo functional assays were performed following SLC9A9 knockdown in ccRCC cells. In addition, amiloride-based rescue experiments were conducted in SLC9A9-overexpressing cells. Single-cell transcriptomic analysis and transwell-based conditioned media assays were performed to evaluate the role of SLC9A9 in macrophage infiltration and migration. SLC9A9 exhibited aberrant expression across multiple cancer types, with pronounced upregulation and consistent association with poor clinical outcomes in ccRCC. Elevated SLC9A9 expression was closely associated with immune and inflammation related pathways, macrophage infiltration, and tumor immune phenotypes. Single-cell analyses revealed preferential enrichment of SLC9A9 in monocyte and macrophage populations, while spatial transcriptomics demonstrated its localization within tumor cell-dense and macrophage-rich regions. In vitro and in vivo experiments confirmed that SLC9A9 silencing suppressed RCC proliferation and tumor growth, while over expression enhanced proliferation in an amiloride-reversible manner. Single-cell analysis and conditioned-medium assays further supported a functional association with macrophage migration. Our pan-cancer analyses reveal the molecular and clinical landscape of SLC9A9, highlighting its involvement in tumor progression and immune microenvironment modulation, with particular relevance in ccRCC. SLC9A9 may serve as a prognostic biomarker and an immunotherapy-relevant therapeutic target in cancer.