Integrated bulk and single-cell transcriptomics with experimental validation to identify calmodulin-related...

Lung adenocarcinoma (LUAD) exhibits poor prognosis partly due to dysregulated calcium signaling. Here, we integrated bulk RNA-seq (TCGA/LUAD, n = 440) and single-cell sequencing (GSE131907) to identify calmodulin-related prognostic genes. Differential expression analysis and LASSO-Cox regression identified SPHK1 and ASPM as independent predictors of overall survival (HR = 1.52/1.47, P < 0.001). A risk model incorporating these genes stratified patients into high/low-risk groups with distinct clinical outcomes (3-year AUC = 0.83). High-risk patients showed elevated γδ T cells and TIDE scores, while SPHK1/ASPM expression correlated with an immunosuppressive myeloid phenotype and M2-like polarization. Single-cell analysis revealed SPHK1⁺-ASPM⁺ myeloid cells dominated in early differentiation stages, with pseudo-time trajectories indicating SPHK1 downregulation and ASPM upregulation during myeloid maturation. Multiplex immunofluorescence validated co-localization of SPHK1/ASPM with MPO⁺ myeloid cells in tumor tissues. Importantly, the MPO⁺SPHK1⁺/ASPM⁺ myeloid compartment was significantly enriched in anti-PD-1 non-responders (P < 0.05). Our study establishes a calcium signaling-based prognostic signature, uncovers myeloid SPHK1/ASPM as drivers of immunosuppression, and provides a potential biomarker for immunotherapy stratification.