Single-cell and spatial transcriptomic profiling of POU5F1 in Lung Adenocarcinoma: Dynamics, spatial niche, and prognosis via multi-algorithm ML

• POU5F1 genetically associated with lung adenocarcinoma risk. • Spatial co-localization of POU5F1+ tumor cells with CAFs. • Machine learning prognostic model shows high predictive accuracy. • POU5F1 knockdown suppresses tumor growth and metastasis. • Multi-omics integration reveals POU5F1-driven tumor microenvironment. POU5F1 (OCT4), a core regulator of pluripotency, plays an important role in tumor stemness and immune microenvironment remodeling, yet its systematic function and mechanisms in lung adenocarcinoma (LUAD) remain incompletely elucidated. This study integrated genetic causality inference, single‑cell and spatial transcriptomics, RNA velocity analysis, and multi‑algorithm machine learning to systematically investigate the role of POU5F1 in LUAD. Summary‑based Mendelian Randomization (SMR) was used to link GWAS with lung tissue eQTL data; single‑cell and spatial analyses characterized POU5F1 + cell states and their interactions with cancer‑associated fibroblasts (CAFs); a machine‑learning prognostic model was constructed based on POU5F1‑related differentially expressed genes (LDEGs); and in vitro functional experiments were performed to validate its biological functions. POU5F1 was genetically associated with lung cancer risk and enriched in malignant, stem‑like cell clusters. RNA velocity indicated progressive activation along pseudotime trajectories. Spatial transcriptomics confirmed the co‑localization of POU5F1 + tumor cells with CAFs. The LDEG‑based prognostic model showed excellent predictive performance (AUC > 0.9) and was correlated with immune suppression and targeted therapy response. Basic experiments further demonstrated that POU5F1 was specifically highly expressed in LUAD cell lines; knockdown of POU5F1 significantly inhibited cell proliferation, migration and invasion, and induced apoptosis; mechanistically, these effects involved regulation of the Bcl‑2/caspase‑3 apoptotic pathway, modulation of the epithelial‑mesenchymal transition (EMT) process mediated by E‑cadherin/Vimentin, and downregulation of the stemness factor Nanog. POU5F1 promotes LUAD progression by maintaining stemness, reprogramming CAFs, and shaping immune evasion, representing a promising biomarker and therapeutic target.