Hypoxia-Associated Glycolytic–Lipogenic Reprogramming and Impaired Antigen Presentation in SPP1+ Tumor-Associated Macrophages in Ovarian Cancer

Ovarian cancer has an immunosuppressive microenvironment in which SPP1+ tumor-associated macrophages (TAMs) are linked to poor prognosis and immune evasion, yet their molecular features and metabolic mechanisms remain unclear. We therefore set out to determine whether SPP1 marks a discrete TAM subpopulation or a functional state in treatment-naïve high-grade serous ovarian cancer (HGSOC), to define the transcriptional and metabolic program that accompanies it, and to test whether local hypoxia is associated with it. Tumor-derived, treatment-naïve samples from three single-cell datasets (one lesion per patient; 47 patients) yielded 385,504 cells; Seurat v5 and Harmony were used for clustering, annotation, and batch correction. SPP1+/SPP1− macrophages were compared by differential expression, GO/KEGG/GSEA, metabolic scoring, and transcription factor inference, and the definition was retested with within-sample SPP1-high versus SPP1-low comparisons and with MSigDB Hallmark gene sets scored by two independent algorithms. Validation used Visium spatial transcriptomics (GSE263920), TCGA-OV (n = 429), CD8+ T cell exhaustion analysis, CellChat and a syngeneic ID8 model. Of 64,067 macrophages (16.62%), 82.0% were SPP1+; upregulated genes were enriched in glycolysis, monocyte migration and tissue remodeling, whereas MHC class II and neutrophil-related genes were downregulated. At the sample level, glycolysis, fatty acid synthesis and glutaminolysis scores were elevated and the cholesterol synthesis score was reduced, with no evidence of suppressed fatty acid oxidation or oxidative phosphorylation; HIF1A ranked 4th and NR1H3 3rd among 271 transcription factors. All 8 spatial sections showed positive hypoxia–SPP1 correlations, and hotspots showed elevated glycolysis, EMT, immunosuppression and macrophage scores. No robust association was found between SPP1+ TAM infiltration and CD8+ T cell exhaustion (p = 0.315). MDK–LRP1/SDC1 was the major macrophage-directed input. Immunofluorescence showed SPP1 immunoreactivity in a subset of CD68+ macrophages within HIF1α-rich areas of ID8 tumors, without CD8+PD-1+ T cell aggregation. SPP1+ TAMs therefore correspond to a hypoxic functional state with HIF1A activation, a glycolytic–lipogenic transcriptional program, reduced cholesterol synthesis gene expression and a reduced MHC class II program; protein-level and functional validation, and direct tests of HIF1A dependency, remain necessary.