SPP1+ Macrophages Promote Immunosuppression in Post-TACE Hepatocellular Carcinoma Involving Lactate-Induced Lactylation and Potential SPP1-CD44 Signaling

Background: Transarterial chemoembolization (TACE) treats inoperable liver cancer by inducing tumor ischemia, but subsequent hypoxia can promote an immunosuppressive microenvironment. This study explores how TACE-related hypoxia influences immune cells through lactate metabolism and lactylation. Methods: Single-cell RNA sequencing of TACE-treated and newly diagnosed liver cancer samples was performed. Scores were assigned to lactate-related gene sets, and differentially expressed genes were identified. Bioinformatics analysis integrated scRNA-seq, TCGA, and GEO data. Machine learning derived a consensus gene signature, and a prognostic nomogram was constructed. The study evaluated immune infiltration and drug sensitivity. Key findings were validated via spatial transcriptomics, in vitro experiments, and immunofluorescence. Results: We identified seven distinct single-cell types, noting an increase in macrophage infiltration and a decrease in CD8+ T cells within the TACE group. A six-gene consensus signature related to lactate-comprising SPP1, DNASE1L3, HRG, TPX2, LAPTM4B, and G6PD-was identified and found to be significantly associated with poor prognosis. This consensus gene score and T, N staging constructed a prognostic nomogram validated by calibration curves and decision curves. We also found that SPP1+ macrophages were elevated in the TACE treatment group, with increased lactate-related gene set scores, and were associated with hypoxia, glycolysis, and inflammatory pathways. Cell communication analysis suggested a potential regulatory link between SPP1+ macrophages and CD8+ T cells, highlighting the potential involvement of the SPP1-CD44 axis. Cell experiments showed that hypoxic, lactate-rich environments lead to macrophage lactylation, increasing SPP1+ macrophages and decreasing CD8+ IFN-γ+ T cells. Spatial transcriptomics and immunofluorescence staining revealed co-localization of SPP1+ macrophages with CD8+ T cells. Conclusion: We established a lactate-associated six-gene signature and prognostic nomogram. The immunosuppressive microenvironment following TACE in liver cancer is highly correlated with the activation of lactate-related genes and an increase in SPP1+ macrophages. These macrophages are closely associated with CD8+ T cells dysfunction, with the SPP1-CD44 pathway serving as a candidate narrative for this intercellular crosstalk. These findings suggest that targeting lactate-induced lactylation or the potential SPP1-CD44 interaction may offer promising therapeutic opportunities to counteract immune evasion after TACE therapy.