Abstract
Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, and secreted phosphoprotein 1 (SPP-1) is linked to tumor progression and immune evasion. This study investigates whether anti-SPP-1 therapy can enhance the anti-tumor effects of anti-CTLA-4 immunotherapy in HCC and elucidates the underlying molecular mechanisms. Data from The Cancer Genome Atlas (TCGA) comprising 374 HCC patients were analyzed to examine SPP-1 expression and its correlation with clinical prognosis using the Kaplan-Meier survival curve and multivariate Cox regression models. Multiple bioinformatics platforms including TIMER and TISIDB were employed to assess SPP-1's role in the tumor immune microenvironment. In vivo validation was performed in H22 hepatocellular carcinoma syngeneic mouse models treated with anti-SPP-1 and anti-CTLA-4 antibodies, where tumor growth and immune cell infiltration were evaluated using flow cytometry, immunohistochemistry, and molecular interaction studies. In the 374 HCC patient cohort, high SPP-1 expression was significantly associated with advanced TNM staging, pathological grading, and vascular invasion. Lower SPP-1 expression significantly correlated with improved overall survival, disease-specific survival, and progression-free intervals, confirming SPP-1 as an independent prognostic factor in multivariate analysis. Immune infiltration analysis revealed that SPP-1 was positively correlated with immune cells and checkpoint molecules including CTLA-4. In a syngeneic tumor model, combination therapy with anti-SPP-1 and anti-CTLA-4 significantly inhibited tumor growth, increased CD8+ T cell infiltration, and promoted M1 macrophage polarization. Mechanistically, combination therapy significantly reduced IL-6 expression and suppressed STAT3 signaling, while interferon-γ levels were elevated in the combination therapy group. Co-immunoprecipitation experiments confirmed SPP-1/IL-6 protein interaction. SPP-1 serves as both a potential prognostic marker and therapeutic target for HCC. Combined anti-SPP-1 and anti-CTLA-4 therapy synergistically enhances immunotherapy efficacy by suppressing the IL-6/STAT3 pathway, offering a promising translational strategy for HCC treatment.