Targeting macrophage-derived SPP1 enhances CD8 T cell infiltration via ROS-DNA fragment/cGAS-STING/STAT1-mediated CXCL9/10 in tumor microenvironment

Background Elevated levels of SPP1 + tumor-associated macrophages (TAMs) are associated with reduced CD8 + T cell infiltration and poorer prognosis in cancer patients, but direct evidence demonstrating a causal role for SPP1 + TAMs in excluding CD8 + T cells is still missing. The precise mechanisms by which SPP1-activated signaling pathways and macrophage-derived factors regulate CD8 + T cell trafficking remain poorly understood. Methods We established multiple tumor mouse models to study the function of macrophage SPP1 in the tumor environment, especially its role in the relationship between macrophages and CD8 T cells. We combined the single-cell (sc) RNA sequencing data of clinical tumor samples and tumor tissues from Spp1 fl/fl-Lyz2-Cre mice to identify the differences in SPP1-related genes and found that SPP1 could regulate the expression of CXCL9 and CXCL10 in macrophages. Through Western blotting, immunofluorescence staining, and flow cytometry analyses, we elucidated the mechanistic basis by which macrophage-specific SPP1 deficiency suppressed tumorigenesis. Results This study demonstrated that macrophage-derived SPP1 played a crucial role in suppressing CD8 T cell infiltration, promoting tumor progression, and diminishing the effectiveness of immune checkpoint inhibitor (ICI) therapy. Sc-RNA sequencing analysis revealed a marked increase in CD8 T cell populations within tumor tissues of Spp1 fl/fl-Lyz2-Cre mice. Furthermore, a negative correlation was observed between CD8 T cells and SPP1 macrophages in human colorectal cancer specimens. Genetic deletion of SPP1 in macrophages markedly enhanced tumor growth suppression in a manner dependent on CD8 T cell-mediated immunity. Mechanistically, SPP1 deficiency in macrophages led to elevated mitochondrial reactive oxygen species (ROS) production, resulting in the accumulation of cytosolic double-stranded DNA (dsDNA) fragments. This accumulated dsDNA activated the cGAS-STING pathway, leading to subsequent STAT1 phosphorylation. The enhanced STAT1 activity upregulated the expression of chemokines CXCL9 and CXCL10, thereby facilitating CD8 T cell recruitment into the tumor microenvironment. Conclusions Deletion of SPP1 in TAMs upregulates CXCL9/10 production by activating the ROS-DNA fragment/cGAS-STING/STAT1 pathway, thereby enhancing CD8 T cell infiltration, inhibiting tumor progression, and improving ICI treatment outcomes in tumors.