Spatial Zonation of Pro- and Anti-inflammatory Tumor Macrophages After Anti-CD40

Seeking to understand how local tissue factors reinforce these divergent TAM phenotypes, we next investigated their spatial distribution in MC38 tumors following anti-CD40 treatment (days 6 and 8 post-inoculation). We performed high-definition spatial transcriptomics at a spatial resolution of ~2 µm on a formalin-fixed, paraffin-embedded (FFPE) section. H&E-staining revealed dense leukocyte infiltrates and a large necrotic core. Spatial mapping showed a clear partition in macrophage phenotypes. The peripheral zone was rich in antigen-presenting and proinflammatory macrophages, while antiinflammatory macrophages were concentrated close to the necrosis-proximal region. This pattern was consistent with the accumulation of Cd74+ and Cxcl9+ macrophages at the tumor edges and Spp1+ macrophages clustering toward the necrotic center. Cells co-expressing pro- and anti-inflammatory markers (Cxcl9+ / Cd74+ and Spp1+) were scattered between the single-positive populations, suggesting a continuous gradient of macrophage states driven by local stress. Overall design: MC38 cells in culture medium were mixed with Geltrex and injected subcutaneously into the mouse flanks. Agonistic anti-CD40 treatment was administered intravenously according to treatment protocol. Mice were euthanized, and tumors were collected at defined timepoints after antibody administration. The tumor was analyzed using spatial transcriptomics.