CD206 ^Hi macrophages and a lack of neutrophils and inflammatory monocytes are associated with an immunosuppressive TME in RFP regions. (A) Schematic of ZipSeq spatial transcriptomic analysis of mixed-population tumors. Composite stitched image of a 200-µm-thick live section marked with YFP regions (encircled with green lines), RFP regions (red lines), and mixed regions (blue lines) is shown. YFP, RFP, and mixed regions were illuminated individually to allow for the binding of a unique barcode (“zipcode”) to a photocaged oligo–CD45 antibody complex. Zipcode-tagged immune cells were analyzed by scRNA-seq. (B) UMAP representation of zipcode-labeled cells with cluster overlay. n = 3,238 cells. (C) UMAP representation of zipcode-labeled cells with zipcode identity overlaid. n_YFP = 1,236, n_RFP = 1,318, and n_Mixed = 684. (D) Abundance of cells belonging to the T cell cluster (cluster 5), calculated as percentage of total immune cells in each region. (E) Abundance of cells belonging to five macrophage clusters, calculated as percentage of total immune cells in each region. (F) Percentage of cells belonging to neutrophil cluster (cluster 3) in each region and pathway analysis showing the gene families enriched in the neutrophil cluster based on the top 250 differentially expressed genes. (G) Percentage of cells belonging to inflammatory monocyte cluster (cluster 7) and pathway analysis of genes enriched in the monocyte cluster based on the top 31 differentially expressed genes. Data in D–G are a representative data of one tumor out of two tumors analyzed. (H and I) Frequency of total macrophages (H) and three macrophage subsets (see Fig. S4 A for gating scheme) (I) detected by flow cytometric analysis in microdissected YFP, mixed, and RFP regions. n = 8–9. The experiment was performed once. (J) CD8 T cell suppression assay using CD206^Hi macrophages and non-CD206^Hi macrophages (combined Arg1^Hi and MHC-II^Hi macrophages) isolated from CIT6-YFP and CIT9-RFP single-population tumors. n = 3–6. Data are a representation of two independent experiments. Statistical significance was determined by one-way ANOVA (H and I) or two-way ANOVA (J) with Tukey’s correction posttest. *P < 0.05, **P < 0.01, and ***P < 0.001. ILC,  innate lymphoid cell.