Spatial organization of tumor populations drives the spatial organization of immune cells. (A) Cross-section images showing examples of a YFP, mixed, and RFP region of a mixed-population tumor and CD3 stain. The image is representation of at least seven tumors. Blue scale bar = 1 mm, white scale bar = 100 μm. (B) Quantification of CD3⁺ T cells per field, plotted for each region. The data are a representation of eight tumors analyzed in two independent experiments. (C) Correlation of YFP⁺ cell fraction and CD3⁺ T cell frequency in each field. CD3⁺ T cell count was scaled to DAPI⁺ cell count in each field to normalize the difference in cell density between fields. The data are a representation of eight tumors analyzed in two independent experiments. (D) Schematic of immune cell profiling in YFP, mixed, and RFP regions of mixed-population tumors. Each mixed-population tumor was sliced into five to ten 400-µm-thick live sections, and each section was manually microdissected into YFP, mixed, and RFP regions using a surgical scalpel under a fluorescent dissecting microscope. All YFP pieces, all mixed pieces, and all RFP pieces from the same tumor were pooled for immune profiling by flow cytometry. White scale bar = 5 mm, blue scale bar = 1 mm. (E–M) Frequency of T cell subsets, ratio of Treg to IFNγ⁺ CD8 T cells or Th1 cells, and frequency of macrophages in YFP, mixed, and RFP regions. Grey dotted lines in panels E–G and M separate data of mixed-population tumors from the data of single-population tumors. Data are a representation of two independent experiments with ≥6 mice per experiment. Statistical significance was determined by Student’s t test (B and E–M) or Pearson’s correlation test (C). *P < 0.05, **P < 0.01, and ***P < 0.001.