Analysis of the MCL TIME composition using 4-plex IF–based image analysis shows that the distribution of cells varies between spatial regions, but CD3⁺ to CD163⁺ cell ratios remain constant. (A) CD20⁺ cells (tumor), CD163⁺ cells (macrophages), and CD3⁺ cells (T cells) in tumor-rich and tumor-sparse ROIs (99 patients, 191 tissue cores). (B) Heat map showing the distribution of cell density (cell counts per area) for the 3 cell types in the tumor-rich region. (C) Box plot comparison of the cell densities (cell counts per area) in tumor-rich and tumor-sparse regions for each of the 3 cell subtypes (63 patients, 98 tissue cores). (D) Box plot comparison of the cell-to-cell ratios in paired samples in which both tumor-rich and tumor-sparse regions were collected in the same patients (34 patients, 51 tissue cores), showing identical CD163⁺ macrophage to T-cell ratios between the 2 regions. The y-axis is log10 transformed for better visualization. Note that all analyses that compared the distributions and ratios were performed per tissue core but were validated per patient using the mean patient aggregate values (data not shown). (E) A spatial point pattern analysis³³ plot that shows the difference in L function value for CD163⁺ macrophages in tumor-sparse and tumor-rich regions using paired samples (34 patients, 51 tissue cores). The colored lines show tissue-specific L function differences between the 2 spatial regions, and the bold black line shows the average L function difference across all tissue. A clustered pattern is suggested when the average L function value is above 0 with the range of 0 to 50 μm being most important.