Fig 1: Spatial distribution of immune infiltrates and checkpoint proteins.(A) Low-magnification image of a clear cell renal cancer subjected to 12-cycle t-CyCIF (see Supplementary file 4 for a list of antibodies). Regions high in a-smooth muscle actin (a-SMA) correspond to stromal components of the tumor, those low in a-SMA represent regions enriched for malignant cells. (B) Representative images from selected t-CyCIF channels are shown. (C) Quantitative assessment of total lymphocytic cell infiltrates (CD3+ cells), CD8+ T lymphocytes, cells expressing PD-1 or its ligand PD-L1 or the VEGFR2 for the entire tumor or for a-SMAhigh and a-SMAlow regions. VEGFR2 is a protein primarily expressed in endothelial cells and is targeted in the treatment of renal cell cancer. The error bars represent the S.E.M. derived from 100 rounds of bootstrapping. (D) Density plot for CD3 and CD8 expression on single cells in the tumor (left) or stromal domains (right). (E) Centroids of CD3+ or CD3+CD8+ cells in blue or dark blue as well as cells staining as SMAhigh or SMAlow (gray and light-gray, respectively) used to define the stromal and tumor regions. (F) Centroids of PD-1+ and PD-L1+ cells are shown in red and green, respectively. (G) Results of a K-nearest neighbor algorithm used to compute areas in which PD-1+ and PD-L1+ cells lie within ~10 µm of each other and with high spatial density (in yellow) and thus, are potentially positioned to interact at a molecular level.10.7554/eLife.31657.029Figure 9—source data 1.Immune cell counts from bootstrapping in tumor and stroma regions (Figure 9C).10.7554/eLife.31657.030Figure 9—source data 2.Single-cell intensity data used in Figure 9.