Figure 1. Single-cell analysis scheme.

(A) TMA construction, Multiplex-stripping immunofluorescence: 60 cores were obtained from 29 patients, assembled in a Tissue Micro Array, and analysed using the MILAN technique; the immunofluorescence images from one round of staining (three markers/round: S100-blue, CD3-red, CD4-green) of three representative cores from our data set are shown. (B) CD8+ cells were analyzed using image analysis for functionality using an activation parameter derived from multiple activation and exhaustion markers evaluated at single-cell resolution; the CD8+ cells are here digitally reconstructed for each of the above standing cores, preserving their spatial distribution in the tissue section and assigning each of them a color according to the activation status. (C) All the cell populations in the cores were phenotypically identified using consensus between three clustering methods and manual annotation from the pathologists; The heatmaps with the levels of expression of the phenotypic markers per cluster for one of the three clustering methods are shown on the left; on the right, all the inflammatory cells are assigned a color based on their phenotype and the tissue is digitally reconstructed for each of the above standing cores, preserving the spatial distribution of each cell. (D) The social network of the cells was analysed using a permutation test for neighborhood analysis in order to make inferences on cell-cell interactions. The results of the neighborhood analysis are generated as a heatmap were the type and the strength of the interaction is expressed with a color code; to simplify the visualization of the interactions, the different cell types are represented in a circle and connected with lines that clarify the type of relationship between them.