Figure 6. Complete digitalization of a core and practical example of a possible downstream analysis.

Anti-clockwise: (1) images for the markers stained with the MILAN multiplexing technique (for this work 39 markers, but we reached in our laboratory an output of around 100 markers per single section) are acquired and composite images with some selected markers can be prepared. (2) All the markers are used to phenotypically identify all the cell subtypes present in the tissue and the tissue is digitally reconstructed. (3) Studies of the functional status of these cells can be done, for example we could localize exhausted and active cells in the tissue. (4) With neighborhood analysis, it is possible to identify the cells that are in proximity with each other more than chance can suggest, inferring possible interactions between these cell types (in the image, two cells identified as ‘neighbors’ are encircled in red).