H&E slides were viewed by a dermatopathologist to determine representative areas for multispectral image capture at 20× magnification using Mantra™ (PerkinElmer Images were analyzed using inform™ software (PerkinElmer) (Fig. 1A-G, Supplementary Fig. S2-5). Five representative areas were chosen by the dermatopathologist: (i) three areas with tumor and up to 50% stroma and (ii) two areas with tumor only (at least 90% tumor). These images were factored equally into the analysis for each patient. Mantra™ captures spectral information from a multiplexed panel of targets using a multispectral camera. For samples of small size, a minimum of two areas meeting the above criteria were required for inclusion. For spectral unmixing, examples of each fluorophore are taken from single stained slides for each antibody, as well as a representative autofluorescence spectrum from an unstained sample. Images from each of these single-stained and unstained slides were used to create a multispectral library in inform™. Intensity of each fluorescent target was extracted from the multispectral data using linear unmixing (47 (link)).