Formalin-fixed paraffin embedded (FFPE) cancer tissues are labeled with Opal™ (Akoya Biosciences, Inc) fluorophores for multiplex in situ immunofluorescence, with panels developed in-house for specific questions. Vectra® (Akoya Biosciences, Inc.) system is used for the multiplex imaging, and subsequently the image analysis is performed with inForm® (Akoya Biosciences, Inc.) and the data analysis with R; or combined analysis performed with the TME software.