Colorectal cancer is a particularly challenging disease for immunotherapy given tumor heterogeneity. Multiplexed whole slide imaging allows deep contextual analysis of cancer tissue and provides new insights into cancer tissue heterogeneity, including cell-to-cell interactions and the tumor immune microenvironment. In the current study, the Cell DIVE™ multiplexed immunofluorescence solution was used to comprehensively map 36 biomarkers on a single Colon Cancer Tumor Microarray (TMA), with over 96 tissue core samples, enabling multiplexed biomarker labeling of tumors from multiple patients. Stitched images were imported into HALO® image analysis software (www.indicalab.com). For analysis, a subset of 18 biomarkers were used for tissue classification, cell segmentation, cell phenotyping and spatial analysis. Multiplexed tissue immunofluorescence imaging of a multi-patient TMA provides unprecedented and novel insights into cell populations and spatial interactions in the tumor microenvironment across patient samples.