The growth in cancer immunotherapy agents requires an understanding of the immune contexture of the tumor microenvironment (TME). One way to understand immune contexture is to use multiplex staining, imaging, and analysis to obtain multi-marker phenotypes of specific cells and analyze their biodistribution in the TME. Imaging Mass Cytometry™ (IMC) is the method of choice for single-step staining and highplex imaging of FFPE tissues. FFPE tissue is autofluorescent, which limits the utility of immunofluorescence methods, particularly when done without amplification. Lung and colorectal tissue (and bone, skin, etc) are highly autofluorescence, and therefore are a good target for IMC imaging, which has no autofluorescence issues. However, developments in analysis software with a single-package workflow for highplex imagery have not kept pace. We present here a comprehensive workflow in the Oncotopix® Discovery platform designed specifically for highplex IMC image analysis, covering tissue segmentation, cell segmentation based on IMC DNA images, cellular phenotyping, and spatial analyses.

Fabian Schneider¹, Brenna O’Neill¹, Rasmus A. Lyngby¹, Rasmus N. Sørensen¹, Andreas Hussing¹, Alessandro S. Massaro¹, Andrew Quong², Smriti Kala², Sam Lim², Clinton Hupple², Nina Lane², Michelle Macpherson², Jeppe Thagaard¹, Johan Doré Hansen¹, James Mansfield¹

1. Visiopharm, Horsholm, Denmark
2. Standard BioTools, South San Francisco, CA, USA