Background Imaging Mass Cytometry™ (IMC™) is the method of choice for single-step staining and high-plex imaging of tissues while avoiding the complications of autofluorescence and cyclic imaging. IMC has three new imaging modes: Preview Mode (PM), Cell Mode (CM) and Tissue Mode (TM). PM rapidly scans a stained tissue to provide a comprehensive overview, mapping out the distribution of over 40 markers and revealing tissue heterogeneity. This enables researchers to make informed decisions about which areas warrant closer examination. Following PM, regions of interest (ROIs) are selected for high-resolution imaging. This is a critical step that is informed by biomarker expression using automated AI algorithms. CM offers high-resolution imaging for detailed analysis of the ROIs identified during PM, all using the same slide. TM provides fast acquisition of the entire tissue at 5-micron resolution, optimal for quantitative pixel-based analysis. These modes support automated, continuous imaging of more than 40 large tissue samples (400 mm²) weekly.

Methods Tissue sections of colon adenocarcinoma were stained with a 30-marker IMC panel of structural, tumor, stromal, immune cell and immune activation markers. Images were acquired on the Hyperion XTi™ Imaging System (Standard BioTools™), first in PM and then in CM with automatic selection of ROIs using Phenoplex™ software (Visiopharm®). ROIs were automatically selected based on two criteria: 1) actively proliferating and non-proliferating tumor regions; 2) cold and hot tumor regions as identified by immune hotspots within stromal or epithelial tumor regions. An adjacent serial section was acquired in TM.

Single-cell analysis of the images obtained in CM was performed using Phenoplex. Tissue segmentation divided the tissue into tumor epithelial and stromal regions; cell segmentation was based on Iridium DNA channels; and phenotyping was performed using the Guided Workflow. This data was used to compare the immune contexture and spatial distributions via interactive t-SNE plots.

Results A high degree of immune infiltration was observed in the tumor, with significant levels of infiltrating myeloid cells. Hotspots of tumor-associated neutrophils expressing granzyme B were found, implicating their role in the recruitment and activation of intratumor CD4+ and cytotoxic CD8+ T cells.

Conclusions This work demonstrates that Hyperion™ XTi can greatly accelerate the ability of IMC users to gain useful insights from complex biological samples. Phenoplex enables a comprehensive workflow for the analysis of this data, providing automated ROI selection, phenotyping, and spatial analyses of high-resolution IMC images for biological assessment.