Assessing the Practical Application of Imaging Mass Cytometry for Visualizing Tumor Immune Microenvironment Heterogeneity

BACKGROUND Analyses of immune cell subsets and immune indices are gaining in importance. By combining molecular probe and immunohistochemical (IHC) analysis with laser ablation, the imaging mass cytometry (IMC) offers spatially high-dimensional in situ measurements in tissue slides with a cell resolution of 1µm. The most critical challenge is how to implement stringent quality control strategies during application of IMC to facilitate reproducible data analysis. METHODS We discussed the multi-step experimental processing procedures of IMC, including specimen preparation, panel design, antibody selection, lanthanide metal labeling and data pre-processing workflows by IHC and fluorescent multiplex immunohistochemistry (mIHC) analysis. Based on IMC, we developed a standard operation and well-established agreement for 29 BC patients to identify a structured tumor immune microenvironment. RESULTS Metal labeling does not affect the antibodies target specificity (p > 0.05). The 3 µm-thick formalin-fixed and paraffin-embedded (FFPE) and fresh-frozen slides exhibited the least blur (p < 0.01) and the lowest nonspecific adsorption of antibodies (p < 0.01). Detailed analysis of human BC tissues revealed a wide range of differences in the composition and frequency of immune cells within the tumor microenvironment. CONCLUSIONS This study offered optimized proposals on procedures for IMC analysis as well as a standard quality control strategy.