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

Background: Analyses of immune cell subsets and immune indices are becoming increasingly important. By interacting molecular probe and immunohistochemistry (IHC) analysis with laser ablation, imaging mass cytometry (IMC) offers high-dimensional in situ measurements in tissue slides with a spatial resolution of 1 mu m. One of the most significant challenges is to implement stringent quality control strategies during the application of IMC to facilitate reproducible data analysis. We have developed a comprehensive protocol for clinical and experimental use by comparing IMC to standard clinical immunohistochemical approaches. Methods: We discussed the multi-step experimental processing procedures of IMC, including specimen preparation, panel design, antibody selection, lanthanide metal labelling, and data pre-processing workflows by IHC and fluorescent multiplex immunohistochemistry (mIHC) analysis. Based on IMC, we developed a standard operation and a well-established agreement for 29 breast cancer (BC) patients to identify a structured tumor immune microenvironment. Results: Metal labelling has no effect on the specificity of the antibodies' target (p > 0.05). The 3 mu m-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). A detailed analysis of human breast cancer tissues revealed a wide range of differences in the composition and frequency of immune cells within the tumor microenvironment. Conclusions: This study presents optimized proposals on procedures for IMC analysis and a standardized quality control strategy.