Unlocking the Tumor Microenvironment: Innovations in Multiplex Immunohistochemistry

The immune control of cancer growth is an area of active investigation. In this study, we demonstrated the feasibility of using standard immunohistochemistry methods in conjunction with a set of newly developed chromogens to demonstrate immune cell markers in a multiplex staining system. Immune infiltrating cells in breast cancer were identified using antibodies to CD20 (B-cells), CD3 (T-cells), and CD163 (macrophages). Additionally, the tumor compartment was identified using cytokeratin (AE1/AE3), and Ki67 was used to determine the proliferation index. These stains showed a significant immune cell infiltrate surrounding and within the tumors. B-cells, T-cells, and macrophages were abundant at the tumor periphery, particularly in areas where tertiary lymphoid structures were also present. In contrast, B-cells were significantly reduced within the tumor interior compared to an abundant infiltrate of T-cells and macrophages. Patterns of B-cell, T-cell, and macrophage infiltration were identified. Depending upon the particular set of markers chosen for analysis, a simple visual examination, without the aid of computer-assisted imaging systems, was sufficient to differentiate up to five different markers.