# Unlocking the Tumor Microenvironment: Innovations in Multiplex Immunohistochemistry

## TL;DR

This study introduces a new multiplex staining system using standard immunohistochemistry to visualize immune cells in breast cancer tumors.

## Contribution

A novel multiplex staining system using new chromogens enables simultaneous visualization of multiple immune cell markers in tumor tissue.

## Key findings

- B-cells, T-cells, and macrophages are abundant at the tumor periphery, especially near tertiary lymphoid structures.
- B-cells are significantly reduced in the tumor interior compared to T-cells and macrophages.
- Visual examination can distinguish up to five markers without computer-assisted imaging.

## Abstract

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.