The evaluation of breast cancer immune microenvironment has been increasingly used in clinical practice, either by counting tumor infiltrating lymphocytes or assessing programmed death ligand 1 expression. However, the spatiotemporal organization of anti-breast cancer immune response has yet to be fully explored. Multiplex in situ methods with spectral imaging have emerged to deconvolute the different elements of tumor immune microenvironment. In this narrative review, we provide an overview of the impact that those methods have, to characterize spatiotemporal heterogeneity of breast cancer microenvironment at neoadjuvant, adjuvant and metastatic setting. Multiplexing in situ can then be useful for new classifications of tumor microenvironment and discovery of immune-related biomarkers within their spatial niche.

The tumor immune microenvironment (TIME) is an important player in breast cancer pathophysiology. Surrogates for antitumor immune response have been explored as predictive biomarkers to immunotherapy, though with several limitations. Immunohistochemistry for programmed death ligand 1 suffers from analytical problems, immune signatures are devoid of spatial information and histopathological evaluation of tumor infiltrating lymphocytes exhibits interobserver variability. Towards improved understanding of the complex interactions in TIME, several emerging multiplex in situ methods are being developed and gaining much attention for protein detection. They enable the simultaneous evaluation of multiple targets in situ, detection of cell densities/subpopulations as well as estimations of functional states of immune infiltrate. Furthermore, they can characterize spatial organization of TIME—by cell-to-cell interaction analyses and the evaluation of distribution within different regions of interest and tissue compartments—while digital imaging and image analysis software allow for reproducibility of the various assays. In this review, we aim to provide an overview of the different multiplex in situ methods used in cancer research with special focus on breast cancer TIME at the neoadjuvant, adjuvant and metastatic setting. Spatial heterogeneity of TIME and importance of longitudinal evaluation of TIME changes under the pressure of therapy and metastatic progression are also addressed.