Characterization of Bone Marrow-Derived Immune Compartment in Murine Brain Metastases by Spectral Flow Cytometry

Metastatic brain cancer is the most common intracranial cancer among adults, often leading to poor survival outcomes. The incidence of brain metastases is on the rise, likely associated with advanced systemic therapeutics which improve patient survival, providing sufficient time for micrometastases to develop into fully established metastases. Therefore, brain metastasis remains an unmet clinical need. The brain metastatic tumor microenvironment is a complex ecosystem composed of numerous brain, stromal, and immune cells with unique adaptations that represent a formidable challenge for treatment. Importantly, brain metastases are enriched in immune cells, especially myeloid resident and recruited cells considered highly plastic. Thus, it is critical to assess the immune landscape and functional phenotypes longitudinally and across treatment conditions in brain metastasis in a non-biased, comprehensive manner. Here, we outline a protocol to assess immune cell populations in murine brain metastasis samples, using a 23-color panel for spectral flow cytometry.