Intracranial Breast Cancer Brain Metastases Mouse Model for Immune and Stromal Cells Characterization in Meninges and Olfactory Bulb.

The central nervous system (CNS) is surrounded by specialized immune interfaces, including the meninges and the olfactory bulb, which can serve both as permissive routes for metastatic colonization and as entry points for anti-tumor immune responses. Understanding how immune cell dynamics evolve within these structures during metastatic progression is essential for identifying mechanisms that either support or restrict tumor growth in the brain. This protocol details the establishment of a breast cancer brain metastases (BCBM) mouse model through intracranial injection of E0771 breast cancer cells into C57BL/6 mice. After tumor development, the meninges and olfactory bulb are carefully dissected, enzymatically digested, and processed into single-cell suspensions. These populations are then analyzed using multiparametric spectral flow cytometry to comprehensively characterize lymphoid, myeloid, and stromal cell subsets within CNS-associated immune niches. This workflow enables comparative analyses between tumor-bearing and non-tumor controls, identifying alterations within CNS immune interfaces that may underlie tumor immune evasion and represent potential therapeutic targets. Overall, the protocol provides a reproducible approach to study BCBM and interrogate immune regulation at CNS-peripheral immune system interfaces. It can be adapted for additional cancer cell lines, immunotherapies, or genetic mouse models to investigate immune resistance mechanisms and therapeutic response within the brain microenvironment.