OMIP‐120: A 22‐Marker Spectral Flow Cytometry Panel for the Characterization of Major Immune Populations in Murine Bone Marrow and Osteosarcoma Tissue

ABSTRACT Tumors that develop in the bone exhibit poor responses to most treatment options, including advanced immunotherapies. Despite its immune‐rich composition, the bone marrow microenvironment often fails to suppress tumor progression and instead promotes tumor growth and immune evasion, although the underlying mechanisms remain poorly understood. To investigate tumor‐induced remodeling of the bone marrow immune environment and characterize tumor‐infiltrating immune populations, we developed a high‐dimensional 22‐marker spectral flow cytometry panel for use in preclinical models of bone and bone marrow cancers. The panel enables profiling of major immune populations including CD4 + T and CD8 + T cells, B cells, Natural Killer cells, dendritic cells, monocytes, macrophages and neutrophils, and incorporates activation, exhaustion and immune checkpoint markers. Panel optimization was performed using spleen, freshly isolated bone marrow and tibial osteosarcoma tissue from adult BALB/c mice. While optimized for murine osteosarcoma and bone marrow tissue, this panel provides a robust and adaptable platform for high‐dimensional immune profiling across solid tumors and hematological malignancies.