Imaging and Analyzing the Macrophages that Regulate Pathological Hematopoietic Clones in the Mouse Calvarial Bone Marrow

The bone marrow microenvironment has been shown to regulate the progression of myeloid neoplasms. Given that bone remodeling is a dynamic process that releases bone particles as danger molecules into the bone marrow space, different stages of bone turnover across marrow cavities can impose spatial heterogeneity of inflammatory responses that impact both healthy and mutant hematopoietic stem cells (HSCs). Specifically, macrophages, serving as critical members in the HSC niche, can adopt either pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes based on these local signals, which results in distinct surface receptor expression and cytokine secretion. Using intravital microscopy, a murine model of Tet2 deficiency (a prevalent mutation across the spectrum of myeloid disorders), and labeling strategies to track marrow macrophages, our recent work identified compartmentalized inflammatory landscapes in the bone marrow that differentially support expansion of mutant cells. In this chapter, we describe detailed protocols to track pathological clones with respect to marrow macrophage in live mouse calvaria, and protocols of image-guided bone button isolation to analyze pro-inflammatory cytokines from the marrow cavities of interest. These protocols can also be integrated with cell isolation for single-cell transcriptomic profiling or downstream functional assessments. The labeling strategies may be applied to image macrophages in other tissue types.