Intravital Imaging of Early AML Expansion in the Mouse Calvarial Bone Marrow

Normal hematopoiesis and leukemia growth are profoundly influenced by the bone marrow microenvironment. Given that bone formation precedes hematopoiesis, and the endosteal surface is constantly undergoing remodeling, different stages of bone turnover can impose heterogeneity on the bone marrow microenvironment that can impact both healthy and malignant hematopoiesis. To visualize bone remodeling in vivo, we have developed an intravital labeling technique and identified distinct bone marrow cavities based on the features of local bone deposition and resorption. By combining intravital tracking of a HoxA9-Meis1 acute myeloid leukemia (AML) mouse model with these labeling strategies, we observed that HoaxA9-Meis1 leukemic cells expanded almost exclusively in a subset of cavities that underwent active bone remodeling. In this chapter, we describe step-by-step protocols to track early proliferation of AML blasts with respect to local bone remodeling in live mouse calvaria. We summarize labeling strategies and image parameters to allow simultaneous visualization of leukemic cell expansion, vascular organization, and bone remodeling status, as well as give examples of image analysis and quantification workflows. The protocols can be readily applied to study hematopoietic disorders that manifest as clonal expansion in the bone marrow. The findings from intravital imaging can also be integrated with spatially resolved tissue isolation and next-generation sequencing techniques to better understand the regulatory machineries supporting AML progression.