P-195: Heterogeneity of Osteoclasts in Multiple Myeloma Revealed by Single Nucleus RNA Sequencing

Introduction: Osteolytic bone lesions are a hallmark of multiple myeloma (MM), affecting up to 80% of patients and leading to chronic pain, pathological fractures, and neurological impairment. These lesions result from excessive osteoclastic bone resorption coupled with suppressed osteoblastic bone formation. Osteoclasts (OCs) have demonstrated phenotypic and functional diversity in other bone-related diseases such as rheumatoid arthritis. However, comprehensive studies exploring the heterogeneity of OCs in MM are still lacking. Here, we utilized in vitro differentiated OCs from the immunocompetent Vk*MYC MM mouse model to investigate OC diversity.

Methods: To characterize MM bone disease, murine Vk*MYC MM cells were intrafemorally transplanted into the femur of C57BL/6 mice. MM development and bone lesions were confirmed via serum protein electrophoresis and µ-CT imaging. To study OC heterogeneity, an in vitro protocol for differentiating OCs from Vk*MYC bone marrow mononuclear cells (BMMNCs) was developed and validated. BMMNCs were cultured with M-CSF and RANKL for five days to expand and induce fusion of OC precursors (OCPs) to form mature OCs. MM cells were present in the supernatant throughout the entire duration of the OC differentiation culture. Single-nucleus RNA sequencing (snRNA-seq) using the 10x Genomics Chromium 3’ platform was performed on day five OCs from Vk*MYC and PBS-injected mice.

Results: Tartrate-resistant acid phosphatase (TRAP) staining of OCs in femurs revealed an increased number of OCs in Vk*MYC mice relative to PBS-injected mice. OC differentiation from Vk*MYC BMMNCs yielded larger and greater quantities of OCs, as observed under the microscope following TRAP staining, compared to those from PBS-injected mice. On day five of the OC culture system, snRNA-seq revealed high expression of OC marker genes in clusters 1, 5, and 13 in both Vk*MYC and control samples. Notably, genes associated with immune response and phagocytosis pathways were significantly more expressed in Vk*MYC cluster 13 compared to the corresponding control cluster. Additionally, Vk*MYC OC differentiation culture exhibited a higher abundance of M1 macrophages, with significantly increased IL-17 receptor expression compared to the control group.

Conclusions: Differential expression analysis of identified OC clusters within each sample showed no significant differences in OC marker genes, indicating that the observed diversity is not due to different stages of differentiation, as OCPs display markedly lower expression of these markers. Cluster 13 in the Vk*MYC sample may suggest the presence of highly activated OCs. M1 macrophages, involved in chronic inflammatory conditions, can differentiate into OCs, and research has shown that IL-17 promotes OCP differentiation. In summary, OCs are a heterogenous population, and a specific MM-related OC subgroup may exist within the bone marrow microenvironment of MM patients with bone lesions.