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    Myeloma Genomics and Microenvironment and immune profiling

    Category: Myeloma Genomics and Microenvironment and immune profiling

    Single-Nucleus Profiling Reveals Aberrant Osteoclast Differentiation in MM

    (PA-210) Single-Nucleus Profiling Reveals Aberrant Osteoclast Differentiation in MM

    Friday, September 19, 2025
    Introduction: Osteolytic bone lesions are a defining feature of multiple myeloma (MM), affecting up to 80% of patients and leading to chronic pain, pathological fractures, and neurological impairment. These lesions result from an imbalance between bone forming osteoblasts (OBs) and bone resorbing osteoclasts (OCs), driven by MM-induced changes in the bone marrow microenvironment. Current therapies, such as bisphosphonates and RANKL inhibitors, fail to promote bone repair, lead to a low bone turnover state, and carry side effects. A key gap is our limited understanding of how MM alters OC differentiation.

    Methods:

    To model MM bone disease, murine Vk*MYC MM cells were intrafemorally transplanted into C57BL/6 mouse femurs. MM development and bone lesions were confirmed via serum protein electrophoresis and µ-CT imaging. The number and spatial distribution of OCs were assessed using tartrate-resistant acid phosphatase (TRAP) staining in FFPE femur sections. In parallel, an in vitro model was developed and validated to differentiate OCs from bone marrow mononuclear cells (BMMNCs). BMMNCs were cultured with M-CSF and RANKL for five days, promoting the expansion and fusion of OC precursors (OCPs) into mature OCs, while MM cells were present in the supernatant of the culture. Single-nucleus RNA sequencing (snRNA-seq) was optimized and performed at two key time points: day two (OCP stage) and day five (OC stage) cells differentiated from Vk*MYC and PBS-injected (control) mice, using 10X Genomics Chromium 3’ platform to identify transcriptional signatures.

    Results:

    TRAP and H&E staining of Vk*MYC bone sections confirmed aberrant osteoclast distribution, cortical and trabecular bone loss, and extensive MM infiltration. Unlike single-cell RNA-seq, which excludes large multinucleated OCs due to size limitations during droplet encapsulation, snRNA-seq enabled capture of all OCs. No differences were detected in the composition of cell types between Vk*MYC and control samples. Our data analysis indicated that at day two (OCP population), the culture system was primarily composed of macrophage subtypes, along with the presence of immune cells such as B cells and dendritic cells. By day five, distinct macrophage-like pre-OCs and mature OCs had emerged. Differential gene expression analysis revealed that macrophage clusters exhibited increased expression of genes linked to phagocytosis, adhesion, and mononuclear cell differentiation, suggesting accelerated OC differentiation in the presence of MM cells. Furthermore, the Vk*MYC OC cluster exhibited a distinct immune-inflammatory transcriptional signature along with upregulation of ribosome biogenesis pathways, consistent with increased functional activation.

    Conclusions: In summary, we observed similar cell populations within the OC culture system in both Vk*MYC and control samples; however, OCPs and OCs displayed altered gene expression profiles, suggesting accelerated differentiation, increased activity, and an inflammatory signature.