Altered osteoblast populations dynamics underlie myeloma progression from precursors diseases in bone microenvironment

Abstract Uncoupled bone-remodelling process characterizes the development of multiple myeloma (MM)-related bone disease, where impaired osteoblastogenesis promotes osteolytic lesions and creates a permissive bone microenvironment (BME) facilitating malignant plasma cell grow. Early functional and transcriptional remodelling of mesenchymal and osteoblastic cells (OBs) occurs across the progression from precursor disease as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to MM, but mechanisms remain incompletely understood. This study aimed to provide the first single-cell transcriptomic characterization of OB lineage cells from bone biopsies across disease stages to uncover BME alterations and define the progressive remodelling of the osteogenic niche across MM progression. Sixteen bone biopsies of MGUS, SMM, and newly diagnosed MM patients were depleted of CD235a+, CD45+, CD31+, and CD138+ cells to enrich the rare BME non-hemopoietic cells that were analysed by scRNAseq. Data were generated on Chromium 10X Genomics. Scanpy version 1.10.4 package in Python 3.12.2 was used to identify differentially expressed markers across cell types and clinical conditions. Pathway and biological process (BPs) inference was conducted via Over representation Analyses (ORA) and GSVA. Our database of 55000 cells comprehends the transcriptomic profiling of the non-hematopoietic bone cells and patient-matched immune/MM tumor cells and revealed several distinct mesenchymal lineage clusters. We identified 9,565 cells as 3 distinct OB lineage cell types defined by classical OB marker expression. In particular, 2 pre-OB clusters with distinct functional characteristics were identified: the first characterized by enhanced osteoblastogenesis activity and bone mineralization programs and elevated expression of osteoblastogenesis-associated genes, including WISP2, hereafter referred to as “pre-OBs WISP2+”. The second pre-osteoblastic cluster, “pre-OBs” is characterized by OB dysfunction-associated genes (such as CLU, SCRG1, and S100A4), and reduced regulation of bone growth and osteoblast differentiation biological processes, along with a more immunosuppressive phenotype. The third cluster, designated “OBs”, expressed mature osteoblast-associated genes, including PDPN, CHI3L1, CTSK, MMP14, with enriched BPs that not only regulate bone development and osteoclastogenesis but also actively contribute to the production of cytokines and mediators of the immune response. Disease progression dramatically altered OB dynamics: pre-OBs WISP2+ cluster was significantly progressively depleted (18.1% MGUS→10.1% SMM→ 4.8% MM), while dysfunctional pre-OBs were expanded (4.2% MGUS→10.7% SMM→11.7% MM). Intriguingly, the pre-OBs WISP2+ cell proportion strongly inversely correlated with tumor burden (r: -0.74; p:0.0041). Notably, beyond their numerical reduction, in MM compared to SMM/MGUS, pre-OBs WISP2+ exhibited an upregulated senescence-associated secretory phenotype (SASP) concomitant with decreased WISP2 expression, reflecting compromised osteogenic differentiation potential. Moreover, MM expanded pre-OBs showed transcriptional upregulation of osteoblast-inhibitory genes that impair cell differentiation, self-renewal, and mineralization function, as well as upregulation of SUSD2, which mediates galectin-1-dependent immunosuppressive, angiogenic, and hypoxic signalling. Furthermore, functional analyses revealed enhanced enrichment of glutamine and glutamate metabolic processes in SMM and MM pre-OBs, suggesting early metabolic reprogramming accordingly to our previous evidence. In MM, OBs cluster, instead, exhibited reduced expression of extracellular matrix deposition genes, alongside a significant enrichment of endothelial development pathways. In conclusion, for the first time, our single cell analysis on bone biopsies of patients with monoclonal gammopathies deciphers the OB populations complexity, overcoming the incomplete view derived from in vitro/bulk approach, highlighting their altered dynamics through the progression from precursors diseases to MM. Moreover, progressive depletion of the functional, with more bone-mineralization features, pre-OBs WISP2+ cluster strongly correlates with disease burden, while expansion of dysfunctional pre-OBs may be involved in the immunosuppressive, pro-tumoral BME of MM patients.