In Vivo postnatal loss of mature osteoblasts creates an immunosuppressive bone marrow microenvironment to permit the engraftment and proliferation of myeloma cells Free

Osteolineage cells are versatile regulators of hematopoietic cells, including providing niches for quiescence and lineage commitment of hematopoietic progenitors. Myeloma cells suppress the numbers and activity of bone-forming osteoblasts, at least in part, through the secretion of DKK-1. Conversely, increased osteoclast activity leads to the clinical sequalae of osteolytic myeloma bone disease. Tumor cells actively engineer their tumor microenvironment (TME) to support their survival, proliferation, migration, and drug resistance. MM originates in the bone marrow (BM), and many cellular and extracellular matrix components of BM TME contribute to myeloma disease progression. Our prior work showed that inhibiting Activin A signaling in immunodeficient mice injected with human MM cells boosted osteoblast counts and suppressed MM growth, suggesting that osteoblasts control MM progression. In vitro co-culture studies showed that whereas adipocytic cell-line OP9 promotes the proliferation of 5TGM1 MM cells, MC3T3 osteoblasts inhibit it. Here, we hypothesize that the loss of osteoblasts promotes myeloma disease progression due to the loss of the osteoblastic niche for myeloma cells. Our long-term goal is to determine the role of osteoblasts in the survival, proliferation, migration, and drug resistance of myeloma cells.

Unlike the other immunodeficient mouse models of MM, we generated an immunocompetent mouse model for the inducible and reversible postnatal deletion of mature osteoblasts. We expressed diphtheria toxin receptor (DTR) specifically in mature osteoblasts by mating DTR floxed (DTRfloxed) mice with mice expressing Cre-recombinase driven by the osteocalcin-promoter (OC-Cre) to generate OC-Cre/iDTR mice. Littermates lacking Cre and heterozygous for DTR (iDTR) were used as controls. Before Diphtheria Toxin (DT) treatment, OC-Cre/iDTR mice were phenotypically normal compared to controls, showing no differences in immune cell populations by flow cytometry or in skeletal architecture by micro-computed CT (micro-CT). To facilitate non-invasive bioluminescence imaging (BLI) of MM-burden, 5TGM1 murine MM cells were engineered to express luciferase and dTomato (5TGM1-Luc-Tom). We initiated osteoblast deletion by i.p. 50 µg/kg DT injections in 8-week-old OC-Cre/iDTR and control mice, the age for skeletal maturity in mice. Micro-CT analysis showed a significant increase in cortical porosity within one week. After 8 weeks, DT treatment markedly reduced trabecular bone fraction (BV/TV), trabecular number (Tb.N), and bone mineral density (BMD), while significantly increasing trabecular spacing (Tb.Sp). Immunohistochemistry for osteocalcin confirmed a rapid loss of mature and endosteal osteoblasts (N.Ob/T.Ar). This correlated with a sharp drop in serum sclerostin and osteocalcin, indicating reduced osteocytes and mature osteoblasts, respectively. Importantly, osteoclast numbers (N.Oc/T.Ar) and serum CTX levels were unchanged. To study MM progression, 1×10⁶ 5TGM1-Luc-Tom cells were injected into the left tibia 1 week after the start of DT and followed for up to 8 weeks post-injection. Engraftment and proliferation of MM cells in the tibia and migration to distant tissues were assessed by BLI at 1-, 4-, and 8-week post-injection. Flow cytometry revealed a 4-fold increase in 5TGM1-Luc cells in the BM of OC-Cre/iDTR mice versus controls. At 4 weeks, bioluminescence imaging (BLI) showed a 600-fold greater tumor burden in OC-Cre/iDTR mice. Notably, by 8 weeks, BLI detected MM cell dissemination to other long bones only in OC-Cre/iDTR mice. Flow cytometric analyses of the BM TME from the 5TGM1-Luc-Tom injected tibia showed a significant increase in immunosuppressive populations like MDSCs and T-regs.

These findings in immunocompetent mice show that MM cells engraft and proliferate rapidly in the BM TME in the absence of mature osteoblasts and tend to migrate to other bones over time, at least in part, due to the immunosuppressive microenvironment generated following the osteoblast depletion. Restoring the mature osteoblast niche or normalizing the immunosuppressive TME could offer a novel therapeutic path to reduce tumor burden and achieve long-term control. This immunocompetent mouse model that allows rapid proliferation of myeloma cells could also serve as a key tool for understanding the MM osteoblastic niche.