Mature Osteoblasts Promote Multiple Myeloma Survival through Cell-Cell Contact and Immune Modulation Mechanisms
Authors: Fulzele Keertik , Panaroni Cristina , Mukaihara Kenta , Mori Tomoaki , Chattopadhyay Shrikanta , Raje Noopur S. 
Published: November 2019

The bone marrow (BM) microenvironment (TME) is a complex mix of cellular and non-cellular components that has a profound effect on hematopoiesis and hematological malignancies. Multiple myeloma (MM), a tumor of plasma cells that originates in the BM is highly influenced by TME components including stromal cells, osteoclasts, and immune cells. Among these cells, osteoblasts are the most versatile regulators of hematopoiesis and also play an integral role in malignant transformations of leukemia and lung cancer. MM is characterized by osteolytic bone lesions partly due to decreased numbers of osteoblasts. Therapies such as denosumab and zoledronic acid aim to restore bone health by inhibiting osteoclastogenesis. However, the contribution of osteoblasts to the initiation and progression of MM is not well understood. We previously demonstrated that increased osteoblast numbers using Activin A inhibition in an in vivo humanized myeloma model resulted in inhibition of MM growth (Vallet et al., 2010). Given that osteoblast numbers are on the decline in a typical MM patient, we hypothesized that the loss of osteoblasts contributes to the initiation and progression of MM.
To assess the effects of osteoblasts on MM cells, MC3T3 murine pre-osteoblasts were differentiated into mature osteoblasts by supplementing media with ascorbic acid and β-glycerophosphate. Co-culture of 5TGM1 murine MM cells with the osteoblasts showed a significant decrease in the proliferation of MM cells by 40% (N=4). Osteoblasts go through distinct stages of maturation i.e., pre-osteoblasts, committed osteoblasts, and mature osteoblasts. These individual osteoblast populations were FACS sorted from mice to isolate pre-osteoblasts from the long bones of Osterix-GFP+ (Osx+) mice, committed osteoblasts from Collagen 2.3-GFP+ (Col2.3+) mice, and mature osteoblasts from Osteocalcin-YFP+ (OCN+) mice. Each of the osteoblast populations was co-cultured with 5TGM1 MM cells. Although all the osteoblast populationssignificantly suppressed MM proliferation, the OCN+ mature osteoblasts suppressed MM proliferation the most (N=3). Therefore, we focused our studies on the role of mature osteoblasts in MM progression.
We generated mice in which mature osteoblasts could be postnatally deleted in an inducible and reversible manner. To achieve this, mice carrying floxed diphtheria toxin receptor (DTR) alleles were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter (OC-Cre) to generate OC-Cre/iDTR mice. The control mice were littermates lacking the OC-Cre allele. The OC-Cre/iDTR mice were indistinguishable from the controls until treated with diphtheria toxin (DT). To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treatedwith 50 µg/Kg DT once a week beginning at 8-weeks of age. To study MM engraftment and progression, 3x1065TGM1-Luciferase MM cells were injected into the tibia of OC-Cre/iDTR and control mice followed by weeklyinjection of DT for 8-weeks. Bioluminescence imaging (BLI) showed that 4-weeks onwards the OC-iDTR mice, but not the control mice, continued to express and increase the BLI signal (N=6). This data suggeststhatunder physiological conditions, mature osteoblasts activelysuppress MM engraftment and progression.
We hypothesized that osteoblasts provide niche support to MM cells via direct cell-to-cell contact. To begin to identify the molecular mechanisms, we compared gene expression changes in primary murine osteoblasts between the undifferentiated and 30-day osteogenic differentiation time points. We also examined the BM TME by quantitative protein antibody arrays at 2-weeks following 5TGM1MM intratibial injection into OC-Cre/iDTR and control mice. The mature osteoblasts showed a significant increase in the expression of integrins, including integrin α4, and several immunomodulatory markers. The cytokine array analysis showed altered expressions of cell-cell communication proteins MAdCAM1, BAFF-R, TACI, and immunomodulatory factors IL-33, IL-17F, and IL-13.
Taken together, using in vivo and in vitro models, we show that mature osteoblasts may have a negative regulatory impact on MM cells through cell-cell communication or immunomodulatory mechanisms. Expanding the osteoblast niche may provide novel therapeutic avenues to reduce disease burden and create an environment for long term tumor control.

Disclosures
Raje: Merck: Consultancy; Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Janssen: Consultancy.