Suppression of Mesenchymal Stromal Cell (MSC) Differentiation in Multiple Myeloma (MM) Is Restricted to the Osteoblast Lineage
Authors: Esteve Flavi a , Hong Chang Sook , Huston Alissa , Palacios Veronica Garcia , Anderson Judy , Patrene Ken , Roodman David 
Published: April 2020

Abstract
Lytic bone lesions in patients with MM rarely heal even when patients are in complete remission for long periods of time. The mechanism behind this prolonged suppression of osteoblast activity is yet to be elucidated, and when or whether the osteoblast suppression can be reversed is unknown. Several possibilities could explain osteoblast suppression in MM:
quantitative MSCs depletion induced by MM cells; a permanent block in MSC differentiation to the osteoblast lineage; and/or an inability of MSCs to differentiate in a hostile microenvironment.
To distinguish among these possibilities, we developed a murine model of MM bone disease that permits us to assess the time course for development of osteoblast inhibition of MSC differentiation of osteoblasts, if this inhibition is reversible anytime during the process, and if the differentiation is restricted to permanent blockade of osteoblast differentiation.
We generated a murine MM cell line genetically modified to contain the TK gene, which allows ablation of the cells by ganciclovir while sparing hematopoietic and stromal cell progenitors. MM cells also contained GFP for tumor estimation and a blasticidin resistance cassette for selection of transfected cells. Hematopoietic precursors and MSCs were cocultured with the MM cells in media containing ganciclovir to assess for toxic effects of this drug on cell differentiation (bystander effect).
Murine MM cells or saline was also injected into the right tibia of SCID mice on day 0 and tumor lesions were documented by weekly imaging of right tibias by micro-QCT and by measurement of IgG2b in serum. Mice were treated with intraperitoneal ganciclovir or saline for 14 days starting at different time points (group 1 = 1d; group 2 = 8d; or group 3 = 14d) after tumor injection, and were sacrificed at week 5. MSCs were recovered from right tibias, cultured in osteogenic media, and alkaline phosphatase levels determined after 10 days of culture to assess osteoblast activity. MSCs were also cultured in adipogenic media, and the presence of mature adipocytes was visualized by Oil Red O staining.
There was no toxic effect of ganciclovir on hematopoietic colony formation or osteoblast differentiation. Lytic bone lesions were documented in mice injected with MM cells by micro-QCT at 4 weeks in groups 2 and 3 and progressed thereafter, but not in group 1 after intratibial injection of MM cells. MSCs from group 1 mice showed greater osteoblast activity when sacrificed at 5 weeks compared to other groups. Mice in group 1 surviving 5 weeks eventually developed MM bone disease and succumbed to it at a much later time than the other groups (p&lt;.01). Significant elevations of serum IgG2b levels were detectable at week 4 in mice from groups 2 and 3 and correlated with the development of bone lytic lesions. Harvesting cells from tumor bearing tibias yielded similar numbers of MSCs in all groups. Comparable levels of adipocytic differentiation by Oil Red O staining were observed among MSC from all groups of mice.
These results demonstrate that MSC depletion cannot explain the absent osteoblast activity in this model of MM. MSC differentiation appears to be selectively blocked from the osteoblast lineage. Suppression of osteoblast activity required &gt;24hr exposure to MM cells in vivo and correlated with relative tumor burden. Studies are underway to determine if osteoblast suppression is permanent or can be reversed in this model. This model of MM bone disease should permit the further elucidation of the mechanisms responsible for osteoblast suppression in MM, and testing of anabolic agents in a model that does not require treatment of mice with agents to eradicate MM cells and that are toxic to the marrow microenvironment.