Osteocyte Apoptosis Attracts Myeloma Cells to Bone and Supports Progression through Regulation of the Bone Marrow Microenvironment

Multiple Myeloma (MM) pathogenesis and progression is intricately tied to the bone marrow (BM) microenvironment, and understanding this microenvironment is of upmost importance. Osteocytes are the most abundant cell type in bone, comprising over 90% of the cellular population. These cells were long thought to be primarily inert mechanical sensors, however osteocyte regulation of the BM is increasingly appreciated. Still, very little is known about the role of osteocytes in the context of cancer. We initially evaluated MM patient BM biopsies and determined that osteocyte apoptosis is increased compared to MGUS and healthy BM. We further validated this finding in the SCID-hu mouse model - increased osteocyte death was seen in the primary human bone grafts that were injected with aggressive MM cells compared to those injected with less aggressive MM cells. Surprisingly, and more interestingly, increased osteocyte apoptosis/death was also found in the contralateral, un-injected human bone grafts from mice with aggressive MM. Importantly, these changes in the secondary bone grafts occurred prior to detection of MM cells. In the present study, we investigated if and how osteocyte apoptosis at distant bone sites, before MM metastasis, feeds back to MM cells to support dissemination and progression.

We developed a unique, syngeneic mouse model of MM in which osteocytes can be specifically ablated in vivo. Mice expressing the Diphtheria toxin receptor (DTR) in mature osteocytes (RIKEN BioResource Center, Japan) were crossed with C57Bl/KaLwRij mice, a strain that when injected with syngeneic 5TGM1 MM cells develops many characteristics of human MM. The resulting 8-week old progeny expressing the DTR gene and on the C57Bl/KaLwRij background were intraperitoneally (i.p.) injected with Diphtheria toxin (DT,12.5 μg/kg) or PBS as control (n=7 per group). After 7 days, mice were injected intravenously (i.v.) with 2x10⁶ 5TGM1 MM cells expressing firefly Luciferase. Weekly bioluminescent imaging and ELISA for IgG2bκ (secreted by 5TGM1 cells) tracked tumor location and growth over time. Remarkably, compared to control mice (DTR+PBS), osteocyte ablated (DTR+DT) mice showed significantly enhanced tumor homing to bone by bioluminescent imaging and significantly increased total tumor burden by both bioluminescent imaging and sera IgG2bκ ELISA.

To understand mechanistically how osteocyte apoptosis enhances MM-bone homing and tumor progression, we evaluated BM microenvironmental changes after osteocyte apoptosis was induced. Syngeneic DTR mice were injected at 8 weeks with DT or PBS (n=3-4 per group). Mouse legs were collected after 7 days: one leg for sectioning and one leg for isolating BM cells and BM extracellular fluid. Flow cytometry was performed on BM cells and BM extracellular fluid was evaluated using a cytokine/chemokine array. Leg sections were stained via immunohistochemistry (IHC). Cytokine arrays of BM extracellular fluid revealed substantial increases of multiple cytokines/chemokines important for MM dissemination and progression (i.e. IL-6, HGF) in DTR+DT mice. Additionally, bone destruction-associated molecules DKK1, RANKL, and M-CSF were also increased in DTR + DT mice compared to DTR+PBS control mice. IHC showed increased numbers of osteocytes positive for sclerostin, HIF-1α and RANKL. Finally, flow cytometric analysis suggested heightened immune suppression in the BM as multiple immune-suppressive cell populations were enhanced in DTR+DT mice compared to DTR+PBS mice. These include myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and a regulatory B cell subset (Breg).

In conclusion, we have successfully generated a syngeneic model of MM in which osteocytes are selectively ablated in vivo after a single injection of DT. Using this model, our data demonstrate that osteocyte apoptosis supports MM homing and growth in bone, likely as a result of an altered BM microenvironment. Interestingly, these changes include enhanced MM supportive/attractive soluble molecules, support of bone destruction, and enhanced immune suppression. Combined, our data suggests that osteocyte apoptosis is important for developing a pre-metastatic niche and contributes to MM progression in bone. Therefore, preventing osteocyte apoptosis may help stall and prevent dissemination of MM, and possibly other cancers, to bone.