Abstract

Multiple myeloma (MM) bone disease is characterized by increased osteoclast-mediated resorption of bone adjacent to tumor growth. Fusion and activation of osteoclasts (OC), indicated by elevated tartrate-resistant acid phosphatase (TRAP) production, are induced by a myriad of inflammatory factors; e.g. TGF-β, growth factor independent 1 transcription factor (GFI-1), RANKL, and IL-6 that are produced by myeloma cells and cells in the marrow microenvironment. Current therapies reduce tumor burden and block osteoclast formation and activity, thereby decreasing bone destruction. However, MM bones rarely heal. While the anabolic effects of exercise on healthy bone are well-documented, patients with compromised skeletal tissue, such as in MM, may not be able to participate in regimented exercise for fear of developing a fracture. Low intensity vibrations (LIV) that deliver subtle mechanical signals, on the order of those induced by fast-twitch muscle fibers on bone, can enhance mesenchymal stem cell differentiation towards osteoblasts. LIV also significantly preserves trabecular bone while reducing tumor burden in femora of mice harboring U266 MM cells. We hypothesize that LIV is both anti-resorptive and anabolic, through differential effects on MM cells and bone cells. To test this hypothesis, we used a vertically oscillating, vibratory platform that delivered mechanical signals (<1 x Earth's gravitational acceleration; where 1g=9.8m/s2) to culture plates.

5TGM1 murine MM cells (5x105 cells/well) were cultured in RPMI with 10% FCS and subjected to LIV or Sham-LIV. Each 6-well plate received either LIV (frequency=90Hertz, acceleration=0.3g) or Sham-LIV (SH; unpowered, static platform) twice per day for 20min per treatment for 48hrs. MM cell lysates were then collected using RIPA buffer. IL-6, GFI-1, and RANKL levels were determined via Western Blot using specific monoclonal antibodies. GAPDH was used as the loading control. Conditioned media (CM) were isolated from SH- and LIV-treated MM cultures. Purified primary murine OC precursors (non-adherent marrow mononuclear cells) were expanded with 10ng/mL of MCSF for 3 days and then these cells were isolated by trypsinization and replated in 12-well plates at a density of 5x105 cells/well in α-MEM, 10% FCS, and 30% v/v CM. All cultures were then incubated for 36hrs. OC number and morphology were confirmed via TRAP staining (counterstained with hematoxylin) of the cultures. TRAP+ OC's containing at least three nuclei were quantified in the cultures by microscopy.

Expression levels of IL-6 and GFI-1 were 53% (p<0.01) and 50% (p<0.02) lower, respectively, in lysates from LIV-exposed 5TGM1 cells as compared to those that were SH-treated. Expression of RANKL was not significantly different between these groups. TRAP+ OC's were 61% (p<0.005) lower in LIV-treated cultures compared with SH cultures. The morphology of OC's exposed to LIV-conditioned media from MM cultures was distinct from control cultures, with fewer filopodia than OC's in cultures treated with SH-conditioned media. Based on these data we then examined the direct effect of LIV-treatment on committed OC-precursors treated with RANKL (50ng/mL) to induce formation of mature OC's. After 48hrs of LIV-treatment, committed OC-precursors had 36% (p<0.0001) fewer nuclei per OC than in SH-treated cultures. The cultures of committed OC-precursors directly treated with LIV had fewer filopodia in comparison to SH-treated cultures upon examination of OC morphology.

These findings suggest that mechanical signals in the form of LIV may influence the secretion of IL-6 and GFI-1 by myeloma cells, which, in turn, can decrease the capacity of MM cells to induce OC formation. The morphology of OC's and expression of TRAP by OC's exposed to LIV further suggest a distinct mechanosensitive effect on osteoclast formation and fusion that may impact their bone resorbing capacity. Thus, LIV has a significant effect on the capacity of MM cells to secrete inflammatory cytokines as well as directly affect OC formation. These results further suggest that low intensity vibrations may be a useful, non-invasive approach for treating myeloma bone disease.

Disclosures

Roodman: Amgen Denosumab Trial: Membership on an entity's Board of Directors or advisory committees.

Author notes

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Asterisk with author names denotes non-ASH members.