‘Educated’ Osteoblasts Reduce Osteoclastogenesis in a Bone-Tumor Mimetic Microenvironment

SIMPLE SUMMARY: Patients with late-stage bone metastatic breast cancer experience skeletal related events, including osteolytic lesion formation, due to overactive osteoclast bone resorption. It is well-known that osteoclast function is altered by breast cancer cells in bone. Breast cancer cells stimulate osteoblasts to secrete factors that initiate osteoclast differentiation and activation. Our lab has previously identified a novel subpopulation of osteoblasts in the bone-tumor microenvironment called “educated” osteoblasts (EOs) that alter breast cancer cell proliferation. The aim of this study was to identify how osteoclasts are affected by EOs during metastatic breast cancer progression in bone. Our results demonstrated that pre-osteoclast interaction with EOs reduces osteoclast formation and bone resorption in a bone-tumor mimetic microenvironment. Furthermore, we identified that altered osteoclast formation can be modulated, in part, by tumor necrosis factor alpha (TNFα). Overall, our data demonstrate osteoclastogenesis is reduced by EO cells, suggesting EO cells have a protective effect in bone and exert an inhibitory effect on tumor progression. ABSTRACT: Breast cancer (BC) metastases to bone disrupt the balance between osteoblasts and osteoclasts, leading to excessive bone resorption. We identified a novel subpopulation of osteoblasts with tumor-inhibitory properties, called educated osteoblasts (EOs). Here we sought to examine the effect of EOs on osteoclastogenesis during tumor progression. We hypothesized that EOs affect osteoclast development in the bone-tumor niche, leading to suppressed pre-osteoclast fusion and bone resorption. Conditioned media (CM) was analyzed for protein expression of osteoclast factors receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα) via ELISA. EOs were co-cultured with pre-osteoclasts on a bone mimetic matrix to assess osteoclast resorption. Pre-osteoclasts were tri-cultured with EOs plus metastatic BC cells and assessed for tartrate-resistance acid phosphatase (TRAP)-positive, multinucleated (≥3 nuclei), mature osteoclasts. Tumor-bearing murine tibias were stained for TRAP to determine osteoclast number in-vivo. EO CM expressed reduced amounts of soluble TNFα and OPG compared to naïve osteoblast CM. Osteoclasts formed in the presence of EOs were smaller and less in number. Upon co-culture on a mimetic bone matrix, a 50% reduction in the number of TRAP-positive osteoclasts formed in the presence of EOs was observed. The tibia of mice inoculated with BC cells had less osteoclasts per bone surface in bones with increased numbers of EO cells. These data suggest EOs reduce osteoclastogenesis and bone resorption. The data imply EOs provide a protective effect against bone resorption in bone metastatic BC.