Abstract Breast cancer (BC) preferentially disseminates to bone. During BC progression in bone, BC cells communicate with osteoblasts (OBs), osteoclasts (OCs), and other stromal cells to shape the bone-tumor niche. Recently, our lab discovered two distinct subpopulations of OBs in the bone-tumor niche based on protein marker expression. One group ‘educated' osteoblasts (EOs) have engaged in crosstalk with disseminated BC cells, whereas another group, ‘uneducated' osteoblasts (UOBs) have not. We have new evidence that EOs suppress osteoclastogenesis and reduce bone resorption associated with BC cell metastatic progression in bone. Culture supernatant of EOs and OBs was analyzed for soluble protein expression of receptor activator of nuclear factor-kappa β ligand (RANKL) and osteoprotegerin (OPG) via ELISA. EOs were co-cultured with pre-osteoclasts to determine OC maturation and resorption. EOs and pre-osteoclasts were co-cultured on a mimetic bone matrix in the presence of soluble RANKL to assess for OC resorption and pit formation. In addition, pre-osteoclasts were tri-cultured with EOs plus either human metastatic triple negative or estrogen receptor positive (ER+) BC cells to assess the effect of BC cells on the formation of OCs. The culture supernatant of EOs expressed reduced amounts of RANKL and OPG soluble protein compared to UOB supernatant. OCs formed in the presence of EOs were approximately half the size of those formed in the presence of UOBs as visualized by TRAP stain. Furthermore, we found up to a 66% reduction in the number of OCs formed in the presence of EOs when compared to the number of OCs formed in the presence of UOBs. When pre-osteoclasts were co-cultured on a mimetic bone matrix, up to 45% reduction was observed in the number of resorptive pits formed upon co-culture with EOs versus UOBs. Moreover, the tri-cultures of pre-osteoclasts, EOs, and human metastatic BC cells yielded up to a 28% reduction in the number of mature, multi-nucleated OCs formed, as opposed to the number of mature, multi-nucleated OCs formed in tri-cultures of pre-osteoclasts, UOBs, plus human metastatic BC cells. Overall, these data suggest that EOs suppress osteoclastogenesis and reduce bone resorption on a bone mimetic surface. These data imply that EOs provide a protective effect against bone resorption during bone metastatic BC progression. These data underscore the importance of investigating EOs as mediators of bone remodeling during BC progression in bone. Supported by NIH R00 CA178177 and Commonwealth of Pennsylvania-Department of Health SAP 4100072566 for KMB. Citation Format: Alexus D. Kolb, Karen M. Bussard. 'Educated' osteoblasts suppress osteoclastogenesis and bone resorption in a bone mimetic microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3993.