Abstract 3205: Breast cancer cell quiescence in bone is modulated by Osteo-TAFs
Authors: Bussard Karen M. , Marini Frank C. 
Published: August 2015

Abstract
Breast cancer frequently metastasizes to bone. Upon entry to a secondary site, disseminated breast cancer cells (BCCs) can undergo a period of proliferative quiescence for ≤25 years where they remain clinically undetectable and untreatable.
We propose that disseminated BCCs educate osteoblasts (OBs) into a tumor-associated fibroblast (TAF)-like cell that, in turn, orchestrates the proliferative quiescence BCC phenotype in bone. To test this, differentiated MC3T3-E1 murine osteoblasts (OBs) were incubated for 21 days with conditioned medium (CM) from metastatic BCC variants (MDA-231 or metastasis-suppressed MDA-231). OB cell lysates were analyzed for TAF proteins. Importantly, OBs reduced their expression of alkaline phosphatase, F4/80, CD31, and alpha SMA, with the largest change seen in 41-day-old conditioned OBs. These results are consistent with the described aggressive “matrix-remodeling” TAF. On the other hand, metastasis-suppressed BC CM treated OBs increased their expression of IL-6, suggestive of an OB inflammatory response to disseminated BCCs. TAF-OBs also exhibited altered morphology in the form of a thin, spindle-like shape, as opposed to cobblestone appearance.
As a way to better understand how TAF-OBs can induce proliferative quiescence in BCC we sought to examine the roles of gap junctions and exchange of miRs as modulators to this process. Briefly, OBs were differentiated and ad-mixed with 1) GFP-MDA-231 human BCCs, or 2) GFP-MDA-231 BCCs double-labeled with CellTracker Orange CMTMR. Forty-eight hours later, these co-cultures were assessed for the presence of gap junction protein expression (Cx43, representative gap junction), and whether they functioned to exchange information using a cellular CMTMR dye transfer assay. Cx43 expression was localized specifically between OBs and BCCs. Interestingly, we observed direct cell-cell dye transfer occurring between BCCs that distributed dye to OBs several cell distances away.
Next, we assayed supernatants of OBs and BCCs for the presence of exosomes and exosomal miRs, both known mediators of cell-to-cell communication. Both OBs and BCCs produced exosomes as characterized by transmission electron microscopy and a Nanosight 500 System. OB-derived exosomes were visualized in co-culture assays using a CD63-RFP immunolabel. Late differentiated OBs produced over twice the amount of exosomes than did OBs in their growth phase. Given this, we interrogated the contents of the exosomes for miRs that might induce proliferative quiescence in BCCs. miR arrays demonstrated the presence of miRs involved in cell cycle control (miR 193) and cellular dormancy (miR 320a) as produced by TAF-OBs.
Combined, these data suggest that there is extensive crosstalk between OBs and disseminated BCCs in bone. This work highlights the importance of investigating OBs as key players in BCC dormancy in bone. Supported by NIH 1RC1 CA146381, 1R01NS06994, P50 CA083639 for FCM; (NRSA) T32 CA079448, NIH 1 K99 CA178177 for KMB.
Citation Format: Karen M. Bussard, Frank C. Marini. Breast cancer cell quiescence in bone is modulated by Osteo-TAFs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3205. doi:10.1158/1538-7445.AM2015-3205