Abstract 4804: Metastatic breast cancer cells induce an osteoblast-TAF-like phenotype and communicate with osteoblasts via gap junctions and exosomal crosstalk to facilitate breast cancer cell quiescence in bone
Authors: Bussard Karen M. , Marini Frank C. 
Published: May 2015

Breast cancer has a high propensity for bone colonization. Although the precise mechanism underlying preferential metastasis is unknown, bone likely provides a hospitable environment that both attracts breast cancer cells and allows them to colonize and grow.

Metastatic lesions may originate from disseminated tumor cells (DTCs) shed from a primary tumor. Clinical evidence suggests that DTCs undergo a period of proliferative quiescence for ≤25 years. Despite the high prevalence of dormant tumors in humans, this area of research is poorly understood.

MC3T3-E1 murine osteoblasts (OBs) were grown to early differentiation (10 days old), then MDA-MB-231 human breast cancer cells (BCCs) added at a ratio of 1 BCC to 10 OBs. Co-cultures were fixed and stained for connexin 43 expression (Cx43, representative gap junction). Cx43 expression was localized between OBs and BCCs. No Cx43 expression was seen in the control. Furthermore, a parachute assay was performed under similar conditions, where at time 48 hours, CellTracker Orange CMTMR dye transfer was seen between GFP-labeled BCCs and unlabeled OBs. Then, OBs and BCCs were grown to confluence. Individual cultures were treated with media containing exosome-free fetal bovine serum. Forty-eight hours later, culture supernatants were removed and processed through a series of ultra-centrifugation and concentration steps to purify for exosomes. Both OBs and BCCs produced exosomes, which were characterized using a Nanosight 500 System, and analyzed for microRNAs via real-time PCR.

Next, OBs were grown to early differentiation (10 days), and treated with conditioned medium (CM) from BCCs. Twenty-one days later, OB-derived protein expression was assessed. When treated with BCC CM, OBs decreased their expression of alpha smooth muscle actin, but increased their expression of fibroblast activating protein and matrix metalloproteinase 13. These results are consistent with a tumor-associated fibroblast-activated myofibroblast phenotype.

Finally, OBs were grown to 4 days (growth) and 10 days (early differentiation) and incubated with BCC CM. Twenty-four hours later, total RNA was collected, and ABI TaqMan qPCR cytokine arrays were used to assay for changes in OB-derived cytokine expression. OB-derived cytokines were increased in the presence of BCC CM, and included MCP-1 (CCL2), LIX (CXCL5), LIF, and NFATc2.

These data imply that BCCs communicate with OBs by intercellular crosstalk via gap junctions and exosomes that creates a niche permissive for BCC dormancy in bone. This work highlights the importance of investigating OBs in the bone microenvironment as key players in BCC dormancy.

Supported by the NIH; 1RC1 CA146381, 1R01NS06994, P50 CA083639 for FCM; Ruth L. Kirschstein Institutional Research Service Award (NRSA) T32 CA079448 and NIH 1 K99 CA 178177-01 for KMB.

Citation Format: Karen M. Bussard, Frank C. Marini. Metastatic breast cancer cells induce an osteoblast-TAF-like phenotype and communicate with osteoblasts via gap junctions and exosomal crosstalk to facilitate breast cancer cell quiescence in bone. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4804. doi:10.1158/1538-7445.AM2014-4804