Abstract 3976: Tumor-educated osteoblasts regulate breast cancer cell proliferation via extracellular vesicles
Authors: Shupp Alison B. , Bussard Karen M. 
Published: September 2020

Breast cancer commonly traffics to bone, where breast cancer cells (BCCs) can survive undetected for years before metastatic outgrowth. This period of time can be defined as metastatic latency. In bone, BCCs interact with surrounding stromal cells, including osteoblasts (OBs), to shape the metastatic niche. Our lab discovered that there are at least 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 BCCs whereas another group, naive OBs, have not. We have new, novel evidence that EOs regulate BCC proliferation, and may contribute to metastatic latency. The purpose of this study was to determine if extracellular vesicles (EVs) produced by EOs play a role in regulating BCC proliferation. We hypothesized that EVs produced by EOs, would decrease BCC proliferation. We first isolated EO-derived small EVs from cell culture media via ultracentrifugation and characterized the EVs for size, protein marker expression, and density floatation to validate the purity of the EV samples. We next sought to determine the functionality of EO-derived EVs with regards to regulating BCC proliferation. Strikingly, we found that EVs produced by EOs, but not naïve OBs, decreased BCC proliferation in a concentration dependent manner. We have observed this effect in both triple negative and ER-positive BCCs. Furthermore, using an EdU assay, we found that exposure to EO-derived EVs induces BCCs to undergo cell cycle arrest. Interestingly, we found that this cell cycle arrest was maintained as long as the BCCs were cultured in the presence of EO-derived EVs. However, the cell cycle arrest was reversible and BCC proliferation was restored upon removal of EO-derived EVs. We have also seen that exposure to EO-derived EVs leads to increases in BCC expression of the G1 checkpoint proteins, p21 and p27. We next wanted to investigate proliferative signaling pathways that may be deregulated in BCCs following exposure to EO-derived EVs. We found that EO-derived EVs reduce BCC levels of ERK1/2. This is consistent with previous literature indicating that low ERK1/2 levels are implicated in cell cycle arrest and even cancer cell quiescence. Because our data indicate EO-derived EVs induce sustained cell cycle arrest in BCCs, we desired to know if EO-derived EVs protected BCCs from chemotherapy-induced cell death. Excitingly, we have novel preliminary data demonstrating that BCCs exposed to EO-derived EVs and the chemotherapy drug, doxorubicin, have decreased cell death compared to BCCs exposed only to doxorubicin. Altogether, our data suggest that EOs play a crucial role in bone-tumor microenvironment, by regulating BCC proliferation, and may ultimately contribute to metastatic latency. This study underscores the importance of investigating EOs as regulators of breast cancer progression in bone. Supported by NIH R00 CA178177 and Commonwealth of Pennsylvania - Department of Health SAP 4100072566 for KMB.

Citation Format: Alison B. Shupp, Karen M. Bussard. Tumor-educated osteoblasts regulate breast cancer cell proliferation via extracellular vesicles [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 3976.