Hepatic nonparenchymal cells drive metastatic breast cancer outgrowth and partial epithelial to mesenchymal transition

Nearly half of breast carcinoma metastases will become clinically evident five or more years after primary tumor ablation. This implies that metastatic cancer cells survived over an extended timeframe without emerging as detectable nodules. The liver is a common metastatic destination, whose parenchymal hepatocytes have been shown to impart a less invasive, dormant phenotype on metastatic cancer cells. We investigated whether hepatic nonparenchymal cells (NPCs) contributed to metastatic breast cancer cell outgrowth and a mesenchymal phenotypic shift indicative of emergence. Co-culture experiments of primary human hepatocytes, NPCs or endothelial cell lines (TMNK-1 or HMEC-1) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Exposure of carcinoma cells to NPC-conditioned medium isolated soluble factors contributing to outgrowth. To elucidate outgrowth mechanism, epidermal growth factor receptor (EGFR) inhibition co-culture experiments were performed. Flow cytometry analyses and immunofluorescence staining were conducted to quantify breast cancer cell outgrowth and phenotype, respectively. Outgrowth of the MDA-MB-231 cells within primary NPC co-cultures was substantially greater than in hepatocyte-only or hepatocyte+NPC co-cultures. MCF-7 cells co-cultured with human NPCs as well as with the endothelial NPC subtypes grew out significantly more than controls. MCF-7 cells underwent a mesenchymal shift as indicated by spindle morphology, membrane clearance of E-cadherin, and p38 nuclear translocation when in HMEC-1 co-culture. HMEC-1-conditioned medium induced similar results suggesting that secretory factors are responsible for this transition while blocking EGFR blunted the MCF-7 outgrowth. We conclude that NPCs in the metastatic hepatic niche secrete factors that can induce a partial mesenchymal shift in epithelial breast cancer cells thus initiating outgrowth, and that this is in part mediated by EGFR activation. These data suggest that changes in the parenchymal cell and NPC ratios (or activation status) in the liver metastatic microenvironment may contribute to emergence from metastatic dormancy.