Abstract A12: A novel in vitro model to study the role of phagocytic mammary epithelial cells in postpartum breast cancer promotion
Authors: Fornetti Jaime , Henson Peter , Borges Virginia , Schedin Pepper 
Published: August 2014

Women diagnosed with breast cancer within 5-10 years of pregnancy have significantly poorer prognosis than age-matched nulliparous patients. This poor prognosis is specific to postpartum cases, suggesting that an event related to the completion of pregnancy may drive the poor prognosis of postpartum breast cancer. We have identified postpartum mammary gland involution as a tumor-promotional window following pregnancy/lactation. During postpartum involution, 50-80% of the milk-producing mammary epithelial cells (MECs) undergo apoptosis. Additionally, during involution, the MECs themselves become phagocytic and clear the gland of their dying neighbors. Peak phagocytosis precedes an influx of M2 activated macrophages with immunosuppressive characteristics which are thought to directly contribute to the tumor-promotional microenvironment of the postpartum gland. We hypothesize that clearance of apoptotic cells during involution induces secretion of immune-suppressive signals by phagocytic MECs, promoting M2 macrophage maturation and local immune suppression. These host events are anticipated to facilitate breast tumor cell immune evasion and tumor progression. This hypothesis is based on data that demonstrate clearance of apoptotic cells by professional phagocytes promotes M2 macrophages and immune suppression through TGF-β and COX-2. Here, we characterize the phenotype of phagocytic MECs and determine if phagocytic MECs induce tumor-promotional, M2 macrophage activation.

To study the role of phagocytic MECs in promoting M2 macrophages, we developed an in vitro model in which MECs are grown to monolayers with high electrical resistance, modeling the tight cell-cell junctions unique to the lactating epithelium. To mimic involution, these monolayers are then treated with TGF-β3, which disrupts cell junctions and induces phagocytosis. We provide evidence that disruption of cell junctions is required for MECs to switch from a secretory to a phagocytic phenotype. We observe reorganization of cell junctions and E-cadherin cleavage in our in vitro model, and the extent of junction disruption positively correlates with phagocytosis. Similar adherens junction reorganization, E-cadherin cleavage, and β-catenin nuclear localization are observed upon the switch from lactation to involution in vivo in rodents and humans, consistent with a role for junction disruption in the phagocytic switch.

To assess whether phagocytic MECs may contribute to M2 macrophage activation and local immune suppression during involution, bone marrow-derived macrophages (BMDM) were treated with conditioned media from phagocytic or control MEC cultures and expression of the M2 marker arginase-1 (Arg-1) was evaluated. Phagocytic cell conditioned media induced Arg-1, indicating M2 macrophage activation. Importantly, Arg-1 expression in BMDM correlated with COX-2 expression by phagocytic MECs, which suggests phagocytic MECs may promote M2 macrophages through a COX-2 dependent mechanism, as observed in the promotion of M2 macrophages by professional phagocytes. In vivo, COX-2 expression detected by IHC increases in MECs during involution, consistent with a role for phagocytic MEC COX-2 in promoting M2 macrophages. Additionally, CD86, a co-stimulatory molecule that promotes T cell activation, was decreased in BMDM treated with phagocytic MEC conditioned media, further supporting a role for phagocytic MECs in promotion of an immune-suppressive microenvironment.

By further understanding the role of phagocytic MECs in regulating the immune environment in the breast during involution, we hope to identify novel pathways of metastasis, as well as new targets for postpartum breast cancer therapy.

Citation Format: Jaime Fornetti, Peter Henson, Virginia Borges, Pepper Schedin. A novel in vitro model to study the role of phagocytic mammary epithelial cells in postpartum breast cancer promotion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A12.