Cell division regulates macrophage invasion in mammary epithelia

Macrophages survey the mammary epithelium and are found between the myoepithelial and luminal layers of the ducts where they aid in tissue remodeling during branching morphogenesis. These ductal macrophages expand as more bone marrow derived macrophages arrive during puberty and pregnancy, when there is extensive remodeling of the gland, increased cell proliferation, and luminal apoptosis to maintain the ductal lumen. Interestingly, early macrophage infiltration is also observed in triple negative breast cancer (TNBC) and is associated with poor prognosis. TNBC-associated macrophages resemble the polarized state and tissue remodeling function of ductal macrophages during normal mammary development and are not as commonly found in luminal or human epidermal growth factor receptor 2-positive (HER2+) breast cancers. While it is well documented that mammary macrophages are present within the gland, the mechanisms by which macrophages infiltrate epithelial barriers in both developmental and disease contexts remains unknown. Thus, to address this gap in our understanding of macrophage biology, we employ 3D mammary epithelial and macrophage co-cultures compatible with live imaging. We hypothesize that macrophages respond to transient disruption in epithelial integrity-such as those occurring during mitosis or programmed cell death-and use these short-lived losses of epithelial-epithelial adhesions as opportunities to access epithelial compartments. To examine this hypothesis, we have live imaged 3D mammary epithelial spheroids generated from healthy (MCF10A) and TNBC (MDA-MB-231) cells in co-culture with macrophages. Mammary acini form polarized lumen where apoptotic cells are extruded into the luminal space, like in vivo. Mammary spheroids present an in vitro model where apoptosis can be driven pharmacologically, resulting in increased epithelial turnover, allowing us to model dynamic conditions of the mammary duct. Our data support the hypothesis that macrophages traverse epithelial layers at sites of cell division, consistent with a model in which transient junctional instability guides macrophage positioning. During involution, luminal epithelial cells undergo apoptosis and release “find-me” signals into the tissue microenvironment. We hypothesize macrophages respond to these local attractants, exploiting the transient losses in epithelial-epithelial cell adhesion, to traverse the myoepithelial layer. Together, this work aims to define the epithelial cues that regulate macrophage infiltration into mammary tissues and to understand how these processes shape both normal developmental programs and early disease transition in TNBC. Elucidating the mechanisms of macrophage entry into epithelial environments may reveal new opportunities for targeting immune-epithelial interactions in aggressive breast cancers while enriching our understanding of underappreciated immune-epithelial crosstalk.