UC Berkeley

Epithelial to mesenchymal transition (EMT) and collective cell migration events are of interest for understanding cancer progression and metastasis. The use of in vitro cell lines to model these processes are widely used and help to shed light upon the mechanisms that lead to tumorigenesis. Here we use two such models: the human mammary epithelial cell line HMT-3522 and ex vivo mouse mammary primary culture epithelial organoids. The HMT-3522 cell line provides an encapsulated system to study cancer progression, however detailed ultrastructural analyses of this cell line are lacking. To study collective cell migration we used the murine ex vivo epithelial "organoid" system, which upon growth factor stimulation undergoes branching morphogenesis. Growth of these systems in 3D cell culture is an important component in understanding the highly dynamic and complex processes of EMT and collective cell migration. High resolution imaging via transmission electron microscopy and three dimensional scanning electron microscopy provide a way to discern ultrastructural features of both collective cell migration and EMT.

Electron microscopy imaging of the HMT-3522 S1 cell line, grown in 3D to form acini, demonstrated an abundance of membrane protrusions at all interior lateral and apical surfaces. Furthermore, these acinar structures were determined to have a semi-polarized state as defined by the appearance of a basement membrane, but lack all hallmarks of apical polarization. Three dimensional imaging of the membrane protrusions revealed they have a variety of lengths and an interdigitated nature. Determining this semi-polarized state to be more representative of a transitive tissue state, we sought to image the transient terminal end bud (TEB) of organoids undergoing branching morphogenesis. Ultrastructural analysis of in vivo murine mammary gland and ex vivo un-stimulated organoids revealed nearly identical tissue organization, with typical apico-basal polarity and almost no intercellular membrane protrusions. However, data of the stimulated organoids and specifically the TEB revealed a semi-polarized state of the cells very similar to the HMT-3522 S1 acini, with prolific membrane protrusions. Hypothesizing these membrane protrusions were aiding in normal collective cell migration, we investigated whether disruption of actin polymerization via drug treatments within the TEB would recapitulate the ultrastructure of the tumorigenic cell line HMT-3522 T4-2. RhoA kinase inhibition lead to a remarkably similar tissue and cellular morphology between the two cell types.