Hypothesis: Force homeostasis and an intact basement membrane (BM) are decisive for epithelial tissue organization.
Graphical summary of project A2. Design of the experimental approaches, work packages and own preliminary data.
Background: The mammary gland consists of multiple spheroidal acini that are separated from the surrounding connective tissue by a basement membrane (BM). Here, the interplay between acinar cells, the BM, and the ECM regulates tissue homeostasis. Throughout the whole lifetime, contractile myoepithelial cells, the extracellular matrix (ECM), and luminal secretions dynamically deform the acini’s epithelial lining. We have recently used a well-established 3D cell culture model to characterize the BM protein scaffold as a crucial mechanical stabilizer for breast acini 1,2. We further showed that the BM is a physical cell invasion barrier that is compromised upon EMT-like cytoskeleton reorganization and increased cell forces 3,4.
Aims: We aim to decipher the mechanobiological regulation circuits that steer epithelial organization in response to BM disruption and mechanical stresses. We concentrate on the cellular signal processing mechanisms between cells, the BM barrier, and the ECM. For this purpose, we use basoapically polarized and BM-covered spheroids derived from human epithelial mammary gland cell lines and patient-derived breast tumor cells. ECM stiffness and shear stress transmitted by the ECM are systematically analyzed as key biophysical stimuli. The focus lies on the characterization of cellular responses and the underlying signaling cascades that transduce these mechanical cues into morphological and functional cell adaptation. We investigate how extracellular mechanical stress acts on cells and how these cells in turn exert mechanical signals on the surrounding ECM. We aim at an improved understanding of the reciprocal mechanobiological key mechanisms that steer epithelial cell behavior during breast gland formation, maintenance, and tumor cell invasion.
Approach: Breast gland formation and cell invasion are studied in 3D cell cultures of human non-tumorigenic MCF-10A and HMT3522-S1 cell lines, as well as patient-derived tumor spheroids 5-7. The multicellular spheroids show a basoapical polarization with BM formation and defined cell-cell and cell-matrix junctions. Further, we apply cell variants with inducible proto-oncogene activity to modulate the invasive potential in vitro. Based on our extensive experience mechanobiology, we will investigate cell force as a novel non-proteolytical mechanism of BM disruption and its implications for cancer invasion 8-10. The well-documented influence of tumor-associated ECM stiffening on invasion is also explored for this cell-force-mediated mechanism. To this end, cell force microscopy techniques designed in-house are used. The underlying mechanosensitive signaling pathways are characterized by gene expression and protein activation studies. Furthermore, the impact of ECM shear stress on epithelial tissue organization is investigated systematically using special devices recently designed by us. Together, we will elucidate the interconnectivity of mechanotransductive signaling pathways that govern cellular stress responses and finally orchestrate epithelial tissue function.
1. Gaiko-Shcherbak, A., et al., The acinar cage: Basement membranes determine molecule exchange and mechanical stability of human breast cell acini. PLoS ONE, 2015. 10: p. 1-20.
2. Fabris, G., et al., Nanoscale Topography and Poroelastic Properties of Model Tissue Breast Gland Basement Membranes. Biophysical Journal, 2018. 115: p. 1-13.
3. Eschenbruch, J., et al., From Microspikes to Stress Fibers: Actin Remodeling in Breast Acini Drives Myosin II-Mediated Basement Membrane Invasion. Cells, 2021. 10(8).
4. Gaiko-Shcherbak, A., et al., Cell Force-Driven Basement Membrane Disruption Fuels EGF- and Stiffness-Induced Invasive Cell Dissemination from Benign Breast Gland Acini. Int J Mol Sci, 2021. 22(8).
5. Soule, H.D., et al., Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res, 1990. 50(18): p. 6075-86.
6. Briand, P., O.W. Petersen, and B. Van Deurs, A new diploid non-tumorigenic human breast epithelial cell line isolated and propagated in chemically defined medium. In Vitro Cell Dev Biol, 1987. 23(3): p. 181-8.
7. Strietz, J., et al., Human Primary Breast Cancer Stem Cells Are Characterized by Epithelial-Mesenchymal Plasticity. Int J Mol Sci, 2021. 22(4).
8. Cesa, C.M., et al., Micropatterned silicone elastomer substrates for high resolution analysis of cellular force patterns. Rev Sci Instrum, 2007. 78(3): p. 034301.
9. Merkel, R., et al., Cell force microscopy on elastic layers of finite thickness. Biophys J, 2007. 93: p. 3314-3323.
10. Soine, J.R., et al., Measuring cellular traction forces on non-planar substrates. Interface Focus, 2016. 6(5): p. 20160024.