Cellular mechanoresponse to extracellular matrix shear strain and stiffness studied in a cell culture model of breast gland development

Abstract

Mechanobiological regulation is a crucial element for tissue development and homeostasis. Dysregulation of the normal mechanobiological microenvironment of cells is linked to disease and negatively affects cancer patients' clinical outcomes. On the level of individual cells, much is known on sensation and transduction of mechanical signals. However, it remains elusive how cells integrate multiple signals on a tissue level to orchestrate complex differentiation processes of cells and tissues. A promising approach to elucidate these processes is offered by experiments on three-dimensional (3D) cell culture models like breast epithelial spheroids grown in EHS (Engelbreth Holm Swarm) hydrogels.<br /> The breast gland is a mechanically active tissue constantly subjected to shear deformations by external forces, for example, upon normal physiological activity. However, little is known about the role of these forces in mammary epithelium development and homeostasis because tools to study the impact of such mechanical cues are still lacking.<br /> In this work, a novel shear stress device was employed to apply physiological stress to breast epithelial spheroids. This device utilized magnetic coupling to apply tangential force to the surface of EHS-substrate-derived hydrogels. Moreover, it enabled confocal live cell microscopy over extended periods. The resulting strain in gels was thoroughly analyzed by tracking gel-embedded fluorescent microspheres in 3D. These measurements confirmed reproducible formation of pure and constant shear strain throughout the whole analysis volume. Three-dimensional breast epithelial spheroids with nature-like architecture were subsequently embedded into hydrogels to analyze cellular shear stress responses. Confocal microscopy of the cells’ actin cytoskeleton allowed for the determination of shear strain amplitude within gel-embedded spheroids. This analysis revealed a significant decrease in strain compared to the surrounding matrix, implying a mechanical resistance of breast spheroids to extracellular matrix (ECM) strain. Analyses of strain response in spheroids of different developmental stages showed that spheroid architecture and basement membrane (BM) development are crucial for breast epithelial mechanical resistance.<br /> The device was then utilized to cyclically strain spheroids for prolonged periods (22 hours). Cell viability was not compromised by this procedure. Mechanoresponse was studied by live cell analysis of actin cytoskeleton dynamics. In young, low-developed spheroids cellular morphology indicated a loss of cell-cell contacts upon cyclic shear stress. Furthermore,stressed spheroids were extruding cells that subsequently underwent apoptosis as shown by immunocytochemical labeling of the apoptotic effector protein cleaved caspase-3. Quantification revealed that cell extrusion as a cellular response to shear stress application was exclusively occurring in young, immature spheroids with incomplete epithelial differentiation.<br /> During cancer development, the breast gland ECM undergoes intensive remodeling processes. Here, increased stiffness in tumor tissue causes increased malignant behavior and frequency of BM invasion events. However, due to a sparsity of appropriate tools it is only rudimentary known how cell force generation is modified by the extracellular matrix (ECM) in a physiological 3D context.<br /> Therefore, development of a 3D traction force microscopy (TFM) approach that will allow for the investigation of breast spheroid-derived forces was begun by tracking of microsphere displacements. Confocal image stacks were analyzed with dedicated image processing routines to accurately determine microsphere displacement in 3D and over time. This 3D TFM approach enabled measurement and visualization of spheroid-derived matrix deformation patterns in 3D. Surprisingly strong tangential displacements were observed close to the spheroids’ surfaces. Further development of this novel tool will allow for the quantification of mammary epithelial force generation in response to natural and tumor-like microenvironments in 3D.<br /> In essence, this work was concerned with the mechanobiological regulation of breast gland development and homeostasis. Development of a novel 3D TFM approach was initiated with the aim of investigating cellular force generation in mammary epithelial spheroids. By use of a novel device on a cell culture model of the breast gland, it was possible to apply physiological stresses in a nature-like matrix to mimic the complex mechanical microenvironment of breast epithelial tissue and to observe cellular mechanoresponses to this important mechanical cue.