Multicellular architecture of malignant breast epithelia influences mechanics

Abstract

Cell-matrix and cell-cell mechanosensing are important in many cellular processes, particularly for epithelial cells. A crucial question, which remains unexplored, is how the mechanical microenvironment is altered as a result of changes to multicellular tissue structure during cancer progression. In this study, we investigated the influence of the multicellular tissue architecture on mechanical properties of the epithelial component of the mammary acinus. Using creep compression tests on multicellular breast epithelial structures, we found that pre-malignant acini with no lumen (MCF10AT) were significantly stiffer than normal hollow acini (MCF10A) by 60%. This difference depended on structural changes in the pre-malignant acini, as neither single cells nor normal multicellular acini tested before lumen formation exhibited these differences. To understand these differences, we simulated the deformation of the acini with different multicellular architectures and calculated their mechanical properties; our results suggest that lumen filling alone can explain the experimentally observed stiffness increase. We also simulated a single contracting cell in different multicellular architectures and found that lumen filling led to a 20% increase in the "perceived stiffness" of a single contracting cell independent of any changes to matrix mechanics. Our results suggest that lumen filling in carcinogenesis alters the mechanical microenvironment in multicellular epithelial structures, a phenotype that may cause downstream disruptions to mechanosensing.

Figure 1. Background and experimental design.

(A) A mammary epithelial cell grows in a dynamic environment surrounded by extracellular matrix, fluids, and other cells. (B) Mammary epithelial cells grown in laminin-rich extracellular matrix can be extracted and mechanically probed at single and multicellular states using identical trypsin-free extraction methods.

Figure 2. MCF10A and MCF10AT acini have different architectural and mechanical properties.

Confocal immunofluorescence images of (A) non-malignant MCF10A (hollow lumen) and (B) pre-malignant MCF10AT (filled lumen) acini, taken after 15 days of growth. Scale bars 25 µm. (C) Creep compliance (mean ±95% CI) of hollow and filled breast epithelial acini. (N = 32 and 31 acini for A and T respectively) (D) Differences in mechanical response could be due to (1) different properties of single cells (2) changes in connectivity or (3) changes in multicellular architecture.

Figure 3. Single cell mechanics and cell–cell connections do not explain the mechanical differences.

Creep compliance (mean ±95% CI) of MCF10A and MCF10AT cells at (A) single cell state (N = 14 and N = 15 cells for A and T respectively) and (B) 6–8 day state before lumen formation (N = 34 and N = 33 colonies for A and T respectively). Confocal immunofluorescence images of 8 day colonies of (C) MCF10A and (D) MCF10AT; 6–8 day time points were selected for testing because this was before lumina formed; scale bars 25 µm.

Figure 4. Mechanical differences are explained by the difference in multicellular architecture.

(A) Simulation of hollow and filled structures predicts decreased compliance (increased stiffness) of the structure associated with multicellular architecture. (B,C) Visualization of the 3D plate compression simulation environment using in this study to model the MCF10AT and MCF10A acini geometries. The yellow cube represents the simulation domain which is filled with incompressible fluid. The light blue object represents the deforming elastic–viscoelastic acinus, which is compressed between the dark blue plates. (D) Cross-section through 3D simulation of plate for hollow and filled structures. For numerical convenience, the influences of the plate and bottom surface are smoothed out across several layers of grid points, so they appear to overlap with the top and bottom of the acini. Regions of higher pressure are visible at the locations where the plate and bottom surface make contact. In the spherical shell simulation, a region of negative pressure is also visible, as the interior part of the shell is stretched during the deformation.

Figure 5. Multicellular architecture could affect the perceived mechanical microenvironment independent of material properties.

Cross-section through 3D simulations of single cell contraction in (A) filled and (B) hollow structures, showing the magnitude of shear stress, The single cell is shown by the black circle. (C) Perceived stiffness for a single cell in a hollow structure is approximately 20% lower than a filled structure. The dashed horizontal line shows the actual stiffness of the acinus.