Mammary gland ECM remodeling, stiffness, and mechanosignaling in normal development and tumor progression

Abstract

Cells of the mammary gland are in intimate contact with other cells and with the extracellular matrix (ECM), both of which provide not only a biochemical context, but a mechanical context as well. Cell-mediated contraction allows cells to sense the stiffness of their microenvironment, and respond with appropriate mechanosignaling events that regulate gene expression and differentiation. ECM composition and organization are tightly regulated throughout development of the mammary gland, resulting in corresponding regulation of the mechanical environment and proper tissue architecture. Mechanical regulation is also at play during breast carcinoma progression, as changes in ECM deposition, composition, and organization accompany breast carcinoma. These changes result in stiffer matrices that activate mechanosignaling pathways and thereby induce cell proliferation, facilitate local tumor cell invasion, and promote progression. Thus, understanding the role of forces in the mammary gland is crucial to understanding both normal developmental and pathological processes.

Figure 1.

Mammary epithelial cells respond to the stiffness of a collagen matrix. (A) Scanning electron microscopy image of mammary acini showing individual mammary epithelial cells (M) surrounded by oriented collagen fibers (arrows). (SEM image courtesy of Paolo Provenzano.) Bar, 30 um. (B) Elastic modulus of collagen gels increases with increased collagen concentration. Modulus was measured by tension as described (Provenzano et al. 2009). Triangles represent data from Provenzano et al. (2009), squares from Roeder et al. (2002). Data was fitted and the resulting equation shown. (C) MCF10A cells cultured in a low-density (1.0 mg/ml) collagen gel form polarized acinar structures (a), while the same cells cultured in a high-density (2.0 mg/ml) collagen gel proliferate in a disorganized manner (c). Addition of HGF to low-density cultures results in tubule formation (b), while HGF added to cells in high-density culture results in an invasive phenotype (d). (Panels B and C reprinted from Provenzano et al. 2009 with permission from Nature Publishing Group © 2009.)

Figure 2.

Anisotropic organization of matrix is an intrinsic property of MECs and is developmentally regulated. (A) Macro-patterning of anisotropic ECM by mammary epithelial cells in 3D culture. Bar, 15 um. (B) Collagen fibers in blue (Trichrome stain) show increased deposition in regressing mammary lobules (white asterisks) in comparison to lactating lobules (black star). Bar, 100 µm. (C) Comparison of fibronectin and laminin ratios between rat mammary ECM and EHS reconstituted basement membrane (Matrigel). Lane 1: 10.8 µg EHS tumor matrix. Lane 2: 10.8 µg Day 4 involution rat mammary matrix. Based on scanning densitometry, rat mammary matrix has ∼five-fold more FN per μg protein than EHS matrix, whereas EHS matrix has ∼10-fold more LN per μg protein than rat mammary matrix, with a FN/LN ratio of 50 or higher in rat mammary matrix compared to EHS matrix.

Figure 3.

Mechanical signaling pathways. (A) Mammary cells in a compliant matrix organize into polarized acinar and ductal structures. Local deposition and remodeling of extracellular matrices accompanies tumor progression (green = collagen fibers). (B) Diagram of signaling pathways related to focal contacts and cellular contractility. (T) talin, (V) vinculin. (C) Diagrams representing conformational changes in putative mechanosensors: filamin, talin, and integrins.

Figure 4.

Collagen alignment facilitates invasion. (A) Parallel alignment of collagen fibers (arrows) around a non-invasive tumor (T), imaged in a live mammary tumor ex vivo by multiphoton laser scanning microscopy and second harmonic generation (SHG) to image collagen. (B) Diagram of TACS-2 (example in A), in which collagen fibers are denoted by tan-colored lines. (C) Perpendicular alignment of collagen fibers (arrows) at a tumor (T) boundary, which is depicted by the red line. (Panel reprinted from Provenzano et al. 2006 with permission from BioMed Central Ltd. © 2006.) Live tumor was imaged as in A. (D) Diagram of TACS-3 (example in C), with cells invading out from the tumor along aligned collagen fibers. (E) Human T47D breast carcinoma cells (yellow) aligns relative to collagen fibers (green) within an engineered collagen matrix. The orientation of collagen fibers is depicted by the arrow. Live cell in matrix was imaged as in A.