(D-126) Reciprocal intra- and extra-cellular polarity enables deep mechanosensing through layered matrices

Cell migration is an important process that regulates development and tissue maintenance, dictating organ health and functionality. While stiffer surfaces are known to enhance cell migration, it remains unclear whether cells sense basal stiff environments buried under softer, fibrous matrix. During these processes, cells interact with their surrounding extracellular matrix (ECM) and respond to its chemical and mechanical composition through adapting cellular adhesions, cell shape, and force generation via actin-myosin contractility. Specifically, it has been shown that direct contact with higher stiffness substrates can lead to greater reinforced cell adhesions and cell-generated forces polarized in frontward direction, resulting in cell migration. However, the cell niche is a complex environment consisting of many distinct layers, with unique compositions and mechanical properties. Currently, it is unknown whether cells can sense beneath their immediate, adhered matrix to sense the underlying layers of its environment. If true, it’s possible that the mechanical properties of distant ECMs could regulate cell migration.

To investigate whether cells can sense basal matrix stiffness deeply through collagen, we fabricated layered, hybrid extracellular matrix platforms. The base consisted of polyacrylamide (PA) gels of two distinct stiffnesses; ~0.5 kPa (soft) and ~110 kPa (stiff). For control gels (further referred to as direct ECMs), a collagen type I monolayer was coated on both PA formulations. For layered matrices, we polymerized and covalently bonded a collagen type I gel (~10 μm thick) to the PA surface via Sulfo-SANPAH crosslinker (Fig. 1A). We then utilized these platforms to study the behavior of two widely studied cell types due to their distinct phenotypes – MCF-10A human mammary epithelial cells and MDA-MB-231 triple-negative breast cancer cells, which we refer to as ‘normal’ and ‘cancer’ cells, respectively, for simplicity. We then analyzed a number of characteristic behaviors, including cell morphological outcomes; motility; and cell and ECM stiffness changes, in order to determine if cells exhibited mechanosensitive behaviors characteristic of cells in direct contact with the underlying PA substrate.

We found that while normal cells showed no response to distant ECM stiffness, cancer cells showed drastic differences in both morphological and migratory behavior, determined by distant basal PA stiffness. Namely, cancer cells on stiff layered ECMs maintained polarized morphologies through stable, coordinated protrusions, resulting in fast, persistent migration speeds (Fig. 1B-F). We found that the interplay and coordination of intra- and extracellular polarity was the predominant mechanism through which cancer cells sensed the distant ECM deeply through the top collagen layer, with important factors such as cell polarity, protrusion stabilization, and matrix deformability. Loss of any of these key factors, whether it be through cellular inhibitions, such as reducing contractility and destabilizing actin, or extracellular inhibitions of matrix crosslinking or disconnection, resulted in suppression of this ‘depth-sensing’ through layered ECMs. Furthermore, these behaviors were conserved among other cell types with similar representative phenotypes compared to MDA-MB-231 cells. Discovery of both the phenomenon of distant ECM sensing and the mechanisms through which it is possible reveal further insights into the treatment of certain diseases and disorders with both spatially and temporally evolving tissue stiffness. For example, metastasizing cancer cells navigate through heterogeneous, layered environments as they attempt to enter and exit circulation. The state of the ECM is interpreted by the cell and the cellular response can often be seen through behavioral changes, such as altered morphology and strong migratory responses. It is possible that more than just the cell’s immediate environment influences their behavior and instigates deeper invasion through healthy tissue. Correspondingly, this work could also provide new ideas for therapeutic targets specific to front-rear cell polarity and F-actin branching that regulate depth-sensitive cell migration. The data for this work has been published (https://doi.org/10.1016/j.celrep.2023.112362).¹

1. Walter, C., Mathur, J., & Pathak, A. (2023). Reciprocal intra-and extra-cellular polarity enables deep mechanosensing through layered matrices. Cell Reports, 42(4).