Dynamically stiffened matrix promotes malignant transformation of mammary epithelial cells via collective mechanical signaling

Abstract

Breast cancer development is associated with increasing tissue stiffness over years. To more accurately mimic the onset of gradual matrix stiffening, which is not feasible with conventional static hydrogels, mammary epithelial cells (MECs) were cultured on methacrylated hyaluronic acid hydrogels whose stiffness can be dynamically modulated from “normal” (<150 Pascals) to “malignant” (>3,000 Pascals) via two-stage polymerization. MECs form and remain as spheroids, but begin to lose epithelial characteristics and gain mesenchymal morphology upon matrix stiffening. However, both the degree of matrix stiffening and culture time before stiffening play important roles in regulating this conversion as, in both cases, a subset of mammary spheroids remained insensitive to local matrix stiffness. This conversion depended neither on colony size nor cell density, and MECs did not exhibit “memory” of prior niche when serially cultured through cycles of compliant and stiff matrices. Instead, the transcription factor Twist1, transforming growth factor β (TGFβ), and YAP activation appeared to modulate stiffness-mediated signaling; when stiffness-mediated signals were blocked, collective MEC phenotypes were reduced in favor of single MECs migrating away from spheroids. These data indicate a more complex interplay of time-dependent stiffness signaling, spheroid structure, and soluble cues that regulates MEC plasticity than suggested by previous models.

<p>HIF-1α, TWIST-1 and ITGB-1, associated with Tumor Stiffness, as Novel Predictive Markers for the Pathological Response to Neoadjuvant Chemotherapy in Breast Cancer</p>

“…11 Recent studies show that high matrix stiffness could induce epithelial to mesenchymal transition (EMT) and tumor progression by activating Twist family BHLH transcription factor 1 (TWIST-1). 12,13 In addition, TWIST-1 overexpression is associated with short survival and a poor response to chemotherapy in patients with cancer. 2,14,15 Hypoxia-inducible factor 1-alpha (HIF-1α), which mediates adaptation to hypoxia in cells, has been shown to be associated with matrix stiffness.…”

H‐Ras Transformation of Mammary Epithelial Cells Induces ERK‐Mediated Spreading on Low Stiffness Matrix

“…To further explore the upstream EMT activators, we measured mRNA expression of five key transcription factors associated with EMT, Snail, Slug, Zeb1, Zeb2, and Twist1. This showed no significant differences in expression levels between groups (Figure S2B, Supporting Information), although it has been noted that transcriptional activity of some markers requires nuclear localization . Thus we documented localization changes of Twist1, finding that in stiff conditions, it became nuclear localized for all cell lines (Figure 2B,C and Figure S2C, Supporting Information).…”

“…However, for cells on soft substrates, 10AT was the only line to indicate Twist1 localization, albeit with remnants of spheroids exhibiting significant cytoplasmic Twist1 (Figure 2B,C). These data suggest that TWIST localization is not being driven by ECM stiffness in this context, so we examined other signaling molecules associated with the induction of EMT that have previously been implicated in mechanotransduction . No increase in TGF‐β mediated SMAD2/3 localization was detected within spread regions and TGF‐β inhibition via Galunisertib did not prevent cells from spreading (Figure S3A, Supporting Information).…”

“…These changes increase ECM stiffness, which induces depolarization of mammary acini and invasion of MECs into the microenvironment . A variety of potentially redundant mechanisms have been identified, namely epithelial to mesenchymal transition (EMT) through localization of the basic helix loop helix transcription factor Twist1 as well as activation of other mechanosensitive proteins such as YAP/TAZ and SMAD 2/3 . More recently, 3D culture of MECs on a soft but dynamic hydrogel results in acinar polarization that can be induced later to undergo EMT by stiffening the matrix; such dynamics more accurately models in vivo behavior and dramatic changes in morphology, gene expression, and response to drugs .…”

Photoinduced directional domain sliding motion in peptide hydrogels promotes ectodermal differentiation of embryonic stem cells

“…Mechanical cues present within cell local microenvironment regulate cell behavior and fate due to their critical role in cell communication [1][2][3]. In particular, mechanical signals in the stem cell niche resulting from intracellular processes or external force sources significantly affect the basic functions of stem cells, such as selfrenewal and differentiation [4][5][6][7][8]. This fact stimulates the development of biocompatible hydrogels exhibiting extrinsic or intrinsic mechanical signals as biomimetic extracellular matrices (ECM) for stem cell-based organogenesis, thus leading to great potency in tissue engineering and regeneration [9][10][11][12][13].…”