Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome

Tissue Stiffness Drives Breast Cancer Malignant Progression

Breast cancer is the most common cancer of women worldwide and the second leading cause of cancer death in women in the United States. Despite new therapies that show efficacy in treating the primary tumor there is a significant lack of therapies that treat metastasis, the most common cause of death. This deficit underscores the necessity for a new perspective in the study of breast cancer metastasis. Breast cancer malignant progression coincides with drastic tissue extracellular matrix (ECM) remodeling and stiffening. Previous work in our lab has shown that ECM stiffening is both necessary and sufficient to drive tumor aggression and metastasis in vivo. Consistently, our lab has demonstrated ECM mechanical properties act through integrin focal adhesions to regulate cell functions critical to tumor aggression such as cell proliferation, survival, and motility. Yet, how ECM structural changes occur with tumor progression and which signaling mechanisms downstream of cell-ECM adhesions are critical for tumor aggression remains unclear. In this thesis I address the overarching hypothesis that ECM mechanical properties influence tumor malignant progression through integrin focal adhesion signaling and subsequent downstream signaling affecting tumor motility and invasion, tumor metabolism, and immune cell response. To address this hypothesis I used a combination of isolated mammary epithelial cells with 2D and 3D mechanically tunable substrates ex vivo, in vivo breast cancer models, and human breast cancer tissues samples to robustly quantify the physical and structural ECM changes with tumor progression, identify through which integrin signaling mechanisms tumor cells sense these changes, and determine how tumor cell mechanosensing alters cell signaling to regulate tumor metastasis. From this work I found that tumor cell malignant phenotype is enhanced by ECM stiffness specifically via α5β1 – FN integrin binding which enhances pro-tumorigenic cell signaling to enhance tumor cell invasion and migration and alter tumor cell metabolism. Additionally, ECM stiffness alters tumor cytokine secretion and inflammatory signaling to regulate immune system response which I show in both in vivo models and human samples plays a critical role in tumor malignant progression. Thus, this work provides new mechanistic insight into how the physical microenvironment regulates tumor malignant progression

Tissue Stiffness Drives Breast Cancer Malignant Progression

Breast cancer is the most common cancer of women worldwide and the second leading cause of cancer death in women in the United States. Despite new therapies that show efficacy in treating the primary tumor there is a significant lack of therapies that treat metastasis, the most common cause of death. This deficit underscores the necessity for a new perspective in the study of breast cancer metastasis. Breast cancer malignant progression coincides with drastic tissue extracellular matrix (ECM) remodeling and stiffening. Previous work in our lab has shown that ECM stiffening is both necessary and sufficient to drive tumor aggression and metastasis in vivo. Consistently, our lab has demonstrated ECM mechanical properties act through integrin focal adhesions to regulate cell functions critical to tumor aggression such as cell proliferation, survival, and motility. Yet, how ECM structural changes occur with tumor progression and which signaling mechanisms downstream of cell-ECM adhesions are critical for tumor aggression remains unclear. In this thesis I address the overarching hypothesis that ECM mechanical properties influence tumor malignant progression through integrin focal adhesion signaling and subsequent downstream signaling affecting tumor motility and invasion, tumor metabolism, and immune cell response. To address this hypothesis I used a combination of isolated mammary epithelial cells with 2D and 3D mechanically tunable substrates ex vivo, in vivo breast cancer models, and human breast cancer tissues samples to robustly quantify the physical and structural ECM changes with tumor progression, identify through which integrin signaling mechanisms tumor cells sense these changes, and determine how tumor cell mechanosensing alters cell signaling to regulate tumor metastasis. From this work I found that tumor cell malignant phenotype is enhanced by ECM stiffness specifically via α5β1 – FN integrin binding which enhances pro-tumorigenic cell signaling to enhance tumor cell invasion and migration and alter tumor cell metabolism. Additionally, ECM stiffness alters tumor cytokine secretion and inflammatory signaling to regulate immune system response which I show in both in vivo models and human samples plays a critical role in tumor malignant progression. Thus, this work provides new mechanistic insight into how the physical microenvironment regulates tumor malignant progression

A 3D tension bioreactor platform to study the interplay between ECM stiffness and tumor phenotype

Extracellular matrix (ECM) structure, composition, and stiffness have profound effects on tissue development and pathologies such as cardiovascular disease and cancer. Accordingly, a variety of synthetic hydrogel systems have been designed to study the impact of ECM composition, density, mechanics, and topography on cell and tissue phenotype. However, these synthetic systems fail to accurately recapitulate the biological properties and structure of the native tissue ECM. Natural three dimensional (3D) ECM hydrogels, such as collagen or hyaluronic acid, feature many of the chemical and physical properties of tissue, yet, these systems have limitations including the inability to independently control biophysical properties such as stiffness and pore size. Here, we present a 3D tension bioreactor system that permits precise mechanical tuning of collagen hydrogel stiffness, while maintaining consistent composition and pore size. We achieve this by mechanically loading collagen hydrogels covalently-conjugated to a polydimethylsiloxane (PDMS) membrane to induce hydrogel stiffening. We validated the biological application of this system with oncogenically transformed mammary epithelial cell organoids embedded in a 3D collagen I hydrogel, either uniformly stiffened or calibrated to create a gradient of ECM stiffening, to visually demonstrate the impact of ECM stiffening on transformation and tumor cell invasion. As such, this bioreactor presents the first tunable 3D natural hydrogel system that is capable of independently assessing the role of ECM stiffness on tissue phenotype

Rapid disorganization of mechanically interacting systems of mammary acini

Cells and multicellular structures can mechanically align and concentrate fibers in their ECM environment and can sense and respond to mechanical cues by differentiating, branching, or disorganizing. Here we show that mammary acini with compromised structural integrity can interconnect by forming long collagen lines. These collagen lines then coordinate and accelerate transition to an invasive phenotype. Interacting acini begin to disorganize within 12.5 ± 4.7 h in a spatially coordinated manner, whereas acini that do not interact mechanically with other acini disorganize more slowly (in 21.8 ± 4.1 h) and to a lesser extent (P < 0.0001). When the directed mechanical connections between acini were cut with a laser, the acini reverted to a slowly disorganizing phenotype. When acini were fully mechanically isolated from other acini and also from the bulk gel by box-cuts with a side length <900 μm, transition to an invasive phenotype was blocked in 20 of 20 experiments, regardless of waiting time. Thus, pairs or groups of mammary acini can interact mechanically over long distances through the collagen matrix, and these directed mechanical interactions facilitate transition to an invasive phenotype