Mechanotransduction of interstitial fluid stresses and effects on tumor cell migration

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2013."September 2013." Cataloged from PDF version of thesis.Includes bibliographical references (pages 93-106).Breast cancer incidence in the United States is I in 8, and over 90% of breast cancer related deaths are due to metastases, secondary tumors at a site distant from the primary tumor. Metastasis formation requires carcinoma cells to navigate through the tumor microenvironment and invade the surrounding stroma. Migration is a highly orchestrated process in which cells are guided by both internal signals and signals from the microenvironment. Hence, understanding the mechanisms that guide cell migration in response to various stimuli in the tumor and stromal microenvironments is key to developing therapies that prevent tumor cell migration and render cancer more treatable. Osmotic and hydrostatic pressure gradients within the extracellular matrix (ECM) drive flow of interstitial fluid through the ECM. Elevated osmotic pressure, lymphatic collapse, solid stress, and increased microvascular permeability contribute to elevated interstitial fluid pressure (IFP) during carcinoma progression, and high intratumoral IFP leads to pressure gradients at the tumor margin, which drive fluid flow that emanates from the tumor core to drain in the surrounding stroma. In this thesis, we explore the effect of interstitial flow (IF) on tumor cell migration. We developed a microfluidic platform to apply repeatable, robust IF through tissue constructs consisting of human metastatic breast cancer cells embedded within a 3D collagen type I matrix. We implemented the microfluidic device to validate CCR7-mediated autologous chemotaxis as a mechanism that guides downstream migration in response to IF. However, we identified a separate competing pathway that drives cell migration upstream (rheotaxis). Rheotaxis results from asymmetry in matrix adhesion stress that is required to balance fluid drag imparted by IF on tumor cells. Thus, autologous chemotaxis, mediated by chemical transport, and rheotaxis, mediated by fluid stresses, compete to direct cell migration downstream or upstream in response to IF. Our results provide insight into mechanotransduction in 3D porous media and into the mechanisms by which asymmetries in matrix adhesion tension guide cell migration. Furthermore, our results demonstrate that the consideration of IF is crucial for understanding and treating metastatic disease. Key words: Interstitial flow, mechanotransduction, tumor cell migration, microfluidics.by William J. Polacheck.Ph. D

Effects of interstitial flow on tumor cell migration

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 80-84).Interstitial flow is the convective transport of fluid through tissue extracellular matrix. This creeping fluid flow has been shown to affect the morphology and migration of cells such as fibroblasts, cancer cells, endothelial cells, and mesenchymal stem cells. However, due to limitations in experimental procedures and apparatuses, the mechanism by which cells detect flow and the details and dynamics of the cellular response remain largely unknown. We developed a microfluidic cell culture system in which we can apply stable pressure gradients and fluid flow, and in which we can observe transient responses of breast cancer cells seeded in a 3D collagen type I scaffold. We employed this system to examine cell migration in the presence of interstitial flow to address the hypothesis that interstitial flow increases the metastatic potential of breast cancer cells. By varying the concentration of chemoattractants, we decoupled the mechanisms that provide the migratory stimulus and the directional stimulus to migrating breast cancer cells in the presence of a flow field. We found that cells migrated along streamlines in the presence of flow and that the strength of the flow field determined directional bias of migration along the streamline. We provide evidence that CCR7-dependent autologous chemotaxis is the mechanism by which cells migrate with the flow, while a competing CCR7-independent mechanism leads to migration against the flow. Furthermore, we demonstrate these competing mechanisms are a powerful migrational stimulus, which likely play an important role in development of metastatic disease.by William J. Polacheck.S.M

Integrins in Ovarian Cancer: Survival Pathways, Malignant Ascites and Targeted Photochemistry

Integrins are surface adhesion molecules that, upon binding to ligands, cluster to form adhesion complexes. These adhesion complexes are comprised of structural and regulatory proteins that modulate a variety of cellular behaviors including differentiation, growth, and migration through bidirectional signaling activities. Aberrant integrin expression and activation in ovarian cancer plays a key role in the detachment of cancer cells from primary sites as well as migration, invasion, and spheroid formation. An emerging area is the activation or rearrangement of integrins due to mechanical stress in the tumor microenvironment, particularly in response to fluid shear stress imparted by currents of malignant ascites. This chapter describes the role of integrins in ovarian cancer with an emphasis on crosstalk with survival pathways, the effect of malignant ascites, and discusses the literature on integrin-targeting approaches in ovarian cancer, including targeted photochemistry for therapy and imaging

Inhibition of αvβ5 Integrin Attenuates Vascular Permeability and Protects against Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is a leading cause of AKI. This common clinical complication lacks effective therapies and can lead to the development of CKD. The αvβ5 integrin may have an important role in acute injury, including septic shock and acute lung injury. To examine its function in AKI, we utilized a specific function-blocking antibody to inhibit αvβ5 in a rat model of renal IRI. Pretreatment with this anti-αvβ5 antibody significantly reduced serum creatinine levels, diminished renal damage detected by histopathologic evaluation, and decreased levels of injury biomarkers. Notably, therapeutic treatment with the αvβ5 antibody 8 hours after IRI also provided protection from injury. Global gene expression profiling of post-ischemic kidneys showed that αvβ5 inhibition affected established injury markers and induced pathway alterations previously shown to be protective. Intravital imaging of post-ischemic kidneys revealed reduced vascular leak with αvβ5 antibody treatment. Immunostaining for αvβ5 in the kidney detected evident expression in perivascular cells, with negligible expression in the endothelium. Studies in a three-dimensional microfluidics system identified a pericyte-dependent role for αvβ5 in modulating vascular leak. Additional studies showed αvβ5 functions in the adhesion and migration of kidney pericytes in vitro Initial studies monitoring renal blood flow after IRI did not find significant effects with αvβ5 inhibition; however, future studies should explore the contribution of vasomotor effects. These studies identify a role for αvβ5 in modulating injury-induced renal vascular leak, possibly through effects on pericyte adhesion and migration, and reveal αvβ5 inhibition as a promising therapeutic strategy for AKI

Probabilistic Voxel-Fe model for single cell motility in 3D

Background: Cells respond to a variety of external stimuli regulated by the environment conditions. Mechanical, chemical and biological factors are of great interest and have been deeply studied. Furthermore, mathematical and computational models have been rapidly growing over the past few years, permitting researches to run complex scenarios saving time and resources. Usually these models focus on specific features of cell migration, making them only suitable to study restricted phenomena. Methods: Here we present a versatile finite element (FE) cell-scale 3D migration model based on probabilities depending in turn on ECM mechanical properties, chemical, fluid and boundary conditions. Results: With this approach we are able to capture important outcomes of cell migration such as: velocities, trajectories, cell shape and aspect ratio, cell stress or ECM displacements. Conclusions: The modular form of the model will allow us to constantly update and redefine it as advancements are made in clarifying how cellular events take place.European Research Council (Project ERC-2012-StG 306751)Spain. Ministerio de Economia y Competividad (DPI2012-38090-C03-01)Spain. Ministerio de Economia y Competividad (FPI Grant BES-2010-029927)Singapore-MIT Alliance for Research and Technolog

Microfabricated blood vessels for modeling the vascular transport barrier

The vascular endothelium forms the inner lining of blood vessels and actively regulates vascular permeability in response to chemical and physical stimuli. Understanding the molecular pathways and mechanisms that regulate the permeability of blood vessels is of critical importance for developing therapies for cardiovascular dysfunction and disease. Recently, we developed a novel microfluidic human engineered microvessel (hEMV) platform to enable controlled blood flow through a human endothelial lumen within a physiologic 3D extracellular matrix (ECM) into which pericytes and other stromal cells can be introduced to recapitulate tissue-specific microvascular physiology. This protocol describes how to design and fabricate the silicon hEMV device master molds (takes ~1 week) and elastomeric substrates (takes 3 d); how to seed, culture, and apply calibrated fluid shear stress to hEMVs (takes 1-7 d); and how to assess vascular barrier function (takes 1 d) and perform immunofluorescence imaging (takes 3 d)

Mechanotransduction of fluid stresses governs 3D cell migration

Solid tumors are characterized by high interstitial fluid pressure, which drives fluid efflux from the tumor core. Tumor-associated interstitial flow (IF) at a rate of ∼3 µm/s has been shown to induce cell migration in the upstream direction (rheotaxis). However, the molecular biophysical mechanism that underlies upstream cell polarization and rheotaxis remains unclear. We developed a microfluidic platform to investigate the effects of IF fluid stresses imparted on cells embedded within a collagen type I hydrogel, and we demonstrate that IF stresses result in a transcellular gradient in β1-integrin activation with vinculin, focal adhesion kinase (FAK), FAK[superscript PY397], F actin, and paxillin-dependent protrusion formation localizing to the upstream side of the cell, where matrix adhesions are under maximum tension. This previously unknown mechanism is the result of a force balance between fluid drag on the cell and matrix adhesion tension and is therefore a fundamental, but previously unknown, stimulus for directing cell movement within porous extracellular matrix.National Science Foundation (U.S.). Graduate Research Fellowshi

Matrix degradability controls multicellularity of 3D cell migration

A major challenge in tissue engineering is the development of materials that can support angiogenesis, wherein endothelial cells from existing vasculature invade the surrounding matrix to form new vascular structures. To identify material properties that impact angiogenesis, here we have developed an in vitro model whereby molded tubular channels inside a synthetic hydrogel are seeded with endothelial cells and subjected to chemokine gradients within a microfluidic device. To accomplish precision molding of hydrogels and successful integration with microfluidics, we developed a class of hydrogels that could be macromolded and micromolded with high shape and size fidelity by eliminating swelling after polymerization. Using this material, we demonstrate that matrix degradability switches three-dimensional endothelial cell invasion between two distinct modes: single-cell migration and the multicellular, strand-like invasion required for angiogenesis. The ability to incorporate these tunable hydrogels into geometrically constrained settings will enable a wide range of previously inaccessible biomedical applications

Synthetic extracellular matrices with tailored adhesiveness and degradability support lumen formation during angiogenic sprouting

Current tissue engineering strategies lack materials that promote angiogenesis. Here the authors develop a microfluidic in vitro model in which chemokine-guided endothelial cell sprouting into a tunable hydrogel is followed by the formation of perfusable lumens to determine the material properties that regulate angiogenesis

A non-canonical Notch complex regulates adherens junctions and vascular barrier function

The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and hemostasis1. Hemodynamic shear stress plays a critical role in maintaining endothelial barrier function2, but how this occurs remains unknown. Here, using an engineered organotypic model of perfused microvessels and confirming in mouse models, we identify that activation of the Notch1 transmembrane receptor directly regulates vascular barrier function through a non-canonical, transcription independent signaling mechanism that drives adherens junction assembly. Shear stress triggers Dll4-dependent proteolytic activation of Notch1 to reveal the Notch1 transmembrane domain – the key domain that mediates barrier establishment. Expression of the Notch1 transmembrane domain is sufficient to rescue Notch1 knockout-induced defects in barrier function, and does so by catalyzing the formation of a novel receptor complex in the plasma membrane consisting of VE-cadherin, the transmembrane protein tyrosine phosphatase LAR, and the Rac1 GEF Trio. This complex activates Rac1 to drive adherens junction assembly and establish barrier function. Canonical Notch transcriptional signaling is highly conserved throughout metazoans and is required for many processes in vascular development, including arterial-venous differentiation3, angiogenesis4, and remodeling5; here, we establish the existence of a previously unappreciated non-canonical cortical signaling pathway for Notch1 that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodeling