Tissue engineering complex and mechanically active microenvironments for studies in asthma and cancer

The goals of tissue engineering include recapitulating specific tissue functions for regenerative medicine and developing in vitro models of human tissues to study human physiology and pathophysiology and for testing and screening drugs before expensive clinical trials. Success of these goals relies on the capability to recapitulate the complex interactions between cells and their microenvironments in vitro, which requires interdisciplinary approaches. Tissue engineering has evolved greatly in the past two decades, with notable progress in replacing function in tissues such as cartilage, bone, arteries, skin, and pancreatic islets. What is lacking, however, are examples of, and design principles for, engineering complex microenvironments to understand disease progression. The two particular examples addressed in this thesis are (1) airway remodeling in asthma and (2) tumor invasion and metastasis in cancer. These diseases involve complex interactions between different cell types as well as extracellular matrix remodeling, which, together with the mechanical environment that affects cell-cell and cell-matrix interactions and that can change in a diseased state (or in some cases induce the disease state), collectively cause the pathology. Such disease states are difficult to study in vivo because (a) one cannot easily tease out mechanisms or have much control over these complex interactions in an animal model, and (b) there are many differences between animal and human cells. The aim of this thesis was to engineer mechanically dynamic microenvironments for the study of two disease processes: viral infection in asthmatic remodeled airways and cancer metastasis to the lymph node. These involved multidisciplinary approaches and drew tools and methods from mechanobiology, materials science, cell biology, and molecular biology. First, we developed and characterized a relevant 3D in vitro model of the bronchial mucosa with a functional epithelial barrier, including improved epithelial differentiation, ciliogenesis, and mucociliary function and showed for the first time that dynamic compression, such as that seen in asthmatic bronchoconstriction, enhances viral infection of epithelial and subepithelial cells using lentiviral technology and may help explaining why asthmatic patients are more susceptible to viral infection. However, the strain device had deficiencies in reproducibility, ease of use, time to assemble, and most importantly, homogeneous distribution of strain (because collagen and other natural extracellular matrices are mechanically weak since they have >99% water and cannot retain residual strain). To address these problems, we engineered a completely different dynamic strain device, which includes an elastic porous superstructure to contain the collagen and which can modulate stress/strain to the entire culture system. We characterized this device and showed that we could impose dynamic strains to 3D soft, hydrated cultures over relatively long periods of time and with variable amplitude and frequency in a uniform way. The device has the potential to be used in a wide spectrum of applications, including the possibility to study the correlation between mechanostimulation and metastatic potential of tumor cells in the context of lung metastasis of many cancer types. Finally, we used this material as a supporting structure for tissue-generated stress such as that generated by myofibroblasts, and in particular the ones that build up the lymph node stroma in the paracortex. The importance of these cells relies on their ability to form conduits for chemokine transport to high endothelial venules, where lymphocytes enter the node, the ability to form a platform for directing immune cell migration and interactions, and the ability to secrete two chemokines that are important to regulate lymphocyte adhesion and migration: CCL19 and CCL21. Specifically, we found that lymph node stromal cells use this supporting composite to remodel and reorganize their surrounding matrix into physiologically relevant bundles. By giving these cells the relevant stimuli (matrix microenvironment and flow), we could recapitulate certain features of the lymph node microenvironment, including a physiological stromal structure and chemokine profile, that may be critical for understanding immune and cancer cell interactions there. Using this model we demonstrated for the first time that slow flow through the lymph node stroma can itself drive cell and stromal organization and upregulates CCL21 production. Finally, we incorporated this engineered lymph node model into a larger model of the tumor-lymphatic-lymph node pathway to model the relative modulation of tumor growth, migration within a lymphatic "channel" towards the lymph node, and behavior within this lymph node environment. We used human lymphatic endothelial cells and a mass of transduced tumor cells with engineered chemokine profiles. We showed how CCR7 signaling correlates with the ability of tumor cells to metastasize to the lymph node and to interact with lymph node stromal cells once they reach the node. Our findings demonstrate how a multidisciplinary tissue engineering approach allows creating complex microenvironments in vitro that include both physical and chemical cues and allows recapitulating tissue function in vitro and investigating dynamic pathophysiological diseases like asthma and cancer metastasis. It combines both the development of these model systems and some examples of their usefulness to gain insight into asthma pathophysiology and cancer biology.