Engineered models of the lymphatic stroma to study cell and fluid transport

The lymphatic system plays essential roles in regulating fluid balance and immunosurveillance. Across the body, local lymphatic vessels collect waste in the form of lymph and deliver it to nearby lymph nodes (LNs) to be filtered and screened for pathogens. With broad implications in adaptive immunity, cancer metastasis, and cancer treatment, developing novel in vitro models will provide new platforms to explore lymphatic function in health and disease. This dissertation sought to develop tissue-specific engineered models of the LN stroma and the meningeal lymphatics to examine the transport of cells and fluid. Within the LN, fibroblastic reticular cells (FRCs) maintain a network of extracellular matrix conduits that guide varying rates of interstitial fluid flow (IFF) based on inflammatory state. Eventually, that flow exits the LN through the afferent lymphatics, consisting of lymphatic endothelial cells (LECs). We first developed a spatially organized model of the LN stroma consisting of a monolayer of LECs on the underside of a tissue culture insert and an FRC-laden hydrogel within. We demonstrate that high magnitude IFF (3.0 µm/s) had positive impacts on FRCs but disrupted the integrity of the LEC barrier, and these effects were accompanied by increased secretion of a variety of inflammatory chemokines. We also show that IFF of any magnitude decreased T cell egress from the model. Next, we sought to apply the LN stroma model toward understanding metastasis. LN metastasis is the most important prognostic factor in breast cancer, with size of metastasis informing treatment plan. Metastasis greatly alters the structure of the LN, which in turn alters transport. However, the impact of altered transport on cancer progression is not well understood. We added different numbers of breast cancer cells to our LN stroma model to examine tumor burden. We found that tumor cells invaded the LEC barrier at similar numbers regardless of initial burden. Additionally, at the highest tumor burden, diffusivity in the stroma was significantly decreased. Most excitingly, flow velocity was positively correlated with FRC spread in the hydrogel, demonstrating the contributions of FRCs to transport. Finally, we looked to the central nervous system (CNS). The meningeal lymphatics are responsible for draining cerebrospinal fluid to the cervical lymph nodes for CNS immunosurveillance. We developed a simple model of a meningeal lymphatic vessel lumen consisting of a monolayer of LECs on the underside of a tissue culture insert and a monolayer of meningeal fibroblasts within. This is, to our knowledge, the very first in vitro model of the meningeal lymphatics. We demonstrate that our model has barrier function and is capable of immune cell transmigration and egress. We examined how systemic chemotherapy for breast cancer could cause off-target disruption of the meningeal lymphatics and found that docetaxel was significantly deleterious. We further began to explore leukemia cell behavior in our LN stroma and meningeal lymphatics model. Throughout this dissertation, we emphasize the importance of incorporating fluid and cell transport into engineered models of immunity. These models represent a step toward building up the complexity of in vitro lymphatic models to improve pre-clinical screening and understand pathophysiology.