Engineering Ex Vivo Models for Probing Tumor, Lymphatic, and Immune Interactions in Metastasis and Immunotherapy

My thesis work has been based on developing a model of the tumor, lymphatic, and immune landscape. I've utilized unconventional materials, such as paper, recycled pipette tip holders, and double-sided tape, to fabricate a system that is both easy to build and easy to adapt. In this effort, I've modeled several biological processes that would otherwise be difficult to observe using traditional in vitro methods. In chapter 1, I provide an overview of the complexities of the tumor-immune microenvironment. I describe a complicated relationship between vascular endothelial growth factor C (VEGFC), metastasis, and immune education. I also describe current methods for modeling biological interactions in vitro and the limitations of these methods. In chapter 2, I describe the design and optimization of two devices for the culture of ex vivo tissue. In the vertical flow design, we see long-term viability of several cell lines and demonstrate how this device may be used for studying drug resistance. In the horizontal flow design, we have the ability to include upstream and downstream cell populations. In both devices, we utilize materials that can be easily modified and translated to other labs. In chapter 3, we use the horizontal flow design to study the formation of a pre-metastatic niche. Here, we culture lymph nodes that have been pre-educated in vivo by tumors either with or without VEGFC expression. We find that wild-type melanoma cells survive better in lymph nodes pre-educated by the VEGFC expressing tumor. With the addition of collagen-binding IL-12, we see a dampened pro-survival effect in the VEGFC educated tumor-draining lymph node. Finally, we visualize migration towards a pre-metastatic niche. Tumor draining lymph nodes pre-educated by VEGFC expressing tumors promote the migration of cancer cells ex vivo. In chapter 4, we probe the effects of combinatorial immunotherapy, checkpoint inhibitors anti-PD-1 and anti-CTLA-4 with collagen-binding IL-12, on tumor draining lymph nodes and spleens pre-educated by VEGFC expressing tumors. In both the lymph node and the spleen we observe a stronger immunotherapy effect when a tumor expresses VEGFC. This suggests VEGFC may act to potentiate immunotherapy through the pre-education of secondary lymphoid organs with tumor specific antigens. In chapter 5, we explore the impact that lymphatic transport has in another disease indication, lipedema. Here, I present a continuation of work performed in the Swartz Lab, where we purified extracellular vesicles from lipedema patient lipoaspirate. We find 31 differentially expressed miRNAs in stage 3 lipedema patients. I also describe early efforts to characterize changes in lipedema skin lymphatic vessel structure, fibrin clotting, and NETosis. Finally, in chapter 6, I offer a brief overview of my findings. I also suggest future directions that should be further explored in modeling tumor, lymphatic, and immune interactions.