A microvessel-on-a-chip for studying how tumor-derived factors prime the endothelium for extravasation
Date
2022
Authors
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Publisher
University of Delaware
Abstract
Metastasis is a leading cause of disability and death from cancer. Each year, 50% of new cancer patients are diagnosed with metastasis and must undergo immediate and aggressive treatment. Within 5 years, 60% of these patients will die. ☐ These statistics translate to staggering mortality. In 2020 alone, 5.8 million patients died from metastasis. By 2040, this mortality will increase to an estimated 8.4 million deaths. ☐ Yet few effective treatments for metastasis exist. One reason is poor understanding about how circulating tumor cells escape through the endothelium into the surrounding tissue of distant organs. This process, called extravasation, is critical. If extravasation does not occur, distant metastases cannot form and 5-year survival increases from 25% to 56%. Better understanding extravasation is therefore key to stopping distant metastasis and reducing the mortality of metastasis overall. ☐ However, studying extravasation is challenging. One major roadblock is modeling extravasation and its underlying pathophysiology in vitro. Current culture models lack mechanical cues fundamental to producing an endothelial monolayer that replicates the endothelium in vivo. Improved modeling of extravasation in vitro could provide better understanding of its pathophysiology in vivo. This dissertation thus aimed to create a microvessel-on-a-chip model for studying extravasation and its underlying processes in vitro with physiological relevance and high spatiotemporal resolution not possible with traditional culture models. ☐ The first objective was developing the model. This consisted of reviewing existing microvessel-on-a-chip models for their advantages and disadvantages, then using this information to design the model, and finally developing processes to fabricate the model and form endothelial microvessels inside it. ☐ The second objective was developing tools to quantify key metrics of endothelial phenotype and function from the model. This consisted of developing a toolbox of image processing pipelines and corresponding ImageJ macros for quantifying permeability, patency, protein expression, and morphology. This toolbox provides a semi-automated method to obtain high-resolution data that is laborious, cumbersome, and often inaccurate to obtain via manual means while also offering new ways to quantify the aforementioned properties. ☐ The third objective was validating if the model can produce endothelial microvessels that replicate key properties of the endothelium in vivo. This consisted of quantifying the permeability, patency, protein expression, and morphology of microvessels cultured under static, flow, and inflammatory stimuli. Results show that flow is necessary to replicate the desired properties from in vivo. Furthermore, the microvessels exhibit very low permeabilities to 70kDa (~1E-08 cm/s) and 10kDa (~1E-07 cm/s) dextrans, few-to-no focal leaks (<1 leak/mm), dense and continuous adherens junctions, cell elongation and orientation with flow, cytoskeletal alignment with flow, and an appropriate response to inflammatory stimuli. ☐ The fourth and final objective was to apply the model to study how tumor proteins and tumor extracellular vesicles can prime the endothelium to facilitate extravasation. Results show that tumor proteins have a strong capacity to disrupt and inflame the endothelium: they increased permeability, decreased patency, and shifted endothelial cells to a more mesenchymal phenotype. In contrast, tumor extracellular vesicles showed limited capacity, if any, to disrupt or inflame the endothelium. While preliminary, these results raise important questions the current paradigm that tumor extracellular vesicles prime the endothelium for extravasation and highlight the utility of the microvessel-on-a-chip for studying this process. ☐ In total, this dissertation presents a microvessel-on-a-chip model for studying extravasation and its underlying processes in vitro. This model provides a promising addition to existing culture models and affords newfound physiological relevance and spatiotemporal resolution. By doing so, it can advance understanding of extravasation and thereby help improve treatments for metastasis.
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Keywords
Microvessel, Tumor, Endothelium, Extravasation