Summary:

Blood vessels are central to the treatment of many cancers. On the one hand, they form an obstacle to the effective passage of therapies from the bloodstream to solid tumours. On the other hand, they are also at the origin of the formation of new vessels via angiogenesis, a process that promotes tumour development and is the target of several anti-cancer therapies currently in clinical use. The tumour microenvironment plays an essential role in vessel morphology. In particular, the composition and physical properties of the extracellular matrix and perivascular cells strongly influence the physiology of endothelial cells and the permeability of the vascular barrier. In the context of inflammation, vascular hyperpermeability is a public health problem and a well-known side-effect of certain anti-cancer treatments. To study these vascular phenomena, we are proposing a new model combining microfabrication, tissue engineering and microfluidic technologies. These techniques combine 3D cell culture and perfusion approaches, enabling the construction of organs-on-a-chip. We have designed vessel-on-a-chip devices in which an initial channel is created in a collagen-based hydrogel using a microfluidic approach that relies on the viscosity properties of collagen. This initial channel, which constitutes the future lumen of the vessel, is then seeded with primary human endothelial cells which then form a confluent and cohesive monolayer, so as to mimic the inner surface of a blood vessel. This technique can be carried out once or twice, allowing the diameter of the final vessel to be varied as required, as well as creating a double layer of cells. The integrity of the endothelial barrier was studied by assessing the quality of the distribution of vascular endothelial-cadherin at adherens junctions and zona-occludens-1 for tight junctions. In addition, the permeability of the endothelium was studied using a videomicroscopy approach to quantify the real-time diffusion of a dextran-fluorescent agent through the vessel endothelium. The ability of endothelial cells to be activated by pro-inflammatory cytokines and their capacity to induce adhesion of immune cells and cancer cells to the inner surface of the vessel lumen were assessed. The biomimetic approach of this model was enriched by forming two distinct and concentric cell layers made up of endothelial cells and perivascular fibroblasts in order to approximate even more closely the structure and composition of a natural blood vessel and to observe the influence of interactions between the two cell types on vessel permeability. For example, the presence of perivascular fibroblasts significantly strengthens the endothelial barrier, particularly in response to thrombin. These vessels-on-a-chip are designed in a standardised multi-well plate format for use in robotic, high-throughput drug screening. Microfluidic devices are a technological milestone for answering complex biological questions. They aim to bridge the gap between overly simple 2D in vitro tests and costly, time-consuming and species-specific animal models. Here we propose a device suitable for screening molecules that effect vascular activation and permeability, particularly in the context of anti-cancer treatments.

Abstract:

Blood vessels are central to the treatment of many cancers. On the one hand, they form a barrier to the efficient passage of therapies from the bloodstream to solid tumors. On the other hand, they are also responsible for the formation of new vessels via angiogenesis, a process that promotes tumor development and is the target of several anti-cancer therapies currently used in the clinic. The tumor microenvironment plays an essential role in vessel morphology. In particular, the composition and physical properties of the extracellular matrix and perivascular cells strongly direct the physiology of endothelial cells and influence the permeability of the vascular barrier. In the context of inflammation, vascular hyperpermeability is a public health concern and a well-known side effect of some anti-cancer treatments. To study these vascular phenomena, we propose a new model combining microfabrication, tissue engineering and microfluidic technologies. These techniques combine 3D cell culture and perfusion approaches, allowing the construction of organs-on-a-chip. We have designed vessel-on-chip devices in which an initial channel is created in a collagen-based hydrogel using a microfluidic approach that relies on the viscosity properties of collagen. This initial channel, which constitutes the future lumen of the vessel, is then seeded with primary human endothelial cells which then form a confluent and cohesive monolayer, so as to mimic the inner surface of a blood vessel. This technique can be performed once or twice and allows the diameter of the final vessel to be varied as desired and also to create a double cell layer. The integrity of the endothelial barrier was studied by evaluating the quality of the distribution of vascular endothelial-cadherin at adherens junctions and zona-occludens-1 for tight junctions. On the other hand, the permeability of the endothelium was studied by implementing a videomicroscopy approach to quantify the real-time diffusion of a dextran-fluorescent across the vessel endothelium. The ability of endothelial cells to be activated by pro-inflammatory cytokines and their ability to induce adhesion of immune cells and cancer cells to the inner face of the vessel lumen were evaluated. The biomimetic approach of this model was enriched by forming two distinct and concentric cell layers consisting of endothelial cells and perivascular fibroblasts in order to more closely approximate the structure and composition of a natural blood vessel and to observe the influence of interactions between the two cell types on vessel permeability. Thus, the presence of perivascular fibroblasts significantly reinforces the endothelial barrier, particularly in response to thrombin. These vessels-on-a-chip are designed in a standardized multi-well plate format with the aim of being used for robotic and high-throughput drug screening. Microfluidic devices are a technological turning point for answering complex biological questions. They aim at bridging the gap between too simple 2D in vitro assays and expensive, time-consuming and species-specific animal models. We propose here a device adapted to the screening of effector molecules of vascular activation and permeability, especially in the context of anti-cancer treatments.