An all-human ex vivo model for inflammation and tumour cell metastasis: high resolution imaging of cellular interactions and molecular events at the blood vessel wall.

An all-human ex vivo model for inflammation and tumour cell metastasis: high resolution imaging of cellular interactions and molecular events at the blood vessel wall. Increasing numbers of animals are used for molecular and cellular analysis of inflammation and tumor cell metastasis. This is further accelerated by the growing number of transgenic and knock-out mouse and rat strains currently available, which allows direct testing of the lack or mutation of a specific gene for a particular physiological process. In parallel, the intravital imaging of cellular traffic, such as analysis of lymphocyte transendothelial- or intranodal migration or the metastasis of tumor cells, has received increasing interest. Due to rapid developments in instrumentation, this imaging has become accessible to many laboratories. This further contributes to increasing numbers of animals being used for these experiments. In vitro alternatives for studies on inflammation and metastasis are mainly based on the use of monolayers of human endothelial or epithelial cells, cultured on porous substrates or microscopy slides for imaging. This approach provides many means to investigate molecular processes in leukocytes, in tumor cells or in the endothelium, linked to cell adhesion and transmigration. However, it is also known that endothelial cells loose their tissue-specific features upon isolation and in vitro culture. As a result, in vitro models for inflammation or metastasis are commonly criticized for their limited physiological relevance. This proposal aims to develop an ex vivo assay that approximates the physiological situation in humans to reduce the number of animal studies on inflammation and cancer. The assay is based on the use of intact human blood vessels allowing high-resolution imaging of cellular traffic and subcellular events in human endothelial cells. In this set-up the endothelium is still attached to the underlying basement membrane and in the vicinity of vascular pericytes and smooth muscle cells, cell types in the vascular wall that are known to affect endothelial cell behavior. Dependent on vessel wall thickness, we will use wide-field or confocal fluorescent microscopy to image intact human blood vessels (‘whole mount’), or use human vessels that are opened longitudinally to allow ‘en face’ imaging. To set up the assay we will use arteries and veins from human umbilical cords. The vessels will be cannulated (for ‘whole mount imaging’) or mounted in a flow chamber (for ‘en face’ imaging). This allows the application of physiological flow in combination with high resolution imaging. Directly-labelled fluorescent antibodies, e.g. to ICAM-1 or to VE-cadherin, will be used to image surface molecules and to monitor junctional integrity as a measure for healthy endothelium. Subsequently, we will image adhesion and transendothelial migration of primary human neutrophils or monocytes, routinely isolated in our institute, as a model for inflammation and we will use human invasive tumor cell lines (e.g. the MDA-MB-231 cell line of which a stable GFP- or RFP-transfectant is commercially available, or the A2058 invasive human melanoma cell line) to model tumor cell metastasis. Finally, viral transduction will be used to express fluorescently tagged proteins or shRNA in the vascular wall. This will allow us to monitor protein dynamics and function in the context of leukocyte or cancer cell adhesion and transmigration. All techniques are running in the lab and our recent data show that these approaches are technically well feasible. Once the assay has been optimized using blood vessels from the umbilical cord, the next step will be to use, in collaboration with the department of Pathology, AMC, Amsterdam, blood vessels from the mesentery of cancer- or IBD-(Inflammatory Bowel disease) patients. Together, these approaches will result in establishing a relevant ex vivo alternative for both in vitro experiments with isolated human endothelial cells and intravital studies on live animals.