In vitro Studies of Transendothelial Migration for Biological and Drug Discovery

Abstract

Inflammatory diseases and cancer metastases lack concrete pharmaceuticals for their effective treatment despite great strides in advancing our understanding of disease progression. One feature of these disease pathogeneses that remains to be fully explored, both biologically and pharmaceutically, is the passage of cancer and immune cells from the blood to the underlying tissue in the process of extravasation. Regardless of migratory cell type, all steps in extravasation involve molecular interactions that serve as a rich landscape of targets for pharmaceutical inhibition or promotion. Transendothelial migration (TEM), or the migration of the cell through the vascular endothelium, is a particularly promising area of interest as it constitutes the final and most involved step in the extravasation cascade. While in vivo models of cancer metastasis and inflammatory diseases have contributed to our current understanding of TEM, the knowledge surrounding this phenomenon would be significantly lacking without the use of in vitro platforms. In addition to the ease of use, low cost, and high controllability, in vitro platforms permit the use of human cell lines to represent certain features of disease pathology better, as seen in the clinic. These benefits over traditional pre-clinical models for efficacy and toxicity testing are especially important in the modern pursuit of novel drug candidates. Here, we review the cellular and molecular events involved in leukocyte and cancer cell extravasation, with a keen focus on TEM, as discovered by seminal and progressive in vitro platforms. In vitro studies of TEM, specifically, showcase the great experimental progress at the lab bench and highlight the historical success of in vitro platforms for biological discovery. This success shows the potential for applying these platforms for pharmaceutical compound screening. In addition to immune and cancer cell TEM, we discuss the promise of hepatocyte transplantation, a process in which systemically delivered hepatocytes must transmigrate across the liver sinusoidal endothelium to successfully engraft and restore liver function. Lastly, we concisely summarize the evolving field of porous membranes for the study of TEM.

Keywords: drug discovery; extravasation; hepatocyte transplantation; in vitro platforms; leukocytes; metastasis; porous membranes; transendothelial migration.

Figure 1

The leukocyte extravasation cascade is an elaborate process understood in part due to the use of well-controlled in vitro studies. Platforms incorporating vascular endothelial cells and physiological environmental conditions (e.g., shear stress) permit the highly controlled study of all steps of the leukocyte extravasation cascade: capture, rolling, arrest, intravascular crawling, and both paracellular and transcellular transendothelial migration. P and E selectin bind leukocytes to aid in the capture and rolling phases of extravasation. LFA-1/Mac-1: ICAM-1 and VLA-4: VCAM-1 form high affinity/avidity integrin/ligand interactions to halt leukocytes on the apical endothelial cell surface during arrest. Integrin activation is aided by chemokine signaling (neutrophil chemokine IL-8 and its receptors CXCR1/2 pictured here). In addition to leukocyte arrest and intravascular crawling, LFA-1/Mac-1: ICAM-1 interactions function to signal VE-cadherin junctional turnover and opening of the endothelial cell-cell junctions. Additionally, PECAM-1 and CD99 homophilic interactions between leukocytes and endothelial cells function to drive membrane mobilization from the lateral border recycling compartment (LBRC) to increase membrane surface area around the transmigrating leukocyte. The understanding of each of these components of leukocyte extravasation have been guided by the use of simple and modern in vitro systems. These same devices can, in turn, function as early stage drug discovery platforms for preventing devastating inflammatory diseases.

Figure 2

From local invasion to micrometastasis formation, metastatic cancer cells take a complex, and strenuous path to complete the metastasis cascade. While the percentage of cancer cells able to successfully form a secondary tumor is low, metastasis accounts for 90% of cancer mortality, thus making it one of the most important oncological processes that can be exploited for drug discovery. Cancer cells begin the metastasis cascade by transitioning from a static, epithelial phenotype to a dynamic, mesenchymal phenotype (EMT), and invading surrounding tissue. Once on the basal endothelial cell surface, metastatic cancer cells may intravasate into circulation, moving across the endothelium from the abluminal to luminal surface. Shear blood flow releases the cancer cell from the luminal vascular wall where it is carried to a secondary location. Size restriction in the capillaries halts the cancer cell, where β₁ integrins can attach and facilitate transendothelial migration. In some cases, P and E selectin as well as β₂ integrins have been implicated in cancer cell extravasation, however, more studies are needed to understand the role these proteins play. Given the ridged nature of the cell nucleus, all contents of a transmigrating cancer cell are pushed into the basement membrane until the last minute when which the nucleus is squeezed through. Lamin A/C phosphorylation permits nuclear softening and severe deformation, aiding in the push across the endothelium. Successful transendothelial migration constitutes the final step in forming a secondary tumor. In vitro models have greatly contributed to these fresh understandings of metastasis and are opening the door to novel drug targets.

Figure 3

Emerging topics in the study of transendothelial migration (TEM). Hepatocyte transplantation is a promising treatment for liver disease and failure. In this process, donor hepatocytes are perfused into the portal vein where they may translocate to the liver sinusoids. Similar to cancer cells, size restrictions in the liver microvasculature, as well as potential integrin interactions, trap the hepatocytes at the apical endothelial cell surface. Entrapment resulting in ischemia-reperfusion events and Kupffer cell activation lead to liver sinusoidal endothelium disruption. Additionally, vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF), hepatocyte growth factor (HGF), and other factors released by native hepatocytes in the liver plate further drive endothelium disruption, permitting passage of transfused hepatocytes. Once across, donor hepatocytes integrate with native hepatocytes as mediated through the activation of matrix proteases and separation of gap junctions (146). The ultimate goal of this process is to engraft these donor hepatocytes into the liver parenchyma and restore liver function. While there has been some success using this treatment strategy, an improved understanding of the hepatocyte TEM process may lead to discoveries promoting enhanced engraftment of hepatocytes in the liver. Using in vitro devices to breakdown and study this process is key to this progress.

Figure 4

Porous cell culture support membranes are a vital and ever evolving feature of vascular mimetics for transmigration studies. The three main categories of porous membranes are conventional track-etched, silicon-based, and novel polymeric. Each of the main categories have pros (+) and cons (–) that may be weighed in the early stages of planning experimentation. Conventional track-etched membranes (polycarbonate filter shown here) have aided in years of transmigration studies, however, the onboarding of silicon-based (porous silicon nitride shown here) and novel polymeric (parylene membrane shown here) membranes has given way to many options for use within this field. Balancing usability and affordability will be key for drug discovery platforms.