January 1, 2008 (Vol. 28, No. 1)

New Assay Facilitates Functional Secondary Drug Screening

Compound efficacy, specificity, accuracy, and in vivo relevance are key drivers in the decision-making process for progressing drug candidates through the lead-optimization phase into clinical trials. Tools that deliver pertinent data in vitro facilitate better decisions and the selection of the best drug candidates. Understanding how cells adhere and move is a rapidly growing area of research, yet an assay to successfully replicate in vivo conditions has, until now, proved elusive.

Cellix’ (www.cellixltd.com) VenaFlux™ platform is an automated system capable of mimicking cellular behavior in human capillaries. Validated, dynamic, live-cell, flow assays deliver quantitative analysis of drug candidates in cell rolling, arrest, adhesion, and migration in the presence of potential drugs in numerous life science areas, in particular respiratory (asthma and COPD), cardiovascular (thrombosis and anti-inflammatory), autoimmune (rheumatoid arthritis and psoriasis), and oncology (angiogenesis and lymphoma).

Cellix’ ability to mimic human capillaries for executing continuous-flow assays in disposable, plastic biochips has been coupled with a pumping system and detection software to provide customers with a useful cell-based assay platform. This tool and others like it can provide solutions to the current bottlenecks in the drug discovery pipeline by offering an efficient means of screening drugs, eliminating false leads, and thereby potentially saving pharmaceutical companies millions of dollars.

The movement toward miniaturization, in order to increase throughput, accuracy, and efficiency in the development of new drugs, has lead to a demand for instruments and tools capable of handling microliter quantities of biological fluids and reagents. Cellix’ Microfluidic SP series and the VenaFlux platform meet such demands.

The VenaFlux platform (Figure 1) was designed to provide the researcher with a complete solution for dynamic studies of cell adhesion under well-defined shear stresses. The Microfluidics SP series includes the Mirus™ nanopump, Vena™ biochips, and DucoCell™ analysis software. Cellix provides a range of biochips (Vena8 and VenaEC).

The Vena8 biochips (Figure 2) contain eight parallel, enclosed microcapillaries for cell-based assays. Each microcapillary mimics the dimensions of a postvenule capillary and may be coated with a range of adhesion molecules. VenaEC biochips facilitate the growth of endothelial cells resulting in a microcapillary lined with these cells, which enables researchers to be one step closer to the true in vivo scenario.

The physicochemical behavior of liquids confined to microenvironments enables new strategies for delivering compounds to cells on a subcellular level. With all the biochips, cell suspensions may then be injected utilizing the Mirus nanopump, which supports a range of shear stresses simulating physiological flow. The Mirus nanopump is computer controlled via FlowAssay™ software. The biochips sit in a frame on an inverted microscope encased in a temperature- and CO2-controlled incubator.

DucoCell, a cell-analysis software package, provides reliable cell counting and analysis of several morphological parameters of cells within an image including area, diameter, perimeter, ellipticity, and form-factor. Analyses of several images may be executed automatically and subsequently exported to an Excel spreadsheet while simultaneously generating a graph from the results obtained.

Figure 1. VenaFlux Platform

VenaFlux Applications

As animal and bacterial genomes and proteomes are being fully probed with DNA chips and a wide array of analytical techniques, a picture of cells with dauntingly complex inner workings is emerging. VenaFlux allows the researcher to mimic the biochemical and biophysical complexity of the cellular microenvironment, which is beneficial in many different areas of study.

Immunology—Circulating leukocytes reach their tissue destinations through sequential stages, which include transition from flow to rolling along the endothelium, firm adhesion, and subsequent integrin-dependent transendothelial migration. These sequential processes constitute the currently accepted multistep leukocyte navigation paradigm, which is broadly applicable to different tissues, although the signals imposed by specific microenvironment conditions may vary.

An alteration to any of these stages can lead to autoimmune diseases or conditions that require immunomodulatory compounds to restore homeostasis. Several studies, including work done by Damon Lowes and Helen Galley at the Institute of Medical Sciences, University of Aberdeen, Scotland, have recognized the benefit of statin therapy to reduce the incidence of sepsis in ICU patients, while other studies have documented a benefit in reducing mortality. The potential protective effect of statin therapy is probably due to the immunomodulatory mechanisms of these compounds, which are capable of inhibiting the inflammatory pathways involved in sepsis.

LFA-1 is an integrin that is critical for T-cell adhesion, T-cell priming, and cytokine secretion. Statins inhibit LFA-1 interaction with ICAM. Data generated using the VenaFlux platform has shown that physiologically relevant concentrations of fluvastatin, mevastatin, lovastatin, simvastatin, or pravastatin markedly inhibit T-cell adhesion to ICAM equally (Figure 3).

Figure 2. Vena8 biochips

Therefore, the reported clinical outcome of septic patients receiving statin therapy can at least be partially associated with T cell-ICAM binding activities.

Cardiovascular—Platelet adhesion is an essential function in response to vascular injury and is generally viewed as the first step during which single platelets bind through specific membrane receptors to cellular and extracellular matrix constituents of the vessel wall and tissues. This response initiates thrombus formation that arrests hemorrhage and permits wound healing.

Pathological conditions that cause vascular alterations and blood-flow disturbances may turn this beneficial process into a disease mechanism that results in arterial occlusion, most frequently in atherosclerotic vessels of the heart and brain. Besides their relevant role in hemostasis and thrombosis, platelet-adhesive properties are central to a variety of pathophysiological processes that extend from inflammation to immune-mediated host defenses and pathogenic mechanisms as well as cancer metastasis.

All of these activities depend on the ability of platelets to circulate in the blood as sentinels of vascular integrity, adhere where alterations are detected, and signal the abnormality to other platelets and blood cells. In this respect, therefore, platelet adhesion to vascular wall structures, to one another (aggregation), or to other blood cells represent different aspects of the same fundamental biological process.

Detailed studies by many investigators, Dermot Kenny and Ger Meade of the Royal College of Surgeons in Ireland (RCSI) in Dublin among them, have been aimed at dissecting the complexity of these functions, and the results obtained now permit an attempt to integrate all the available information into a picture that highlights the balanced diversity and synergy of distinct platelet adhesive interactions.

VenaFlux allowed researchers at RCSI to mimic blood flow and monitor thrombus formation. Rolling/adhesion of platelets on Vena8 biochips, coated with adhesion molecule vwf, occurs at lower shear stress (4 and 40 dyne/cm2), whereas at higher shear stress (60 and 120 dyne/cm2) platelet thrombus formation appears. A threshold limit of approximately 60 dyne/cm2 is required for thrombus formation, conditions that occur in microcirculation.

Oncology—Although the spread of tumors is poorly understood, mainly due to the inavailability of an appropriate animal model, recent developments have provided insight into the complex cascade of specific events required to establish a metastatic deposit. As knowledge unfolds, cancer adjuvant therapy will be directed increasingly toward the prevention of the devastation caused by metastases.

Blood flow has traditionally been accepted as the only determinant of the site of a metastatic deposit. In metastasis, cells are spread from a primary tumor to a distant site, where they arrest and grow to form a secondary tumor. Interaction with endothelial cells at the initial and later stages of the metastatic cascade is mandatory for the passage of tumor cells into vessels at the primary site and exit from vessels at the metastatic site.

Several factors, including certain members of the immunoglobulin and selectin families, facilitate adherence of tumor cells to endothelium. Platelet-fibrin thrombi influence the arrest of tumor cells. Chemotactic factors released by endothelial cells enhance tumor-cell mobility.

VenaFlux can visualize and quantify the movement of tumor cells and their interactions with the endothelium as they travel through metastatic pathways within the body to arrest at secondary sites. It can also be used to quantify the morphology and functional capacity of tumor microvasculature, as well as the timing and dynamic effects of drugs targeted to disrupt tumor vasculaturization.

With the development of new fluorescent probes and reporter genes, VenaFlux has the potential to provide evidence of the timing and location of metabolic processes within the metastatic cascade that may serve as specific targets for the treatment of cancer.

Figure 3

Kate Fitzgerald, Ph.D., is applications specialist for Cellix. Web: www.cellixltd.com. Phone: 353 1 896 4016. E-mail: [email protected].

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