More than a decade after Judah Folkman suggested that anti-angiogenesis could be a new approach to treating cancer, drug makers have been trying to develop therapeutics that inhibit the supply of blood to solid tumors.
More recently, a small number of cell-based assay and high-content screening developers have been trying to apply their technologies and methodologies to the angiogenesis space in the hopes that pharmas will use their tools in drug discovery [see sidebar].
At least five cell-based assay vendors and drug makers have developed tools designed to help researchers observe angiogenesis, and scores of academic scientists have been quietly building their own entrants into the market. Yet in spite of these efforts, and despite the widely held notion that HCS technologies are particularly well-suited to angiogenesis, there are no reliable assays on the market today for the indication.
Now, a team of researchers from the University of Kentucky has entered the mix and is developing a cell-based screening platform for anti-angiogenesis drugs as well as angiogenesis stimulators.
Using internally developed software and a one-year, $177,000 grant from the National Institute of Neurological Disorders and Stroke, the researchers aim to create an automated high-content and high-throughput imaging system to analyze three-dimensional growth patterns of vascular endothelial cells in a collagen matrix.
"Should we be successful, we would look for partners" to develop a commercial platform, which could have broader applications than just vascular growth, for example growth of neuron dendrites.
Royce Mohan, an assistant professor at the department of ophthalmology at the university who is leading the research, likened these growth patterns to human fingerprints, which, like the growth of blood vessels, are influenced by the local environment and differ, for example, between identical twins.
According to Mohan, anti-angiogenesis drugs can alter the local environment in different ways, even though this would not be predictable from the chemical structure of the drugs.
For the sprouting assay, the researchers have created spheroids of endothelial cells growing on beads, which can be placed into microtiter plates and stimulated to grow. What is especially tricky is to produce uniform spheroids with equal numbers of cells, Mohan said. "A lot of quality control has gone into this part," he said.
So far, the scientists have been using 96-well plates but are hoping to move up to 384-well plates soon.
To image the growth patterns, the researchers will be using a Nikon TE2000 inverted microscope, which is also used for other purposes than high-content screening in the department. Nikon representatives have helped with the instrument's various applications, but "they are not technically involved in the development of the platform," according to Mohan.
Mohan said the "power of the approach" lies in the internally developed image-analysis software, which was developed by collaborator Daniel Lau, an assistant professor in the department of electrical engineering and computer science at the University of Kentucky. Lau originally developed imaging software for security purposes that could be used, for example, to analyze the three-dimensional structure of fingerprints and for three-dimensional face recognition. He and Moran have begun collaborating just recently, Mohan said.
Angiogenesis Applications Remain Obvious
Several years after drug makers began studying ways to develop cancer drugs that block the supply of blood to solid tumors, there are no reliable high-content assays on the market that can help them perform this kind of research.
This has been a challenge to the nascent high-content screening and cell-based assay research community because in the years that followed Judah Folkman's angiogenesis discoveries, HCS scientists have believed that these kinds of tools can lend themselves particularly well to angiogenesis research and drug development.
But that doesn't mean HCS tool vendors aren't trying.
Ivan Baines, scientific coordinator and director of services and facilities at the Max Planck Institute for Molecular Cell Biology and Genetics, in July said he believes a classic example of assays that his center is developing involves angiogenesis, for which reliable screening assays have been long sought after but difficult to achieve for the pharmaceutical industry.
Another company attempting to tackle the angiogenesis space is Definiens, whose Cellenger technology helped customer Novartis Pharma to develop its high-content assay for the indication. According to Definiens' web site, this in vitro assay, which enables image-analysis of induced in vitro vascular cell growth, comprises smooth muscle cells and vascular endothelial cells that "are used to measure the influence of possible drug candidates on cell growth."
In the meantime, contract research organization Phylonix has obtained an NCI grant in 2002 to study angiogenesis in zebrafish-based assays. More recently, in March of this year, Xenogen, an Alameda, Calif.-based biotech specializing in small-animal in vivo imaging assays, won a US patent granting it protection for screening assays both in live cells and small animals for angiogenesis-modulating compounds.
The imaging of the three-dimensional vascular sprouts is done in two dimensions, but because collagen is transparent, the researchers will sample images of sprouts in different planes. "This is where the proprietary software plays an important role," according to Mohan.
The researchers have not yet signed any agreements with vendors, Mohan said, but are protecting the intellectual property for the software and its applications. "Should we be successful, we would look for partners" to develop a commercial platform, Mohan said. "We see opportunities with the growing use of high-content screening … as well as with stand-alone software applications." Such a platform could have broader applications than just vascular growth, he added, for example growth of neuron dendrites.
As part of the NIH-funded project, the scientists are planning to validate their assay and image-analysis software by performing a number of small-scale drug screens with academic collaborators who Mohan said have ties to pharmaceutical companies. So far, there have not been any direct collaborations with drug companies, but "once we start to demonstrate [the system's] applications, there will probably be a lot more interest," he said.
According to Mohan, vendors have been focusing on isolated steps of the assay, such as invasion or migration. However, "biology is that much more complex, each step integrated with the next," he said. "It's better to have an assay that represents many of the steps."
The reason why others have not taken this approach? "It's difficult," he said.