Part one of a two-part series.
QUEBEC CITY, Canada — Flow cytometry, long regarded as a useful method for clinical diagnostics and basic biological research, has been slow to take hold as a tool for high-throughput cell-based drug screening, despite having qualities that would seemingly make it attractive for such an application.
Recently, however, scientists from academia and industry have begun to consider flow cytometry more seriously as a high-throughput drug-discovery tool, as evidenced by an increase in the number of presentations devoted to the subject at the International Society for Analytical Cytometry congress, held here this week.
In fact, some of these presenters feel that flow cytometry used as a drug-screening tool has a lot in common with high-content cell-based screening in terms of capabilities and challenges. This, in turn, has led people in the industry to believe that drug makers will increasingly adopt flow cytometry in the way that they have HCS over the past decade.
The number of presentations on flow cytometry for drug discovery was noticeably larger at this year's ISAC meeting as compared with the last ISAC meeting, held in 2004 in Montpelier, France. Still, only a relatively small percentage of the overall presentations at ISAC covered the topic.
"No one believes that there is this capability [to use flow cytometry as a drug-screening tool]. It's a stretch for many people because they're just not accustomed to thinking this way."
Flow cytometry has several built-in attributes that make it attractive for drug screening: the ability to perform single-cell and multiplexed analyses, its high degree of sensitivity, and extremely fast data acquisition. But the technology also has several limitations — including weak throughput, overwhelming data-analysis needs, and a lack of standards — that have made pharma and academia think twice about using it for screening applications, preferring instead to stick with basic research, clinical diagnostics, and even biochemical assays.
"My supervisors have asked me for years whether flow cytometry would ever be used for drug discovery," Phil Marder, a researcher advisor for Eli Lilly, said at the conference. "My answer has always been, 'not in its present form.'" Marder made his comments during his introduction of a presentation by Larry Sklar, a University of New Mexico researcher attempting to commercialize a high-throughput sampling system for flow-based drug discovery (see related story, "UNM-Luminex Alliance …", this issue). In his introduction, Marder described Sklar's work as a "promising new method" that may help change that thinking.
But Marder and others' thinking is slowly evolving. For example, Marder himself has recently begun to champion flow cytometry as a tool for drug activity biomarker assays, and co-authored multiple posters or other presentations at the conference covering topics such as the development of a flow-based assay for drug inhibition or stimulation of the p38 MAP kinase signaling cascade in monocytes, which has implications in inflammatory diseases and cancer.
Garry Nolan of Stanford University is another researcher whose recent work may help open the door for flow cytometry as a drug-screening tool. In an ISAC lecture, Nolan discussed how he uses flow cytometric analysis to detect activated kinases and phosphoproteins in primary-cell signaling pathways.
This work has been enabled by a "barcoding" technique for multiplexed flow cytometry developed by Nolan and graduate student Peter Krutzik that was published in a recent issue of Nature Methods (see CBA News, 4/28/2006). By combining this method with a form of machine learning, Nolan and Krutzik have been able to develop signaling system network graphs in primary cells, and determine the effects of kinase inhibitors on the pathways of specific cell subsets — work that has broad implications in drug screening and personalized medicine.
In another lecture, Mario Roederer, chief of the Immunotechnology section in the laboratory of immunology at the National Institutes of Health's Vaccine Research Center, discussed how his lab has been using flow cytometry to evaluate T-cell response to various HIV-related challenges, which is an active front in HIV vaccine development.
"Flow is the most sensitive way to measure a response. It's a single-cell analysis, so each cell gets measured individually, as opposed to a population average, which is what you get in other assays."
Specifically, Roederer and colleagues use flow cytometry to measure functional responses, such as cytokine profile, cytotoxicity, and proliferation from individual T-cells, with the aim of discovering a combination of challenges that would provoke an ideal immunoprotective T-cell response. Such advances could become a starting point for vaccine development.
But these kinds of drug-screening projects are still few and far between.
"I've gone through the [ISAC] program, and there is only a handful of abstracts where people are actually setting up the type of biological system that would even be amenable to [a flow-based] screen," Sklar told CBA News. "No one believes that there is this capability. It's a stretch for many people, because they're just not accustomed to thinking this way."
Sklar said that through his research he has been attempting to address one of the biggest issues holding flow cytometry back as a drug-discovery tool: sample delivery from well plates, the format to which the pharma industry is most accustomed.
"The major issue is how to deliver samples to a flow cytometer at a rate that is compatible with screening," Sklar said. "Historically, the types of deliveries that have been available were manual, tube-to-tube. There have been a number of commercial projects over the years that have delivered samples at one to two per minute, and have appeared on Becton Dickinson and Beckman Coulter instruments, but for a 384-well plate, it could still take hours.
"These types of sampling rates have not been compatible with screening as is required for a discovery operation, unless the libraries are highly focused," he added.
Flow's 'Most Sensitive' Advantage
Some pharmas have already made headway. Novasite Pharmaceuticals is one of the only existing drug-discovery operations whose screening platform is exclusively based on flow cytometry, and focused small-molecule libraries are exactly the reason why, Teresa Bennett, associate director for drug discovery at the company, told CBA News.
Like Sklar's group, Novasite has developed a proprietary sample-handling technique that allows it to run cell-based functional screens for allosteric modulators of GPCRs from 96-well plates.
"We don't do high-throughput screening," Bennett said. "At the moment, we screen maybe 300 compounds a day … and we screen very focused libraries. Instead of doing massive amounts of screening, we concentrate on specific chemistries, have generated a small screening library, and we screen against peptide receptors."
Bennett said flow cytometry has inherent advantages for cell-based drug screening, in particular its sensitivity.
"Flow is the most sensitive way to measure a response," she said. "It's a single-cell analysis, so each cell gets measured individually, as opposed to a population average, which is what you get in other assays.
"So we can detect a rare event," Bennett added. "If 10 percent of the cells are responding, we detect that, and you can miss that in other assays. We have about a 50-fold signal-to-background resolution, so we find things that other people miss. We've been given libraries that nobody finds anything in, and we've found hits."
Bennett added that the hits are not likely to be lead compounds because of their low potency, "but they give you a starting point to identify new scaffolds. Then you can go back to your chemists, work off of that, screen again, and build from there."
Another reason Novasite chooses to use flow cytometry for its discovery platform is its multiplexing capabilities. Standard flow cytometers can measure anywhere from a handful to a dozen or more parameters, with some of the newest models able to assess as many as 25 parameters simultaneously.
This is a double-edged sword, however, as multiplexing of such magnitude produces an enormous amount of data, requiring sophisticated and dedicated data-analysis methods.
"We get a load of information from each screen, and how to manage the data is still a question," Bennett said. "We had to write our own software because it's not available [elsewhere]. One of the sessions [at ISAC] was addressing that. It's getting to the point where maybe standards should be discussed."
If these roadblocks to wider drug-screening adoption sound familiar, they should: The same problems slowed the adoption of high-content imaging as a drug-discovery tool. And although those challenges have not been completely solved for HCS, enough progress has been made so that almost every pharmaceutical company now owns and operates at least one, if not several, high-content imaging platforms for drug discovery.
— Ben Butkus ([email protected])
Part two of this article will appear in the June 2 issue of CBA News. It will cover the slow adoption of flow cytometry as a drug-discovery tool; the role — or lack thereof — of flow cytometry vendors in that process; other companies developing approaches for applying flow-based platforms to drug discovery; and where flow cytometry might be best marketed for drug discovery.