Second in a two-part series. Click here to read part one, which appears in the May 26 issue of Cell-Based Assay News.
Used as drug-discovery tools, high-content imagers and flow cytometers have some similarities — in particular, they can both perform highly multiplexed assays to interrogate physiologically relevant pathways using cells.
But they have one major difference: Their evolving marketing strategies have helped usher high-content imagers into drug-discovery labs while leaving flow cytometers to labor in basic research and diagnostics.
Flow cytometry has been around since the 1960s, but its use at pharmaceutical companies has been limited primarily to basic research applications, albeit high-throughput ones. But that may be changing — as those hoping to push flow cytometry into the drug-discovery arena may now have a template to follow in high-content imaging.
Imaging has been around even longer than flow cytometry, and had also traditionally been a basic research tool. But this began to change in the late 1980s and early 1990s with the advent of automated, or high-content, imaging.
Still, many feel that it took a significant targeted marketing charge to begin placing high-content imagers at pharmaceutical companies — a charge that most agree was led by the founders of Cellomics.
Meanwhile, a handful of researchers including the University of New Mexico's Larry Sklar were also eyeing flow cytometry as a drug-discovery tool — as were big tool vendors like Becton Dickinson and Beckman Coulter, although from a safe distance, and with the comfort of already having a significant market for flow cytometry in basic research and diagnostics.
"They were the leaders, and were the people out there with the early adopters and developing the systems. Those systems created enough pull in pharma and biotech that other manufacturers jumped in."
To Found or Not to Found?
"Cellomics was founded by three people with extraordinary capabilities in fluorescence imaging agents, cell biology, and engineering," Sklar said, referring to Cellomics co-founders Alan Waggoner, Lansing Taylor, and Terry Dunlay, respectively. "They were automating cell imaging as a laboratory tool, much in the same way that we were automating sampling for flow cytometry as a laboratory tool.
"They realized in the late 1990s, however, that there was in fact a drug-discovery [component], and I believe it was specifically their vision that drove imaging systems into pharma," Sklar added. "They were the leaders, and were the people out there with the early adopters and developing the systems. Those systems created enough pull in pharma and biotech that other manufacturers jumped in."
Sklar's group was similarly developing rapid sampling technology for flow cytometry that might enable high-throughput drug discovery, but it took a different tack. When Cellomics' founders were getting their business off the ground, Sklar and his team decided to remain in academia, choosing instead to partner with companies that could market their technology for them.
"We had the same vision at the same time," Sklar said. "Lans Taylor and Alan Waggoner left their academic positions and started companies, but our vision drove us not to start a company but to partner with other companies."
Sklar's decision, he said, turned out to be a mistake. First of all, the UNM Science and Technology Corporation, the university tech-transfer group that manages Sklar's IP, couldn't convince big boys like BD and BC to buy in as distributors or to incorporate the technology into their existing flow platforms.
Later, a few smaller biotechs took some interest in the technology, but according to Sklar, UNMSTC became embroiled in a dispute between two undisclosed partnering companies: one that wanted to use the technology exclusively and another that sought to distribute it. "Those visions were mutually incompatible," Sklar said. He and his team sided with the first company.
"We got caught up on one side and thought that we were likely to do better by associating ourselves with the company that was going to use it for discovery rather than the company that was going to distribute it," Sklar said. "That didn't work."
It turns out that there just wasn't enough of a buzz in drug-discovery circles to promote adoption of the technology — or to even consider flow as a drug-discovery tool at all.
"We have, over the years, licensed variations and copies of our technology a handful of times," Sklar said. "None of those have penetrated the marketplace. We misunderstood the driving forces in the tech market before it crashed.
"In a sense, you can blame me," Sklar added. "There were already well-established companies in flow cytometry that had a huge clinical pull driven by sales of antibodies and reagents, and these other applications that would be relevant for drug discovery didn't really emerge."
Into the Big House
So why did BD and BC take a pass on Sklar's technology — or not look into developing their own flow-based drug-discovery products? Besides maintaining their focus on a relatively successful basic research and diagnostic market, they too were fearful that flow did not have the chops to succeed as a drug-discovery tool.
John Dunne, associate scientific director at BD Biosciences, told CBA News that his company, among others, had been aware of the potential power of flow for drug discovery for many years, but as for-profit companies they also recognized that product development is influenced by end-user demands.
"To those who are deeply involved in flow — both among vendors and experts in the pharma flow labs — flow has always been an interesting possibility as a drug-screening platform, and some attempts have been made to drive adoption," Dunne said. "Some substantial traditional challenges have certainly kept flow out of the game: sample management, throughput, and data management being primary."
Only now are some of those challenges being addressed, according to Dunne.
"The drug-screening market has been only marginally informed or interested [in flow cytometry]," Dunne said. "In the early screening days, no one wanted to mess with complex data or cells, two of flow's strengths. As the screening paradigm has broadened to include both, and as novel flow implementation strategies have developed, the fit is definitely improved.
"In the last few years, flow has addressed some of the more obvious [technology] gaps," Dunne added. "Now we have lots of plate-based cytometers, and we've seen some development of robust assay configurations. It still takes an expert to work up the data robustly, but here again the tools have improved with regard to ease of use and throughput."
Still, there are only a handful of scientists and companies putting effort into commercializing or exploiting flow for drug discovery. These include Sklar's group at UNM, which has now recruited the help of Cellomics co-founder Dunlay to commercialize its technology; Novasite Pharmaceuticals; and Luminex, which plays primarily in bead-based biochemical assays and collaborates with UNM in this area (see CBA News, 5/26/2006, and part one of this article).
A Killer App for Flow?
Emerging flow cytometry applications are increasingly bound with a clinical component, so perhaps flow will find its killer application in areas where drug screening and clinical analyses intersect. Such intersections are growing as pharma and biotech companies increase their use of stem cells and cells from human patients in their drug-discovery programs.
One company meeting drug makers at this intersection is Amnis. The Seattle-based start-up markets the ImageStream 100, which combines certain strengths of flow cytometry with high-content automated imaging.
"The goal with our system — and I don't care if you want to call it a flow cytometer or an imaging device — was to extract as much information as possible from the cell," Amnis COO Bill Ortyn told CBA News. "We do that by multiple modes of imagery, which give you different bits of information.
"It will do immunofluorescence like a flow cytometer," Ortyn explained. "But a lot of people in pharma want to look at the distribution of molecules and movement of molecules from one cellular compartment to another, and you can't see that without high-resolution, high-sensitivity imagery. A flow cytometer might say something is near or on the cell, but if a molecule moves to the cytoplasm from the nucleus, it all looks the same in flow."
Perhaps not surprisingly, Amnis' sales are split nearly evenly between academic, government, biotech, and pharma labs, with most of its instruments placed in core flow cytometry facilities.
Tad George, principal scientist at Amnis, said that nuclear localization assays are one of the most common drug-discovery applications of the ImageStream. These types of assays are also the bread and butter of high-content plate-based imaging systems, but — and here's where the flow component comes in — "we're the only one that will do this well in cells that are not adherent, which is a lot of clinical-type samples, blood-based assays, or cells that are less amenable to plates," George said.
"The other subset is antibody drug conjugates, like Rituxin, where you have a target drug, and you want to know how a tumor cell responds," George said. "How does it shuttle into the endosome or lysosome pathways? What is it about the tumor cells that make it resistant to this drug?"
Ortyn and George said that the ImageStream is also well-suited to morphological feature classification of cells, in particular for monitoring stem cell differentiation; and for high-throughput studies of the cellular infection processes of viruses, bacteria, or parasites.
BD's Dunne believes that flow cytometry will continue to have an impact in two areas, in particular.
"As a preparative technology, flow sorting is extremely valuable [for] generating engineered cell lines optimized for all kinds of assay formats," Dunne said. "Screening cells at thousands of cells per second and automatically cloning the best signal-to-noise phenotypes is often the most important step in building robust cell-based assays.
"As an analytical tool, flow is unbeaten for speed — not in wells per second, but in cells scrutinized per second — and in terms of quantitative information per cell it's different but competitive with imaging," he added. "Assay paradigms [that] benefit from the natural diversity of cell populations, especially primary cell assays, really show off the power of single-cell analyses like flow and imaging, so the two platforms feed each other's adoption."
— Ben Butkus ([email protected])