Researchers from 3M Pharmaceuticals have published research describing the use of high-content screening with cell-based TUNEL staining to evaluate the apoptotic response of cancerous and normal human cells to small-molecule receptor agonists.
The research demonstrates one way that HCS can be employed as a secondary screening tool in the drug-discovery process and, in particular, how it can be used to improve the accuracy and speed of well-characterized, but relatively low-throughput, cell-based assays.
Furthermore, the work suggests a possible future niche for HCS in the clinic — specifically, as a tool for tailoring chemotherapy to specific patients by assessing cytotoxicity at the bedside.
As described in the June 7 online edition of the Journal of Biomolecular Screening, the 3M scientists investigated the apoptotic response of squamous carcinoma cells and normal human keratinocytes to cancer drug actinomycin D; imiquimod, a toll-like receptor [TLR] agonist used in skin cancer treatment; and several structurally related TLR agonists.
To assess apoptotic activity of the drugs, they measured DNA fragmentation in the cells in 96-well plates using terminal deoxynucleotidyl trasnferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining and nuclear counterstaining. To quantitate the intensity and localization of the fluorescence signals from single cells, the researchers used a Cellomics ArrayScan platform.
"The determination of apoptosis is extremely important in today's pharmaceutical industry, and to do so with HCS is a perfect match."
Interestingly, Cellomics does not currently offer an image-analysis algorithm specifically for quantitation of TUNEL staining, so the researchers had to get creative with their platform.
Cellomics used to advertise a multiparameter apoptosis bioapplication that included TUNEL stain analysis, but has since removed it from its offerings, Jon Inglefield, lead author on the paper and a scientist in 3M Pharmaceuticals' department of pathology and toxicology, told CBA News this week.
"I don't think they discontinued it because there wasn't an interest, I think it was a first-generation application and they retired it when they offered second- and third-generation bioapplications," Inglefield said.
The company now offers a general cell-health profiling bioapplication, which Inglefield said doesn't give specific information about whether a cell died by necrosis or apoptosis.
"But we wanted to find out if novel chemical entities were killing cancer cells specifically by way of apoptosis," Inglefield said. "We leveraged the HCS platform to do that, and we had to adapt their target activation bioapplication, which is plastic, and useful for a bunch of different biological applications. Cellomics hadn't specifically mentioned to try this with apoptosis, but we had enough experience — we've been working with this instrument for over four years — to give it a try."
TUNEL vs. Caspase
TUNEL staining is one of two popular methods used in pharmaceutical labs for assaying apoptosis in adherent cells; the other assaying for the activation of caspase-3 enzyme.
However, caspase-3 activation assays present a couple of difficulties for the types of high-throughput, fixed-end-point cell-based assays typically amenable to drug screening, according to Inglefield. The first is that several apoptosis pathways are independent of caspase-3 activation, and thus the enzyme can not always be reliably used as an apoptotic indicator. The second is that "the timing of caspase elaboration is very bouncy," Inglefield said.
"Some cells will undergo caspase protein activation very quickly after a stimulus, and others will wait for a day or two, suddenly express it, transiently activate it, and then the cell lifts off the plate," he said.
TUNEL staining is a solution to this problem because it identifies apoptotic cells regardless of the upstream mechanism. However, it has traditionally not been favored as a drug-screening tool because evaluation of the stain necessitated either manual microscopy, which is tedious, time-consuming, and subjective; or flow cytometry, which requires that adherent cells be trypsinized and released, which is also time-consuming and generally not desirable.
This is where high-content automated imaging comes in handy, the researchers argued in the paper. They compared TUNEL analysis using the Cellomics platform with manual microscopy, and achieved similar results. However, manual microscopy required some four-and-a-half hours to assess a 96-well plate, while the high-content analysis took approximately 40 minutes.
"This one plate comprised approximately one-twentieth of the entire project and underscores the efficiency of the analysis with the HCS imager," the researchers wrote.
Such reduced screen times make HCS useful as a secondary screening tool, Inglefield said, as a way to further characterize drug activity after primary ligand-binding screens that can result in thousands of hits.
Inglefield believes his lab's research may help renew interest in TUNEL staining as a drug-screening assay because of the increased throughput that HCS provides.
The 3M scientists cited papers that described the use of low-throughput imaging with TUNEL stain, and the use of HCS with other apoptotic indicators such as caspase-3, nuclear size, and mitochondrial membrane permeability. But "I'm not aware of research in which HCS has been used TUNEL," Inglefield said. "We designed a new way of quantifying the TUNEL reaction, but the TUNEL reaction itself is old. The determination of apoptosis is extremely important in today's pharmaceutical industry, and to do so with HCS is a perfect match."
The research also points toward the possible use of HCS as a personalized medicine tool by assessing the apoptotic response drug candidates elicit in cells taken from cancer patients.
To underscore this point, the 3M researchers cited a 2005 Methods in Molecular Medicine paper published by researchers from Pittsburgh-based cancer therapy firm Precision Therapeutics. That paper described the company's ChemoFx assay, a phenotype-based cell culture assay for predicting anticancer drug responses in individual cancer patients.
The ChemoFx assay simply assesses the percentage of live cells versus dead cells ex vivo following treatment with escalating doses of chemotherapeutic compounds. Inglefield said that the high-content TUNEL assay might be used in the same way, or integrated with that type of an assay.
"We're using it as a tool for drug discovery, but someone could just as easily have done it for a personalized medicine approach," he said. "The advantage, I think, is coming up with results that aren't subjective — in other words, the machine counts, and helps you to make better decisions."