Cells are miniature laboratories that allow scientists to study key biological processes. Thus, they may provide a more natural way to assess the in vivo effects of drugs.
According to Visiongain, the market for cell-based assays exceeded $2 billion in 2012, and is predicted to quadruple to $8 billion by 2022.
New developments in this arena include getting back to basics by utilizing phenotypic screening for drug development. Additionally, employing patient cells and differentiating stem cells into defined cellular lineages are emerging as key strategies for assessing therapeutic responses. Technological advances include novel approaches for tackling the barrier of how to differentiate stem cells into specific cell types as well as integrating the speed and multiparameter assessments of flow cytometry with digital microscopy to perform phenotypic and functional analyses simultaneously.
Despite the rapid advances and focus on target-based drug discovery, the lion’s share of new first-in-class small molecule drugs (approved 1999–2008) were identified using the earlier method of phenotypic screening, notes Gary Allenby, Ph.D., business director at Aurelia Bioscience, a bioassay screening and development CRO in the U.K.
“Some compare phenotypic drug discovery versus target-based drug discovery as serendipity versus constrained research. In reality, examining the pharmacological properties of the same compound in different assays can allow greater insight into the mechanism of action to better understand the target’s biology. We employ a number of different phenotypic assays,” says Dr. Allenby.
“Label-free assays for screening are a very useful example. In the last five years, more investigators have become interested and excited about this technology. We see this as being implemented more and more, especially to differentiate between G-coupled protein receptors (GPCRs). One can measure responses in native or physiologically relevant systems including primary human or animal cells.
“You don’t have to overexpress or amplify via genetic modifications. This is a great help for understanding the molecular pharmacology of a receptor in its natural environment. Also a recombinant system doesn’t necessarily have the natural coupling or even engage the correct signaling pathways. Label-free assays allow measurements such as changes in cell shape, movement, and translocation of proteins into or out of the measurement window (~100 nanometers). The criticism of these assays is that it’s a black-box approach and one doesn’t know if the ligand causes direct or indirect effects.”
Another cell-based assay Aurelia employs is the fluorescent imaging plate reader (FLIPR) technology. “We have employed FLIPR in a range of assay formats, readouts, and targets, including GPCRs. These can be evaluated using fluorescent methodologies in 96-, 384-, and 1,536-well plates,” adds Dr. Allenby.
For the future, Dr. Allenby says that he finds the use of microfluidics combined with label-free cell biological assays to be an intriguing type of cell-based assay. “This would allow much more in-depth exploration of the boundaries of how physiological compounds affect the cell environment.”
Although the last 20 years of drug development has focused primarily on target-based discovery, a more powerful and productive approach would be to look at the biological responses of individual patient cells, says Rod Benson, Ph.D., COO of Imagen Biotech.
“The industry has been caught on a wrong paradigm that has blocked efficient development of new therapeutics for many years. Instead of just looking for targets, why not assess the cells you are trying to treat? As part of that change in direction, our company is focusing new R&D efforts on traditional cell-based assays but using cells derived from patients themselves. While we are employing high-content assays to screen for therapeutic responses, the primary tissue is isolated from individual cancer patients. This is a major step in accomplishing personalized medicine,” says Dr. Benson.
The company has procured grant monies and other funding to carry out its studies. It will initially target types of cancers in which cells are easily isolated and purified.
“We have begun formal collaborations with clinicians in which biopsies are performed and cells are isolated and plated for high-content screening. Using 384-well plates, we perform dose responses of single and combinations of specific therapeutics followed by assessment of cellular apoptosis or death. Even if two drugs are equally effective against a cancer of a particular patient, it is important to see at what level they are effective. Obviously, if one works at 1 nanomolar concentration and the other at 100 nanomolar, the drug that works at the lowest concentration would be preferable,” adds Dr. Benson.
The first phases of this new approach will assess many different types of cancers and select those that are easier to culture. Next, a host of therapeutics will be evaluated. Although initially targeting apoptotic responses, Dr. Benson sees the possibility of using many other cell-based assays.
“One could measure signs of inflammation in cells (such as stickiness), assess phenotypic endpoints, or employ any traditional high-content, cell-based assay. Another strategy to consider for the future is using phenotypic screens of patient cells to test natural products for their ability to treat cancer or other diseases. For example, products derived from plants and marine sources could also be assessed to identify candidates that are more natural therapeutics for individual patients.”
High-Content Flow Cytometry
Plate-based, high-content imaging readers provide a sophisticated analysis of adherent cells. However, many cells are not adherent. A new technology geared especially for the analysis of nonadherent cell populations combines the high-throughput functional insights of microscopy with the robust multi-parameter assessments of flow cytometry.
“Our company recently acquired the technology of Amnis that included the ImageStream® Mark II and FlowSight® imaging flow cytometers,” reports Blaine Armbruster, Ph.D., senior product manager, discovery and development solutions at EMD Millipore, a division of Merck KGaA.
“This is a breakthrough technology that combines the power of digital fluorescence microscopy with the sensitivity and speed of flow cytometry to allow applications that weren’t possible before.” Dr. Armbruster notes that suspension cells can be imaged at up to 60× magnification to analyze highly heterogeneous samples as well as rare sub-cell populations at speeds up to 5,000 cells per second. The technology allows both phenotypic and functional analyses to be performed simultaneously using up to seven lasers and 12 images per cell.
There are a number of important applications. “One of the challenges of nonadherent cell populations, especially those being analyzed from blood, is determining individual versus clumps of cells,” says Dr. Armbruster. “Sometimes cells just aggregate, but at other times an immune response stimulates cell–cell interactions that are important to identify and characterize. This technology not only allows easy differentiation among such cell populations but also tumor cells that may be a more rare population in blood. It also can be applied to study co-localization of proteins within cells. This has not been possible with flow cytometry alone.”
For the thousands of images generated, the company’s INSPIRE® and IDEAS® software are utilized for data mining. “Data analysis is designed to be intuitive and straightforward,” explains Dr. Armbruster. “For example, one can click on a dot in any plot to see the corresponding image of the cell or click on a histogram bin to view all the cells within that area. One can also gate specific populations seen in dot plots to hone in on the resulting populations to validate results. Overall, it provides a great cell-based assay for generating key information on populations of cells not accessible before.”