Reading Cells in Complex Assays
Eberhard Krausz, Ph.D., director for assay development and target validation at Janssen Research & Development (a division of Janssen Pharmaceutica), discussed two research tracks, one focused on protein-protein interactions and the other on the use of a 3-D co-culture assay for tumor microenvironment assessments.
The kind of assay approach he described as “more accurate and increasingly important” is to monitor cell viability concurrently (not serially or in parallel) with target binding events.
For instance, several current approaches for protein-protein interactions “show directly in cells how compounds penetrate, find the target, and interact with other targets,” he said. One particular approach Dr. Krausz presented is a high-content screen that anchors one of the two proteins under study, either in the nucleus or other specific locations such as at a particular DNA site.
Simultaneous assessment of “stressinduced phenotypical change such as morphological changes give an idea that the cell is either under stress or apoptotic. In particular HCS allows multiparametric data analysis to extract the direct target information as well as information on any kind of influence of ‘cellular happiness’ that could indicate off-target effects that would create potential problems later in follow up and development of any compound that comes out of such screen.”
Janssen R&D has also developed a 3-D tumor-growth assay that “has certain advantages over 2-D classical cell-proliferation assays,” Dr. Krausz described. “We know from gene-expression studies that tumor cells under assay exhibit changed behavior in ways similar to tumors in human patients. Ideally then you would want to have tumor cells in co-culture that mimic the in vivo tumor microenvironment, which would then include normal, nontumor cells such as fibroblasts and other cell types.
“Typically there are interactions between tumor cells and nontumor cells. Disruption of these interactions could allow us to exploit new options for targeted therapies that far exceed typical antiproliferative strategies,” he said.
“A very important point with this assay is that we can discriminate between simple cytotoxic compounds and those that show promising potential mechanisms of action through our ability to count individual cells or colonies and discriminate according to colony size. Essentially we seed tumor cells along with primary mesenchymal stem cells in a matrix, treat them, incubate, and then count colonies.
“Individual tumor cells will either divide to form minitumors or remain in the singlecell status. With noncytotoxic compounds, the total number of colonies does not change because the cells experience only growth inhibition,” which Dr. Krausz described as a particular strength of this assay approach.
Dr. Krausz sees developments in live-cell assays, such as 3-D assays and co-culture, as having a “bright future on many fronts,” not only for disease areas but also in ADMET. Ideally, he posited, their utility would come with a drastic reduction in the number of compounds actually being screened but in more complex assay platforms.