Scaling HCS to HTS
Fabio Gasparri, Ph.D., principal research scientist at Nerviano Medical Sciences, stressed the importance of tending to basics. Nerviano conducts HCS assays to evaluate mechanisms of action of lead compounds affecting regulation of cell cycle or apoptosis.
Cytostatic or cytotoxic effects are determined through specific marker assessments. For confounding compound classes, such as those inducing polynuclear formation or that increase cell volume, which in turn can affect monoparametric readouts such as ATP content, his lab applies alternative methods such as cell count to better determine the real effects of lead compounds.
Currently, the lab employs time-lapse microscopy among its tools. “The time variable is crucial for better evaluation and good characterization of lead compounds. Focusing only on endpoint assays has some limitations; for instance, they may not clearly distinguish between cytostatic and cytotoxic effects.”
Dr. Gasparri is looking toward adoption of technologies that will further strengthen the robustness of Nerviano's R&D efforts. He said image-based technologies, like TAP Biosystems' cell- IQ® platform or impedence-based technology such as Roche's xCELLigence system for monitoring real-time cell proliferation in plates, “could be useful to specifically monitor cytostatic or cytotoxic effects and improve early characterization studies.”
Dr. Gasparri's lab currently conducts highcontent mechanism-based assays using the Thermo Fisher Scientific Cellomics ArrayScan HCS reader.
“In particular, we monitor the regulation of the cell cycle in response to targets we've identified at Nerviano related to cell-cycle control and to signal transduction. We employ assays based on immunofluorescence or reporter cell lines stably transfected with GFP proteins. Recently, we've been performing primary HC screening assays and are integrating these to analyze multiparametric data arising across different HC primary screens.”
Ultimately, this is all intended to increase the predictive contribution of earlier in vitro data gathering toward in vivo outcomes. After all, he insisted, when discussing in vivo work, “we are talking about models, so even as we try to increase the relevance of our models, they remain models.”