High-content phenotypic screening, bringing physiological relevance back to early stage drug discovery

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31 August, 2015

blog: reviewing the advances in laser scanning imaging cytometry (LSIC)

so, how do you discover a potential blockbuster?
You throw a large amount of money into a black hole and hope that, via serendipity, the one molecule or identified mechanism wins you the Nobel Prize 20 years down the road – if you have managed to pursue it for that long!

For a long time, drug discovery operated like this: a potential target was identified and large libraries of compounds were screened in biochemical assays. Hits were assessed through lead optimization before a selection of drug candidates proceeded to be tested within a biological setting. It is therefore, not surprising, that lack of efficacy or toxicity led to frequent late-stage and expensive failures.

Something had to change. The industry embarked on a mission to improve the physiological relevance of early stage drug discovery. High-Content Screening (HCS) technology was perfectly positioned to facilitate this change. The ability of HCS to measure multiple features of individual cells, colonies, or organisms has made it a powerful tool for improving compound efficacy, and toxicity predictions.

Dr. Paul Wylie (TTP Labtech) has recently reviewed the latest technologies in HCS describing laser scanning imaging cytometry (LSIC) and how it is being applied to specific applications within drug discovery (Wylie, P.G. et al, (2015) Assay Drug Dev Technol 13(2): 66-78).

advanced technologies for HCS
A decade ago, HCS was largely synonymous with automated microscope-based charge-coupled (CCD) imagers. These CCD imagers have proven their effectiveness within many stages of drug discovery, notably target identification, validation and screening. However, their implementation within a screening environment creates challenges, such as the requirement for large storage solutions to manage the mountains of image-based data. Additionally, sample throughput for HCS is generally lower when compared to more traditional spectrophotometric screening platforms.

TTP Labtech’s acumen® Cellista laser scanning imaging cytometer provides an alternative solution that combines the value of an HCS approach to primary screening, within an HTS friendly format.

Using a series of photomultiplier tubes (PMTs) for fluorescence detection in combination with a Cartesian coordinate system that defines the image resolution, LSIC boasts fast, highly sensitive, whole-well HCS with the additional benefit of small data files. A key advancement of this technology is its ability to scan large 3D objects precisely and without time penalties, using an F-theta lens with a large depth of field.

TTP Labtech’s acumen Cellista is compatible with several styles of phenotypic screening workflows. acumen may be used in isolation (cytometric analysis) for high-throughput HCS, facilitated by Cellista software to remove the data burden and adoption barriers associated with image analysis. acumen may also be used in combination with traditional imagers as a triaging tool to reduce throughput bottlenecks. Here, large compound libraries are screened by acumen Cellista to identify hit wells. Only hit wells are interrogated further (wells-to-cells). Additionally, acumen may be used as a rapid image capture device, where whole well TIFF images are exported for analysis using third-party image analysis packages.

expanded applications for LSIC technology
HCS gained an initial foothold in oncology research, due to the early development of applications for measuring cell proliferation and apoptosis. Now, HCS is routinely used for a plethora of different applications within many other therapeutic areas. Within the review, a range of applications suited to LSIC screening is described in detail.

cell health
Late stage failures due to toxicity, have been a major problem for promising drug candidates. Screening out potential adverse effects early in the drug discovery process is now mainstream practice but many high-throughput assays only measure a single parameter, which could lead to toxic effects being missed. Results from a recent study, measuring four cellular parameters for each compound on TTP Labtech’s acumen Cellista, demonstrated that high-throughput multiplexing is essential for understanding the complexity of cellular toxicity.

cell cycle analysis
Defects in cell cycle regulation are a characteristic feature of tumor cells, and mutations in the genes involved in controlling the cell cycle are extremely common in cancer. Traditionally nuclear content was measured using flow cytometry to measure changes in DNA content. This uses a large number of cells, is slow and is limited to cells in suspension. The use of LSIC has increased the throughput and in the case of acumen Cellista, an entire 384- or 1536- well plate can be read in less than 8 minutes. As this method can be applied to cells in situ, live cells can be analyzed allowing morphological effects to be preserved.

3D cell culture models
It has become clear that 2D cultured cells are poor preclinical models for tumor growth and progression, with limited predictability for accurately reporting compound efficacy. Cells grown in 3D environments are being evaluated to establish if they can provide a more representative screening model. LSIC using acumen Cellista is particularly well suited to this application. In minutes, whole wells can be scanned without image stitching and depth of field information can be obtained through the F-theta lens without the need to Z-section.

where’s this all heading?
It is expected that the drive to develop more refined cellular models of disease will continue, with primary cells, induced pluripotent cells, cellular co-cultures and pseudotissues playing an increasingly important role in phenotypic screening. With these developments it is more likely that inappropriate drug candidates will be eliminated early, whilst those proceeding through the drug discovery process rely less on serendipity to succeed.

TTP Labtech’s acumen Cellista is suited to primary screening, delivering robust high-content data from target or phenotypic screens with throughputs of over 2 million data points a week!

If you would like access to an eprint of the entire article in Assay and Drug Development Technologies or obtain more information about acumen Cellista and how it is used in HCS applications, please contact TTP Labtech on sales@ttplabtech.com