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Stay in Step with the 'Crazy Pace' of High-Content Screening Advances

Magic. It can happen when people with curious minds gather to digest and dissect a rapidly changing and highly productive field of life sciences discovery and technology. Magic happened in Dresden, Germany, in June of 2016.

The SLAS Europe High-Content Screening Conference drew some 160 high-content screening (HCS) academics and industry professionals to the Max Planck Institute (MPI) of Molecular Cell Biology and Genetics June 27-29 in Dresden. With four sessions – High-Content Screening; Emerging HCS Technologies; Model Systems; and High-Content Analysis/Data Analysis – 24 speakers freely shared details of their latest work in the field.

“High-content screening is a hot topic in drug discovery these days, so SLAS felt it was an opportune time to create a conference to be held at one of the places which was a very early adopter of HCS back in 2004,” explains SLAS Europe Council member Marc Bickle. Bickle is head of the Technology Development Studio (TDS) at Max Planck, co-chair of the SLAS Europe High-Content Screening Conference and a JBS Editorial Board member. “We wanted an event that would connect academia and the pharmaceutical industry. Academia brings the perspective of basic science, and there are a lot of discoveries coming up every year. It’s a pretty crazy pace at the moment with revolutions in technology and computational biology. The pharmaceutical industry is one of the very early adopters of advanced cell culture techniques. This conference helped us all.”

Tuned into the need to provide information exchange in this high-interest area, SLAS further addressed high-content screening by publishing a special collection of scientific reports from the conference in the Journal of Biomolecular Screening; and hosting an SLAS Webinar with one of the featured authors/speakers.

“It is not easy for one person to make all the connections necessary for success in this field, but the Society helps bring those people who offer advanced technologies to the life sciences researchers working in the labs. SLAS has so much to contribute and fills this important role,” Bickle says.

A Power Program

“At the SLAS Europe High-Content Screening Conference, we wanted to offer a broad range of sessions and activities,” Bickle notes. “HCS is a demanding, multidisciplinary field requiring knowledge in biology, image processing, statistics and programming, and the future is likely to demand more of these skills, rather than less. Biologists are often not well trained in mathematics and computation, but it is important that they become familiar with these fields in order to be able to tackle the challenges of HCS in the future.”

The conference chairs note one trend in HCS is the use of more complex cell culture assays, and it was evident in Dresden.

“3D cell culture assays were presented by speakers from academia and industry,” says María Montoya, Ph.D., head of Cellomics Unit at the Centro Nacional de Investigaciones Cardiovasculares, who highlighted presentations by Myles Fennell (Memorial Sloan Kettering Cancer Center), Miguel A. del Pozo (CNIC), Chris Bakal (Institute of Cancer Research), Viswanath Das (Institute of Molecular and Translational Medicine), Stefan Prechtl (Bayer Healthcare), Leo Price (OcellO) and Henriette Lanz (Mimetas). Montoya, along with Thierry Dorval, HCS group leader at Servier in Paris, France, supported Bickle as conference co-chairs.

Montoya pointed to Das’s presentation, “High-Throughput Cell-Based Report System for Screening of Hypomethylating Agents,” as an example. Das is a research fellow from the Institute of Molecular and Translational Medicine in the Czech Republic, and his work focuses on 3D cultures of cancer cells for screening of anticancer drugs and studying spatial dimension- and tumor-stroma-induced resistance to chemotherapeutics. Following previous research on the colorectal cancer epigenome that revealed the majority of patients have aberrant DNA methylation resulting in silencing of tumor suppressing genes, Das presented his lab’s development of a stably-transfected fluorescent reporter system utilizing the silenced FLJ32130 gene for high-content screening of potential DNA hypomethylating agents in 2D and 3D cell culture systems.

Das also published a technical report in the JBS High-Content Screening Special Collection, October 2016, “Reproducibility of Uniform Spheroid Formation in 384-Well Plates – The Effect of Medium Evaporation.” Das and his team examine “the effect of evaporation on the reproducibility of spheroids of tumor and nontumor cell lines in 384-well plates, and show that culture conditions that prevent evaporation-induced medium loss result in the formation of uniform spheroids across the plate.” They also present technical improvements to increase the scalability of the liquid-overlay spheroid culturing technique in microtiter plates, together with a simple software routine for the quantification of spheroid size. “We believe that these cost-effective improvements will aid in further improvement of spheroid cultures for HTS drug discovery,” the team states.

An emerging theme in high-content imaging is intelligent screening/smart microscopy, where the microscope acquisition is controlled by feedback from image analysis, Montoya says. Bickle cites strong work being done in academia in this area and praised conference speakers Rainer Pepperkok, team leader and senior scientist at EMBL Heidelberg, and Daniel Gerlich, senior researcher at the Institute of Molecular Biotechnology in the Austrian Academy of Sciences.

Pepperkok’s presentation, “Using High-Throughput Microscopy to Study Membrane Traffic, Organelle Biogenesis and Disease Mechanisms,” described the microscopy platform they developed to automate and integrate all steps in microscope-based experiments. “The technology also allows automated performance of complex imaging protocols such as FRAP or FCCS experiments at high throughput or the extensive multiplexing of immunocytochemistry in fixed cells,” he shares in the presentation description. “Our data reveal an unexpected relationship between secretory pathway function and genes involved in general metabolic integrity or signal transduction pathways. This provides the basis for an integrative understanding of the global cellular organization and the regulation of the secretory pathway and organelle biogenesis.”

Gerlich’s presentation, “Cellular Phenotype Discovery by Outlier Detection in High-Content Screening,” addressed a problem in widely used screening by automated microscopy – the need for representative phenotype images prior to screening via supervised machine learning. A common fix is to pilot-screen medium-sized perturbation libraries with identification of novel phenotype classes by visual inspection. “This is tedious and may still not cover the entire range of phenotype classes present in the full screening data,” says Gerlich in the presentation description. “To enable phenotype detection without a priori definition of morphology classes, we have developed a method based on outlier detection. Morphology space occupied by negative controls is initially learned without supervision, using a one-class-support-vector machine. The resulting classifier is then applied to the full screening data to identify novel phenotype classes based on their degree of morphological deviation from negative controls. This method accurately detected several distinct mitotic phenotype classes, indicating feasibility of fully automated screening independent of user training. In ongoing experiments, we use the outlier detection method to identify new mitotic chromosome morphology regulators.”

Jan Huisken of the Max Planck Institute of Molecular Cell Biology and Genetics presented “High-Throughput Whole-Embryo Imaging with Contactless Light Sheet Microscopy.” Bickle appreciated learning about his efforts to automate for the highest throughput with microscopy that puts living cells first.

“Jan Huisken presented his automated single-plane illumination microscope (SPIM) for use in screening zebrafish embryos and other 3D objects,” Bickle adds. “His group has developed software solutions to project the data on a 2D map, thereby preserving the 3D information, but discarding the voxels that do not contain information. With this approach it is possible to exploit 3D information and SPIM without running into a computational bottleneck.”

Bickle, Dorval and Montoya were more than pleased with the high quality and innovation in Dresden presentations. “I am very pleased to see that the community is working more and more in the open source field,” says Dorval. “Large pharmaceutical companies have already jumped the gap, or are about to do it. It is more than a matter of cost, but also a way to share experience between the actors of the HCS field that is still highly challenging. I didn’t expect such a change in such a short period of time.”

Furthering Knowledge Exchange

In addition to the Das paper mentioned above, three other JBS original research reports featured in the JBS Special Collection also were mainstays in the conference’s scientific program.

A Novel Automated High-Content Analysis Workflow Capturing Cell Population Dynamics from Induced Pluripotent Stem Cell Live Imaging Data
Maximilian Kerz et al.
A collaborative team from King’s College London, National Institute for Health Research (London) and Farr Institute of Health Informatics Research (London) present “a workflow that incorporates a novel CellProfiler-based image analysis pipeline enabling segmentation of single-channel images with a robust R-based software solution to reduce the dimension of time to a single data point. These two packages combined allow robust segmentation of iPSCs solely on phase-contrast single-channel images and enable live imaging data to be easily integrated to endpoint data sets while retaining the dynamics of cellular responses.”

High-Throughput Platform for Identifying Molecular Factors involved in Phenotypic Stabilization of Primary Human Hepatocytes In Vitro
Jing Shan et al.
Shan of the Harvard–MIT Division of Health Sciences and Technology, MIT, used their high-throughput platform that enables systematic interrogation of liver biology in vitro to identify 12 gene products that may be important for hepatocyte viability and/or liver identity in vitro. “These results represent important first steps in the elucidation of mechanisms instrumental to the phenotypic maintenance of hepatocytes in vitro, and we hope that the tools reported here will empower additional studies in various fields of liver research.”

Development of a 3D Tissue Culture–Based High-Content Screening Platform that Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases
Tijmen H. Booij et al.
A collaborative team from The Netherlands and Hungary describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. They processed 3D image data to measure more than 800 phenotypic parameters; used multiparametric analysis to evaluate the effect of compounds on tissue morphology; then measured the activity and selectivity of inhibitors. Corresponding author Leo S. Price, CEO of OcellO, reported on aspects of this work during the Dresden conference.

Following high interest in his conference session, SLAS invited Price to present an SLAS Webinar, “Compound Screening and Profiling in Cultured Human (3D) Tissues.“ The live webinar was held on Sept. 23, and webinar recordings are available on demand to dues-paid SLAS members.

“The goal of our work at OcellO is to continually strive to get as near to the human situation as possible utilizing tissue-based assays for drug discovery,” Price explains. “In the webinar, I demonstrate how to use technology to get the optimum quality and quantity of information as possible out of 3D assays.”

Price likens the task of performing high-content 3D screening experiments to that of the astronauts and NASA land team in the movie Apollo 13, when tasked with finding a way to power up the command module for safe passage back to earth.

“It’s important to get rid of all the extra stuff you don’t need,” he says. “For these assays, you need to retain the complexity of the biology but otherwise keep the assay as simple as possible to enable automation, analysis and overall assay robustness.”

In his team’s JBS paper, they explain how to use image analysis and profiling to separate out different patterns of drug responses in the selective inhibition of two different and important drug targets – epidermal growth factor receptor (EGFR) and c-Met.

“Both EGFR and c-Met regulate cell behavior. In a cell proliferation assay when you hit these targets, you see pretty much the same response – the inhibition of growth,” Price says. “If you actually look at the tissues and inhibit those targets, you see different patterns of response. Using image analysis, we can separate those out. By analyzing phenotypes in detail and getting maximum information out of the images, you gain much more insight into what those drugs are doing and can classify drugs based on the responses that they induce.”

Price wants SLAS Webinar participants to understand that 3D culture can be applied to pretty much any solid tissue and certainly to solid tumors.

“My webinar examples are related to testing kinase inhibitors in prostate cancer,” Price says. “But 3D culture can be used with any tissue you are studying or any class of therapy that you are developing.”

SLAS Webinars are available at no cost to dues-paying SLAS members on demand.

Scientific Networking Completes the Picture

“It was clear that there was a very good buzz in Dresden,” Bickle says. “I heard very positive feedback from participants – great speakers, great topics and so on. The conference was a good size and maybe the fact that there was limited room in the facility meant interaction was easier and more intense. It was great for getting to know people and talking to people.”

The SLAS Europe High-Content Screening Conference showcased close to 40 poster presentations. Extended coffee and lunch breaks held near the posters allowed time for meaningful discussions with authors and others. A poster competition for students was held and the winner was Sri Teja Mullapudi of the Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany, for “Whole Organism High-Throughput Screening to Identify Modulators for Directed Differentiation of Pancreatic b-Cells.”

“We did not know much about SLAS before the meeting, but we think it's a great platform to give and receive strategies for increasing the throughput of our zebrafish screens – so thanks a lot for organizing the meeting and hosting us,” Mullapudi wrote to Bickle after the conference. “I am really honored at the opportunity to attend SLAS2017 in Washington - thank you again and hope to see you there too.”

“Another successful and different aspect of the conference was having doctoral students – those at the beginning of their careers – do three-minute flash presentations,” Bickle adds. “We asked them to stand up, show a few slides, talk about their work and get attendees interested in their posters. It was great experience for them.”

Completing the knowledge exchange, conference attendees also were able to visit 12 tabletop exhibits and attend tutorial sessions presented by PerkinElmer, Molecular Devices, Wako Automation and Thermo Fisher Scientific. Tuesday morning’s “High-Throughput Sightseeing,” a 7:00 a.m. brisk jog or walk along a nearly 5 km route of historical sites, was another planned networking activity.

“The SLAS Europe office did a very nice job of coordinating logistics, as did the Max Planck event manager,” Bickle says. “As this was not held in a typical conference venue, it was crowded. We could have had many more vendors, but we did not have the space. In the tutorial sessions that were held before and after the event, vendors had the opportunity to sit together with users to discuss details and see how to get the most out of their data and instruments.”

Holding the event at the Max Planck Institute also allowed attendees a first-hand look at some of its labs, and Bickle himself escorted several groups through his areas in the MPI Technology Development Studio.

“This conference was highly successful due to the efforts of Marc Bickle and his co-chairs,” says Brenda Dreier, SLAS interim CEO. “Marc went above and beyond as a volunteer both for the conference and as a JBS Special Collection guest editor. His affiliation with MPI allowed SLAS to take advantage of a working laboratory venue – an interesting and exciting venue for the conference. SLAS is grateful to Marc and his colleagues at MPI for their generous and enthusiastic support.”

Bickle and his co-chairs were pleased and grateful for their chance to be a part of valuable knowledge sharing. “What was striking to me was to see how the community has reached a level of maturity and pragmatism,” Dorval says. “The technologies presented were either already deployed in an industrial context (not only proof of concept anymore) or functional. The period of unrealistic expectations has faded, and now the community understands what can be expected and how to achieve it. Moreover, industry and academia are much more likely to work together, and understand that nothing would happen without the other.”

Next on the Docket: SLAS2017

High-content screening scientists do not have to wait until a future SLAS Europe event to further expand their knowledge. In addition to the SLAS Webinar with Leo Price and the JBS Special Collection, SLAS2017, Feb. 4-8, in Washington, DC, is loaded with HCS sessions of interest. Check the website today.

October 10, 2016