January 15, 2015 (Vol. 35, No. 2)
Lisa Heiden Ph.D. Director of Business Development MyBioSource
Cell-based assay platforms—more physiologically relevant and automated than ever—enhance not just drug discovery but translational medicine, too.
Cell-based assay platforms are evolving to meet diverse challenges—mimicking disease states, preserving signaling pathways, modeling drug responses, and recreating environments conducive to tissue development.
As diverse as these applications may be, the innovations that make them possible may be aligned with just a handful of themes.
These themes, which are expressed in varying degrees in most of the newer cell-based assay platforms, include the following:
- Use of primary rather then established cell lines.
- Development of tools for making physiologically relevant environments.
- Incorporation of Big Data strategies.
- Integration of high-throughput screening (HTS) and automation methods.
- Leveraging of signaling pathway mechanisms.
Be alert to these themes as we consider five different cell-based assay platforms. Three of these platforms are scheduled to be presented at an upcoming event organized by the Society for Laboratory Automation and Screening (SLAS). This event, the SLAS Conference and Exhibition, will take place February 7–11 in Washington, DC. The other two platforms were discussed at an event organized by the SMi Group. This event, Advances in Cell Based Assays, took place late last year, in London.
These platforms demonstrate how today’s cell-based assays are driving translational medicine. These assays are creating opportunities that yesteryear’s scientists could only dream about.
Profiling Signaling Pathways
Maite Romier, a research and development project leader at Cisbio Bioassays, intends to present at the SLAS event. Her presentation, entitled, “New cell-based HTRF assays deserve the exploration of Wnt signaling pathway,” describes how emerging concepts in Wnt/beta-catenin signaling can be translated into practical assays.
“We chose to develop the new HTRF (homogenous time-resolved fluorescence) assay as part of our innovation efforts to provide cell-based solutions for phosphoproteins, and for cell signaling pathway investigation in general,” explains Romier.
The HTRF platform is a complete, well-established assay system that already covers 10 major pathways. Based on an “add and read” homogenous protocol—a robust alternative to ELISA or bead-based technologies—HTRF “simply enables a fast analysis of what’s happening in the cell.”
“Specifically, the cell-based phospho/total beta-catenin assay was undertaken to allow the investigation of the Wnt signaling pathway, an area of growing interest for drug discovery researchers,” continues Romier. “Wnt signaling controls the phosphorylation status of beta-catenin, a critical regulator and key protein in the pathway. This assay serves as a pathway readout when trying to modulate the pathway in the disease state.
“Total beta-catenin is an important co-transcriptional activator of many significant genes involved in differentiation, development, cell proliferation, and hence cancer. Our results showed that the assay can distinguish between pools of cellular endogenous beta-catenin whether phosphorylated or not, which have different important functions in cell signaling. Screening for compounds using the beta-catenin assay seems very promising for discovering new drug mechanisms in the cancer field.”
Mimicking Tumor Hypoxia
Also planning to present at the SLAS event is Samantha Grist, Ph.D. candidate, University of British Columbia. Her talk, entitled “A microfluidic device with integrated oxygen sensors for the cell-based screening of cancer treatments under transient hypoxia,” reflects her translational science perspective: “The multidisciplinary aspect of my project was one of the things that drew me to it.”
“My background is in engineering physics, and I am interested in applying engineering concepts to projects in health sciences,” she says. “My device harnesses the small scales of microfluidic systems to reproduce time scales of transient hypoxia in solid tumors for in vitro drug screening.”
Transient hypoxia in tumors develops when irregular blood vessels in tumors temporarily close and then reopen, leading to cycles of hypoxia and then reperfusion in the tumor. Tumor tissue hypoxia, or inadequate oxygen, is linked to drug resistance.
“I chose the device for my project because it can control the oxygen conditions within a cell culture environment to produce static (chronic) hypoxic conditions and oxygen gradients, as well as time-varying oxygen levels (transient hypoxia),” she continues. “Traditional drug testing platforms like well plates can’t reproduce these kinds of oxygen profiles because of the long distances that oxygen has to diffuse through before reaching the cells.
“I was able to successfully reproduce time-varying oxygen levels on similar time scales to those in the body during transient hypoxia, as well as complex, spatial oxygen gradients like those near tumor blood vessels. Employing this device to mimic in vivo oxygen profiles in vitro could facilitate the development of a screening methodology that better predicts how a given drug will act in the body.”
Identifying JAK Inhibitors
Another impressive project slated for presentation at the SLAS event is “Discovery of an allosteric JAK inhibitor through primary T cell high throughput screening.” The talk will be delivered by Atli Thorarensen, Ph.D., medicinal chemistry project lead at Pfizer. Dr. Thorarensen develops JAK signaling pathway inhibitors. He chose his assay to employ primary cells for HTS and overcome limitations associated with cell-line model systems.
“I think the industry needs to move away from utilizing cell lines as surrogates of pharmacology,” asserts Dr. Thorarensen. “Our learnings have taught us that primary cells are much more valuable as predictors of pharmacology then stable expressed cells, which have forced target expression and may have lost many pathway signaling components.”
Historically, results with JAK inhibitors show a disconcerting disconnect between enzyme and cellular function. For example, in the cell-line model Dr. Thorarensen used there was a “very poor correlation between JAK3 enzyme inhibition with ATP competitive inhibitors and the observed cellular response (measurement of STAT phosphorylation). This indicated that there are artifacts in the cell lines not allowing us to accurately measure inhibition of JAK3 function.
“The cell assay we then developed is a measurement of STAT phosphorylation in primary cells, [which were] PBMCs expanded from a donor. The results showed we could obtain a cellular signal that accurately represented inhibition of JAK3 enzyme activity with ATP competitive inhibitors.”
The sensitivity of the primary-cell-based model system is enabling Dr. Thorarensen to move to the next step and screen for allosteric inhibitors that bind outside of the conserved ATP-binding sites of the JAK enzymes. According to Dr. Thorarensen, the system “should allow for the identification of inhibitors that selectively inhibit specific family members—JAK1, JAK2, JAK3, or TYK2.”
Integrating Live Imaging
Rob Benson, chief operating officer of Imagen Biotech, presented at the SMI event, delivering a talk entitled, “The use of high-content screening of patient samples to add a personal touch to cancer drug discovery.” Another company representative, CSO Gareth Griffiths, Ph.D., agreed to follow up on this talk to contribute to this GEN feature.
“We choose the Thermo Scientific ArrayScan, a high-content fluorescence-based screening machine, for our project,” says Dr. Griffiths. “It automatically captures images and analyzes them with different algorithms; you can teach the machine to extract the information that you want from the assay.”
The drug testing platform employs live cancer cells with live imaging, and it uses the fluorescence of cell death as a readout assay for screening compounds. The red fluorescent is a cell-permeable probe, a death marker, that enters dying but not healthy cells. Fresh ovarian cancer cells or stem cell lines generated from glioblastoma cells, taken directly from patient brain tumors, were used for the assays.
Automation enables rapid analysis of a huge amount of cell data from dose responses from a large number of drugs. “For every hundred hours of work it takes to pull in the samples to analyze, with the software algorithms, [it is possible to] crunch the data in about one hour,” notes Dr. Griffiths.
“The results showed that there is quite a variation in response to the different drugs. You can get ‘super responses’ in some patients, and some drugs will only act in a small proportion of patients,” adds Dr. Griffiths. “This speaks to the future of the target therapy market or personalized medicine. Our rapid screening method should be useful in picking the right subset of patient responders to use in a given clinical trial.”
Recreating the Stem Cell Niche
Another insightful presentation at the SMI event was entitled, “VeraVec endothelial cells recreate the vascular stem cell in vitro for stem cell expansion and drug discovery.” It was delivered by Daniel Nolan, Ph.D., director of research at Angiocrine Bioscience. Dr. Nolan indicated that the VeraVec endothelial cell (EC) platform was developed to overcome historical and pervasive challenges in EC-supported stem cell (SC) expansion.
“Historically everyone culturing ECs has been beholden to irreducibly complex media additives, including various serums and nebulous supplements,” relates Dr. Nolan. “SCs co-cultured with ECs under these conditions do not reliably expand or maintain their SC signatures.”
This traditional system is at odds with biology, he says, because “SCs in vivo sit directly on top of ECs, with ECs actually providing all the growth factors SCs need.”
“Our strategy is a biology approach that enables culture of VeraVec ECs, through Adenovirus E4ORF1 gene expression, without any requirements for irreducibly complex media additives,” elaborates Dr. Nolan. “They are adapted to in vitro conditions amenable to stem cell expansion, and we show that VeraVec EC/SC co-culture successfully recreates the SC niche in vitro. We take various SC populations, including [those of] hematopoietic, hepatic, neural, and cartilaginous [origin], and put them on the VeraVec ECs where SCs expand at a phenomenal pace.
“The beauty of this platform is that it works all the way from basic research through therapy. For example, we are building toward clinical applications and show that when expanded co-cultures are transplanted into animals, VeraVec ECs secrete growth factors and aid in SC-facilitated tissue regeneration.”
Moreover, the fidelity of SC-like phenotypes or signatures characteristic of heterogeneous primary cancer cell cultures can be maintained with VeraVec ECs. In contrast, cancerous cells change their phenotype rapidly in traditional culture.