November 1, 2017 (Vol. 37, No. 19)
GEN Spoke with Several Experts in the Field
GEN: How have flow cytometry methods changed to keep pace with next-generation sequencing technology, and have any new uses for flow cytometry emerged in the past few years in the area of drug discovery?
Dr. Balderas: Flow cytometry is a method that is increasingly being integrated with other technologies to provide richer data for understanding cellular makeup and function. In particular, it is used in conjunction with next-generation sequencing applications such as RNA sequencing (RNA-seq). The integration of cell-sorting technologies, such as index sorting, allows a finely detailed understanding of the cell receptor phenotype for each single cell in an RNA-seq application.
Flow cytometry is used in multiple applications within the drug development and discovery field. These include but are not limited to the use of (1) high-complexity flow cytometry (30 parameters) for deep receptor profiling in precious blood samples; (2) phosphorylation assays to uncover and document key signaling pathways in defined cell populations; (3) cell-cycle and DNA damage and repair mechanisms; and (4) bead technologies for the measurement of soluble proteins expressed in a variety of targeted bodily fluids to define intricate pathways and responses from single cells. Today’s cytometers are easily integrated into high-throughput robotic systems, allowing scientists to use sophisticated flow cytometric barcoding technologies to enhance the speed of discovery in drug development.
Robert Balderas, Ph.D., Vice President, Market Development, BD Biosciences
GEN: How have flow cytometry methods changed to keep pace with next-generation sequencing technology, and have any new uses for flow cytometry emerged in the past few years in the area of drug discovery?
Mr. Wirth: There has been a dramatic shift in the democratization of sequencing over the past 10 years from a specialized technique performed in core laboratories to the individual researcher able to do it in their own lab. At one time, sequencing was expensive, technically challenging, and time consuming. Next-generation sequencing is now easier, less expensive, and faster, making it more assessable.
The flow cytometry field has made similar strides. With the advent of the Guava® easyCyte benchtop flow cytometer, a device introduced by Guava Technologies (now MilliporeSigma), and the Muse® cell analyzer, individual labs have access to easy to use, less expensive instrumentation.
The high-throughput and quantitative analysis of cells or particles that flow cytometry provides has been an ideal technology for drug discovery. Over the past three to four years, there has been an increase in the awareness that extracellular vesicles (EVs) such as exosomes, are important to cellular communication. The EV field is experiencing an exponential increase in the number of publications, and discoveries of biomarkers indicating disease. The flow cytometer, specifically the ImageStream® imaging flow cytometer, which has very high sensitivity due to the CCD detection system, has been useful in detecting and characterizing these small particles.
Potential biomarkers for cardiovascular disease, liver disease, glioma, bladder cancer and pre-eclampsia have been reported, among other diseases. In the search for new drug candidates, these biomarkers could provide the needed indication of effective therapy for future discoveries.
Jean-Charles Wirth, Head of Applied Solutions, MilliporeSigma
GEN: How have flow cytometry methods changed to keep pace with next-generation sequencing technology, and have any new uses for flow cytometry emerged in the past few years in the area of drug discovery?
Ms. Bradford: Most of our understanding of gene expression is based upon bulk population averages. Although informative, this analysis can lead to conclusions that assume averages reflecting the dominant biological mechanism, masking rare or small subpopulations of cells and neglecting cell-to-cell differences within the full population. To fully understand the cellular heterogeneity contribution to biological function, a single-cell approach must be applied.
Flow cytometry is the gold standard for single-cell study of heterogeneous cell populations. For example, with the PrimeFlow™ RNA Assay, researchers can reveal the dynamics of RNA and protein expression simultaneously within millions of cells using flow cytometry. The assay employs fluorescence in situ hybridization (FISH) with branched DNA (bDNA) signal amplification for the simultaneous detection of up to four RNA targets using a standard flow cytometer, and can be combined with intracellular and cell-surface antibody staining.
In the area of drug development, researchers are looking for higher throughput options to reliably run samples using flow cytometry. Having an option for automated plate-based acquisition is now common, and the use of robotics for more efficient automated capability is increasing. The Attune™ NxT Flow Cytometer with Autosampler, combined with the robotics of the Thermo Scientific™ Orbitor™ RS Microplate Mover, enables users to load a series of plates and have them run on the cytometer automatically; the clog-resistant design of the system enables walk-away confidence.
Many researchers in drug development want to monitor multiple cell health parameters at the same time, such as viability, apoptosis, and proliferation. Having robust cell health assay combinations that use minimal sample prep permits streamlined processing, and is compatible with high-throughput instrumentation.
Jolene A. Bradford, Associate Director, Flow Cytometry Systems, Thermo Fisher Scientific
GEN: How have flow cytometry methods changed to keep pace with next-generation sequencing technology, and have any new uses for flow cytometry emerged in the past few years in the area of drug discovery?
Ms. Ovadia: Flow cytometry can be combined with next-generation sequencing (NGS) to not only look at cell phenotype, but also to pair phenotypic data with genomic information coming from the same cell. Using Bio-Rad’s S3e Cell Sorter and the ddSEQ solution, a researcher is able to isolate single cells from a homogenous cell population into individual droplets to prepare each one for NGS, and get full transcript information from each cell. The power to analyze 30 parameters using the ZE5 Cell Analyzer, select for one or two of those markers to generate a pure population, then get a full transcriptome sequence from those cells certainly is changing the pace of research in immuno-oncology.
Immunotherapies such as anti-PD1 and CAR-T cells offer promising opportunities to treat cancer and to improve survival rates in patients with refractory disease. One of the more challenging aspects of developing better drugs and improving upon the successes of immuno-oncology is the need to find and analyze very rare cancer cell populations. Flow cytometry is ideal for this application, and has the ability to detect many rare events in a short period of time because of its fast electronics, which can achieve detection rates of up to 100,000 events per second. Combined with its 30-parameter capability, researchers can simultaneously find rare cells and look at a wide variety of characteristics comprising each single cell. The power to do both improves researchers’ ability to discover biomarkers that can better predict treatment outcomes or disease progression.
Nicole Ovadia, Senior Product Manager, Bio-Rad
