July 1, 2015 (Vol. 35, No. 13)

Microfluidics Is On a Roll and Proteomic Assays On a Cell-By-Cell Basis Is Already Raising the Odds of Clinical Success

Single-cell functional proteomics is the newest technology to migrate to the microchip format, and now that this technology is becoming chip-based, it is speeding toward clinical application. On-chip analysis is rapidly becoming robust and highly reproducible, enabling the analysis of multiple single cells to produce meaningful comparisons and results.

Integration of different functional assays with genomic and proteomic data is particularly advantageous. Such integration is emerging as a means to personalize diagnostics and therapeutics.

Below are just a few single-cell technologies that will be highlighted at an upcoming SelectBio conference that will bring together technology development and clinical diagnostics applications of next-generation sequencing (NGS), single-cell analysis (SCA), and mass spectrometry. The event—NGS, SCA, and Mass Spec: The Road to Diagnostics—is scheduled to take place September 28–30 in San Diego.

The immediate vicinity of cells has a direct effect on cells’ behavior. In particular, cell microenvironment features such as cell surface nanotopography and extracellular matrix composition affect nanoscale interactions such as cellular adhesion, migration, and differentiation.

At the University of California, Los Angeles (UCLA), such natural nanoscale interactions are being mimicked by a technology called NanoVelcro. It exploits nanostructured substrates to capture rare cells from peripheral blood.

“We can achieve significantly enhanced capture of target cells, such as circulating tumor cells (CTCs) and circulating fetal nucleated cells (CFNCs), by using nanowire-coated surfaces,” says Hsian-Rong Tseng, Ph.D., a professor of molecular and medical pharmacology at UCLA. Dr. Tseng’s research team has pioneered several generations of NanoVelcro cell-affinity assays. Notably, these assays are capable of immobilizing target cells with high efficiency. The most advanced versions of these assays incorporate temperature-sensitive polymer brushes.

The polymer brushes are linked with biotin for streptavidin-mediated conjugation of any capture agents. Especially remarkable is this unique technology’s ability to control the accessibility of capture agents via temperature-dependent conformational changes in the polymer brushes. At 37°C, the polymer brushes are coiled, exposing the capture agents on the NanoVelcro chips. As the temperature drops to 4°C, polymer brushes unwind, withdrawing capture agents into the polymer “forest.”

“We demonstrated efficient purification of CTCs and CFNCs from blood samples, followed by subsequent genetic testing,” continues Dr. Tseng. “In principle, the technology can be applied to any type of rare cells.”

The gold standard of prenatal diagnostics is invasive sampling of amniotic fluid and chorionic villi to obtain fetal cells for genetic analysis. According to Dr. Tseng, an alternative approach could take advantage of the NanoVelcro assay. He asserts that it is able to efficiently capture fetal cells present in 5–10 mL of maternal blood. Another relevant technological innovation is the precise isolation of captured cells straight from the chips using laser capture microdissection (LCM). The captured cells can be immediately characterized by conventional techniques such as microarrays and next-generation sequencing.

A UCLA spin-out company, Fetolumina, is gearing up to offer laboratory-developed tests for screening fetal genetic abnormalities, including trisomy. “Our NanoVelcro assay is created using the contemporary lithographic method popularized by the semiconductor industry,” notes Dr. Tseng. “This keeps the cost low, and allows for commercial-scale production.” Fetolumina explores automation of the LCM step to achieve a throughput of nearly 100 samples per day.

Thermoresponsive NanoVelcro assay for purification of circulating tumor cells (CTCs) and circulating fetal nucleated cells (CFNCs). (A) A chip holder is designed to house the NanoVelcro substrate with an overlaid PDMS chaotic mixer. A Peltier cooling/heating pad controls the operating temperature of CTC/CFNC purification system. (B) At 37°C, the overlaid PDMS chaotic mixer is capable of enhancing the contact frequency between the flowing-through CTCs/CFNCs and capture agent-present thermoresponsive NanoVelcro substrate. (C) The conformational changes of polymer brushes alter the accessibility of the capture agents on NanoVelcro substrates.

Monitoring Cellular Interactions

“A cellular functional phenotype is a conglomerate of multiple cellular processes,” says Tania Konry, Ph.D., an assistant professor of pharmaceutical sciences at Northeastern University. “Cellular heterogeneity makes it technically difficult to pinpoint the origin of a specific cellular activity. We developed pico- and nano-sized bioreactors that enable us to follow each individual cell over a period of time.”

These bioreactors are part of a testing platform that makes use of aqueous droplets coated in oil. This platform, which is called ScanDrop, is a portable instrument that incorporates three perpendicular inlet channels that form a nozzle. These channels can carry cells, reagents, and a proprietary oil formulation. As the flow shears in the nozzle, oil breaks the aquaphase, creating droplets containing cells and reagents. Alternatively, two inlets can carry cells of different types such as tumor cells and cells of the immune system. By optimizing the inputs, Dr. Konry is able to generate droplets with precise ratios between different types of cells.

Using this elegant approach, Dr. Konry’s team was able to monitor live immunological synapse (IS) formation between dendritic and T cells. The team loaded the nanodroplets with various ratios of T cells to dendritic cells (DCs), and it was able to monitor T-cell activation in real time. The team demonstrated that due to various stages of maturation not all DCs are able to interact and activate naive T cells.

“Traditional co-culturing of two cellular populations may produce false functionalities because of accumulation of secretory factors indiscriminately affecting all cells in a population,” explains Dr. Konry. “Our nanodroplet reactor uniquely helps us to see which cells are actually active and which are not.”

The technology can be adapted for high-throughput application. For example, with a droplet-generation rate of 200 droplets/sec, ScanDrop can rapidly fill 1,000-well arrays without resorting to advanced robotics.

This kind of efficiency was explored for sensitive detection of single nucleotide polymorphisms in individual cells. Single cells and assay-specific reagents were encapsulated within nanodroplets, and each cell was assayed separately by a creative combination of peptide-nucleic acid DNA “openers,” isothermal rolling circle amplification, and fluorescence-base detection. This approach enabled the team to detect and quantify copies of Epstein-Barr virus that had been incorporated into genomes of B lymphocytes.

Dr. Konry plans to commercialize the ScanDrop platform. The versatility of this approach is poised to generate new insights in many areas of basic biology and drug development.

At Northeastern University, researchers are developing ScanDrop, a lab-on-a-chip biosensor approach that utilizes a well-mixed microfluidic device and a microsphere-based assay capable of performing near real-time diagnostics. ScanDrop has been used to monitor live immunological synapse formation between dendritic cells and T cells.

Tapping Cancer Signaling Networks

“We were able to miniaturize a sandwich-type immunofluorescence assay to a single-cell level and adapt it to a multiplex format,” says Wei Wei, Ph.D., an assistant professor of molecular and medical pharmacology at UCLA. His microchips contain a few hundred to several thousand individual ELISA microchambers. Depending on the application, only 0.1–2 nL reagent volumes are required to complete the assay with a measurement error of just under 10%.

One end of an H-shaped chamber contains multiple strips of single-stranded DNA (single-cell barcode, or SCBC). Since microfluidic patterned oligonucleotides are stable at room temperature, the microchips can be produced on a commercial scale and stored until they are required for use. A cocktail of antibodies labeled with complementary single-stranded DNA oligomers is used to convert the DNA barcode into the antibody barcode just prior to running the assay.

In their current version, the microchips, Dr. Wei asserts, are capable of measuring 42 functional proteins. In the future, Dr. Wei plans to expand this number to several hundred with an antibody array of multidimensional identifiers right in the microchamber. “With such array,” notes Dr. Wei, “this technology would allow discovery level proteomic tests, but with single-cell resolution.”

Proteins are quantified using secondary antibodies conjugated with fluorophores. The SCBC microchip dataset provides three measurements: average analyte levels, the variances in distribution of those levels between the cells, and the correlations between any two analytes.

In a recent study, a team led by Dr. Wei simultaneously interrogated levels of small molecule metabolites and associated metabolic phosphoproteins. The study yielded rich information about cellular metabolic signal regulation in human-derived glioblastoma cancer cells. Moreover, the team was able to reproduce the changes in glucose uptake by glioblastoma cells caused by treatment with erlotinib, as previously observed by PET imaging.

The SCBC microchip was also able to precisely predict the response of signaling networks to changes in partial oxygen pressure, which mimicked hypoxic environment of tumors. Importantly, the data revealed that at specific oxygenation levels, cancer cells are not responsive to certain targeted therapies.

Dr. Wei collaborates with theoretical physicists to create novel algorithms to predict how external perturbations correlate with changes in protein expression. The theoretical framework, based on SCBC chip analytics, is a cornerstone of Dr. Wei’s clinical translational research. Analysis of patient samples is currently underway.

Capturing Circulating Tumor Cells

Steven Soper, Ph.D., a professor of biomedical engineering and chemistry at the University of North Carolina, Chapel Hill, has developed a unique microfluidic system for capturing and analyzing circulating tumor cells (CTCs). “If you study CTCs, there is no other choice but to deploy single-cell technologies,” says Dr. Soper. “Only 1–10 CTCs are present in one milliliter of blood on the background of 106 and 109 white and red blood cells, respectively.”

Dr. Soper’s microfluidic system, which is positioned to study rare events and can be repurposed for rare cells of varied types, consists of three modules—a selection module (for taking CTCs directly from blood), an impedence module (for counting CTCs cell by cell), and an imaging module (for immunophenotyping single cells).

The selection module contains up to 320 microchannels covered with a capture antibody. Each microchannel has a curvilinear shape that assists in cell capture by taking advantage of centrifugal forces that push cells against the microchannels’ walls. The capture antibodies are attached via single-stranded DNAs modified to contain a single uracil residue.

Dr. Soper’s team creatively utilizes the USER™ (uracil-specific excision reagent) system to cleave DNA strands at the uracil residue, releasing the captured CTCs. Released cells are then carried by a fluidic stream to the impedance sensor module. As cells pass through a pair of miniature electrodes, they interrupt the electrical current, allowing for a cell count without labeling.

Next, the cells pass into an imaging module composed of multiple microchannels connected by narrow pores. The cells are pushed against the pores and get stuck, at which point they can be fixed and immunostained directly in the module where they are then imaged by means of a fluorescence technique.

“Because of the unique design and geometry of the modules, we are able to achieve over 85% purity of the cellular preparation,” emphasizes Dr. Soper. “Thirty milliliters of whole blood can be processed in under 20 minutes, without need for preprocessing.”

Dr. Soper chose conventional injection molding fabrication to bring the price down to less than $1 per module. The product was tested with blood samples from patients with confirmed resectable and metastatic pancreatic ductal adenocarcinoma. CTCs were detected in both sets of samples.

Dr. Soper explains that this information could be used to guide the choice of therapy (surgery and chemotherapy versus chemotherapy only). Moreover, the captured CTCs can be subjected to further genetic and proteomic analysis.

Muscling into Western Blot Studies

“Single-cell proteomics is still an emerging field,” reflects Robyn Murphy, Ph.D. an associate professor of biochemistry and genetics at La Trobe University, Melbourne, Australia. “But the Western blot technology is highly accessible, inexpensive, and technically simple.” She adds that adaptation of Western blot to single-cell analysis can reveal whether the protein of interest is present and, if it is, provide details such as the protein’s size and post-translational modifications, as well as information about protein-protein interactions. “You can literally break a cell apart and analyze the components,” Dr. Murphy insists.

Dr. Murphy’s lab pioneered single-cell Western blot analysis by adapting well-established Bio-Rad instrumentation for the task. The lab also focused on applications in Dr. Murphy’s area of expertise, skeletal muscle fibers. Dr. Murphy extended the finding that distinct fiber types are differentiated by isoforms of myosin heavy chain. Specifically, she uncovered fiber-specific protein expression patterns. To do so, Dr. Murphy analyzed individual muscle fibers as well as the fibers’ cellular components.

“The first breakthrough in adapting Western blot to single-cell analysis was in forgoing the enrichment step,” she explains. “Next, we simply dissolved each fiber in sample buffer. It quickly became apparent that using very small amounts of representative sample enhances our ability to identify rare proteins.”

While it may seem that Western blot is not sensitive enough to produce a signal from such minute sample amounts, technological advances have pushed the limits of detection to just a few hundred copies of a target protein with a high-performing antibody. For example, Stain-Free technology from Bio-Rad is an in-gel chemistry incorporating a trihalocompound. When exposed to UV radiation, the trihalocompound links with tryptophans in the proteins resulting in UV-induced fluorescence. A Bio-Rad imaging system, ChemiDoc Touch, can provide high-resolution, high-sensitivity detection.

“The combination of Stain-Free gels and high-sensitivity imaging increased our protein detection abilities a hundred-fold,” says Dr. Murphy. “Sensitivity is no longer a limiting factor.”

Her team uses this approach to examine muscle fibers and explore correlations between physiological changes, such as calcium release, and biochemical properties. Future research will focus on skeletal muscle function in metabolic diseases, such as diabetes, and with aging.

Analysis of the whole tissue does not take into account the “biodiversity” of individual muscle fibers, and their differential contribution to disease. Dr. Murphy believes that understanding the behavior of individual fibers will eventually assist in the development of therapeutics to improve musculoskeletal health.

Bio-Rad’s ChemiDoc Touch Imaging system is designed to provide superior chemiluminescence sensitivity. The system is being used by researchers at La Trobe University in Melbourne, Australia, to enable a stain-free gel application in single muscle cell Western blot analysis.

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