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Nolan Lab Profiles Small-Molecule Inhibitors Using New Multiplexing Method for DVS Sciences' CyTOF

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Stanford University researcher Garry Nolan has developed a cellular barcoding technique for multiplexing mass cytometry experiments on DVS Sciences' CyTOF mass cytometry instrument and has used it to investigate the effects of various small-molecule inhibitors on human peripheral blood mononuclear cells.

Detailed in a paper published in the current edition of Nature Biotechnology, the technique – termed mass-tag cellular barcoding, or MCB – allows for increased throughput and improved reproducibility, Nolan told ProteoMonitor.

DVS's CyTOF mass cytometry platform uses antibodies linked to stable isotopes of elements, which can then be read with high resolution via time-of-flight mass spectrometry, allowing for detection of analytes of interest. Nolan's barcoding technique uses these element-conjugated antibodies to tag cells in distinctive patterns, creating a barcode that can be used to track them over the course of an experiment.

"The essence is that every well in a plate can be given a different tag such that if you were to mix two wells together, you could determine where each cell in the mixed [population] originally came from," he said, noting that this could significantly reduce the time needed for multiplexed CyTOF experiments.

"Previously, if somebody had 10 or 20 96-well plates, that would be an all-day operation for someone, or at the very least a medium-scale robotics experiment that would need a lot of set-up time," he said. "You'd not only have to run each well through the machine individually, but you'd also have to treat them all individually" with metal-tagged antibodies to the targets of interest.

Treating each well individually also increases variability, Nolan noted, due to the difficulty of handling each sample identically.

"That is always the bugaboo in any experiment," he said. "You add 1.1 microliters to one well, you add 1.2 microliters to another… You want to make sure that any given differences you see are due to the biology and not experimental error."

With the MCB method, researchers can multiplex at a rate of 2n where n is the number of metal-conjugated antibodies used for the coding. In their recent research, Nolan's team used seven tags, allowing them to multiplex a full 96-well plate.

The only limit to the multiplexing technique, Nolan said, is having enough distinct metal tags left after performing the barcoding to measure the actual analytes of interest. He noted that with roughly 100 elements currently available for tagging, significant unused capacity still remains.

In the Nature Biotechnology paper, the researchers used the tagging system to perform multiplexed measurements of different peripheral blood mononuclear cell types taken from eight human donors and their responses to 27 small-molecule inhibitors. For each inhibitor, they measured 14 phosphorylation sites in 14 PBMC types at 96 different stimulative conditions, quantifying 18,816 phosphorylation levels from each multiplexed sample.

The study, Nolan said, "shows that there is an underlying global structure to how immune system cells operate, how signaling pathways work in some cells and not in others, and that by collecting enough information like this we can begin to make predictions about relationships in pathways and networks that are cell specific we knew nothing about before."

For instance, he said, similarities between two cell types' signaling data allow researchers to "make reasonable predictions about the utility of drugs [in two distinct cell types] or off-target opportunities or toxicity problems one might expect in a cell type distinct from the supposed target cell."

The research also demonstrated that a number of inhibitors that are commercially available or currently in clinical trials "have nowhere near the specificity that people claim," Nolan said, noting that many of these specificity claims come from studies done in bacterial extracts and cell lines.

"I think that if anything comes out of the … paper, it's that there is mine-able information available to us that if we took advantage of it, we'd be in a much better position to know if drugs are acting correctly or incorrectly," he said. "There is information at the single-cell level that people are leaving on the table."

Beyond its specific findings, the paper offers another example of the potential of the CyTOF instrument, Nolan said. Combining capabilities of flow cytometry and atomic mass spectrometry, the device, which DVS launched in 2009, is able to simultaneously quantify as many as 100 protein biomarkers in individual cells at a rate of roughly 1,000 cells per second, making it a potentially potent competitor to high-throughput flow cytometry systems like BD Biosciences' LSRFortessa.

Last year, Nolan, who owns a less than 1 percent share in DVS and has worked extensively – though independently – on technology and software development for the system – published a paper in Science using the CyTOF for a large signaling study in human hematopoietic cells (PM 5/13/2011).

This recent work represents a further demonstration of the technology, he said, adding that his lab also recently published a paper in Cytometry using the device in a study of the cell cycle and has also applied it to studies of cytokines, immune system transcription factors, and DNA damage markers, and plans next to use it for epigenetic studies.

DVS CEO Joseph Victor told ProteoMonitor that the company has to date sold close to thirty machines worldwide, including to several pharmaceutical and biotech firms. According to Nolan, DVS has placed instruments with Novartis and Eli Lilly, with another unnamed drugmaker currently in the process of buying one.

Given the machine's novelty and roughly $600,000 price tag, "you would expect to see most of the early adoption in academic labs or non-profit institutions," Victor said. "What's pretty exciting is that actually we've seen some early successful penetration into biopharma – both large pharma and some biotech companies."

In July 2011, DVS closed a $14.6 million funding round that included as investors 5AM Ventures, Pfizer Venture Investments, Mohr Davidow, the Roche Venture Fund, and the Ontario Institute for Cancer Research (PM 7/15/2011).

Victor said that the company's commercialization plans are moving ahead as hoped and that it does not "see or plan any need for additional funding."

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