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Stanford Researchers Use Nano-Immunoassay to Study Protein Phosphoisoforms Related to Cancer

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By Adam Bonislawski

Researchers at the Stanford University School of Medicine have developed a nanofluidic immunoassay capable of measuring low-abundance protein phosphoisoforms potentially involved in cancer signaling processes.

With the assay, the scientists have begun examining protein phosphorylation levels in a variety of cancer types. The team has also entered into collaborations with several pharmaceutical firms to investigate the effects of therapeutic agents on protein phosphorylation levels in tumor cells, Dean Felsher, assistant professor of oncology and pathology at Stanford University School of Medicine, told ProteoMonitor.

Using Cell Biosciences' capillary-based Nanopro1000 instrument, the researchers separate tumor cell proteins based on charge via isoelectric focusing and then use antibodies to detect and quantify the proteins of interest. Because the proteins are separated based on charge rather than size, different phosphoisoforms of the same protein register as distinct analytes, and both the total amount of phosphorylated target protein and relative amounts of the various phosphoisoforms can be quantified.

"Typically if you run a Western blot, you'll be lucky if you see one other isomer, because they'll probably be all on top of each other," Felsher said. "But we all know that many of these proteins are phosphorylated on multiple sites, and often in different combinations, so this allows you to actually see the combinations."

With kinase inhibitors making up a considerable portion of the pharmaceutical development pipeline, phosphoproteomics has become a significant area of research interest, with scientists at companies like Merck (PM 08/26/2010) and centers like the University of Texas MD Anderson Cancer Center investigating links between protein phosphorylation levels and disease states (08/27/2010). Less work has been done, however, on linking disease states to levels of proteins' different phosphoisoforms.

"What we're trying to do is classify tumors where we're not just going to say, 'Phosphorylated – yes or no?', but what is the pattern?," Felsher said. "This technology allows you to ask, in my specimen, with four different isomers, what is the exact quantification of each one, and do particular isomers correlate with outcome?"

"That's our hypothesis," he said. "Being able to measure these things much more quantitatively, looking at specific isomers, we'll start finding protein modification states that are really true predictors of biology."

The assay is also useful in that it can work with small samples such as those collected by fine-needle aspirate biopsies, Felsher said, making it potentially useful in studying rare cell populations like circulating tumor cells and cancer stem cells, as well.

The work is still in the early stages, he noted, but the researchers have "shown examples where you can see that a rare isomer changes in a specific way and that correlates with a clinical response."

At last month's American Association for Cancer Research International Conference on Molecular Diagnostics in Cancer Therapeutic Development, Alice Fan, instructor in the division of oncology at Stanford University Medical School and one of Felsher's colleagues, presented results showing that profiles of proteins in the RAS and MAP kinase pathways built using the assay allowed researchers to distinguish tumor cells from normal cells and group different tumor types. They were also able to detect measurable effects on protein phosphorylation in lymphoma and myelodysplactis syndrome patients in response to two novel therapies.

At the American Society of Hematology annual meeting in Orlando in December, Felsher's team will present data from a study done in collaboration with pharmaceutical firm OncoNova Therapeutics on a drug the company has developed for myelodysplastic syndromes targeting the polo-like kinase pathway.

The researchers have also identified phosphoisoform changes linked to clinical response in chronic myelogenous leukemia patients being treated with tyrosine kinase inhibitors and demonstrated changes in phosphorylation patterns of proteins in lymphoma cells in response to treatment with the cholesterol drug Lipitor.

So far, they've developed the assay for about 20 proteins with assay development for another 20 underway — a process, Felsher noted, that is "mainly dependent on having a good antibody."

In total, they've examined some 50 different clinical specimens across eight different tumor types including colon cancer, head and neck cancer, kidney cancer, ovarian cancer, and lymphoma.

"[It's] not enough to make great clinical conclusions, but enough to say that the technology is very robust in that we're able to measure these proteins and phosphorylation states in many different tumors, and it looks like it will be important," Felsher said. "Now what we're doing is trying to get enough data to show that these results are significant."

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To that end, he noted, the researchers are "trying to amass as many samples as we can get." Currently they have several hundred tissue samples they plan to analyze and are looking into ways they might collect additional samples.

They've also entered into collaborations with several drugmakers to investigate the effects of various therapeutic agents, Felsher said, something, he noted, he expects to do more of going forward.

"We think that we're very well poised to really help companies interrogate their drugs, and we're uniquely capable of analyzing the data in an interesting way other than just measuring whether or not a drug targets a molecule," he said.

The assay could also be used to help drug companies select patient cohorts for clinical trials and personalize therapy, he suggested.

"You need a way to make measurements to decide if [a patient is] even a candidate for the therapeutic agent," he said. "Using this technology we can do things like measure oncoproteins in clinical specimens to see if they're present. We'll be able to make decisions on which patients are likely to respond to therapy."

In terms of commercialization plans, Felsher noted that Cell Biosciences has incorporated the technology used in the assay into its Nanopro1000 machine, which his team uses in its research.

David Voehringer, North American sales director for Cell Biosciences told ProteoMonitor that, while the company might be interested in commercial applications based on the research, much of the work "is still in the early R&D phases."

"We all believe that some of those novel post-translational modifications may translate into biomarkers at some point, so from that philosophical standpoint, at some stage developing tools and assays might be something we're interested in," he said.

Voehringer declined to comment on any commercial relationship between Cell Biosciences and the Stanford researchers, but said the two groups were moving to investigate "the broad question of how you really implement this technology in your clinical trials."

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