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Stanford Team Develops High-Content Flow Cytometer to Observe Cells Inside and Out

Researchers at Stanford University have developed a high-content, cell-based tool using phosphospecific flow cytometry that could enable them to screen for inhibitors against multiple endogenous kinase signaling pathways in heterogeneous primary cell populations at the single-cell level.
The researchers, working at the school’s Baxter Laboratory in Genetic Pharmacology, used the natural product library of the National Cancer Institute to identify pathway-selective inhibitors of Jak-Stat and mitogen-activated protein kinase signaling.
Although dose-response experiments in primary cells confirmed pathway selectivity, they also demonstrated differential inhibition of immune cell types, including neutrophils, monocytes, macrophages, dendritic cells, natural killer cells, B-cells, CD4+ cells, and CD8+ cells, and new druggability trends across multiple compounds.
Working on the study were Garry Nolan, an associate professor of microbiology and immunology at Stanford University and the corresponding author on the paper, and Peter Krutzik, a research associate at Stanford and the lead author of the paper.
The two researchers last week described to CBA News how the technology, dubbed phosphoflow, can be used throughout the drug-discovery process because it uses cell-surface markers to identify cell subsets and concurrent measures of intracellular phosphoepitopes to determine pathway specificity. Compounds can therefore be screened in complex primary cell populations, such as human peripheral blood.
The team has licensed the phosphoflow technology to Becton Dickinson.
According to Nolan, “some [undisclosed] pharmaceutical companies have begun to … use it on a limited basis in their pre-clinical trials.”
The study, which used a mouse model to confirm lead compound selectivity in vivo, appears in the February issue of Nature Chemical Biology.

Could you give me a little background on this work?
Garry Nolan: The work grew out of efforts that have been going on in my lab for about five years or more. We were basically looking to enumerate cell signaling in complex populations of cells at the single-cell level.
The objective of this was to bring the power of a relatively established technology, flow cytometry, together with an updated knowledge of biochemical signaling processes. The intersection of that was allowing one to not only delineate, on the surface of cells, cell-type receptors that would indicate, “OK, this is a T-cell, a B-cell, or a macrophage,” but to simultaneously go inside of the cell and measure the signaling proteins as well. That means phospho-signaling cascades using antibodies against phospho-epitopes.
All that being said, it has been a good five years of effort to make the system robust enough so that it not only works on a daily experimental basis, but it could then take the next step and be made into a high-throughput assay that would allow us to do the kind of work that was published in the Nature Chemical Biology paper.
Helping this along were the efforts of Peter Krutzik to really rev up the system’s ability to run multiple samples simultaneously in a more robust manner, and that was our Nature Methods paper on flow-based cellular barcoding, which ran as a cover article about a year-and-a-half ago. This current paper was in many ways the next step for that.
We have been using the technology to look at the standard biochemistry of cells and to do mechanistic studies. But the biggest push, at least from my standpoint in the lab, is to take the kinds of assays that we do and stop doing them in cell lines and mouse models, and start doing them in the kinds of primary cells that we are interested in, meaning human cells directly from patients to qualify drug actions or understand disease mechanisms.
Peter Krutzik: I would say that the major goal of this work was to apply phosphoflow to drug screening and basically just show if we can do this at all, and then if we can extend it into primary cells, and if we can extend it into in vivo pre-clinical models in mice as well, to determine if we see drug activity in mice, or even other animals if you are interested.     
GN: Much of this actually extends from the limitations of what people think of as standard drug discovery.
How does phosphoflow work?
GN: Phosphoflow is really a series of methods that allow you to fix and permeabilize the cells, and in a way expose the internal workings of the cells such that phosphoepitopes can be recognized by antibodies that are conjugated to fluorophores. We add additional antibodies with different colors conjugated to different fluorophores and stain the cells, so that not only on the outside, but also on the inside, we can really read the content in terms of what the cell is trying to tell us.
A couple of additional tricks that we use, which have been laid out in a whole series of papers that we published, is that one of the ways in which you can really reveal what is going on inside a cell is to really perturb the cell, in other words to stimulate the cell, to start an activity or a series of activities.
What that really means is that by getting a network of signaling proteins to start communicating with one another, and by initiating this process and enumerating many of the signaling nodes along those pathways, you get a much clearer picture of the entire cell, or at least an entire signaling network, than looking at any individual component alone.
Seeing many views of the network, rather than just a single view (which has usually been the conventional way that drug screening is done), lets you see what a cell is truly capable of in a disease state or what a drug is capable of doing to a cell in a much more natural environment. The measured environment includes not just the basal state of the cell, but also the effects relevant to perturbations that we introduce.
What is the next step in this work?
PK: I think the next step is expanding on what we demonstrated in this initial paper. This really is the foundation for a lot of future studies that we want to do. A lot of the questions that we tried to answer in the current paper are the basic questions of, ‘Is the technique quantitative? Can we get reproducible IC50 curves? Do known compounds have the predicted effect on known pathways?’
It is validating that the phosphoflow method works not only in cell lines, but also in primary cells. The next step is expanding not only the size of the library that we want to screen, going from a few hundred compounds to a few thousand compounds, but also looking at some more diverse compound libraries, so that we could perhaps identify some more diverse drug leads that might be easier to synthesize in the end.
We would also like to expand the number of measurements that we are taking, so we have an interest in cell cycle work, and expanding the screen into a very large, multiparameter analysis of the cell cycle, which would be great for chemotherapeutic-type drugs, that oftentimes inhibit the cell cycle.
We would also like to look at different pathways that are critical, particularly in the immune system. 
GN: This is a much more holistic way of understanding what drug action is about. We can measure many events at the same time, asking cells to respond to many different input stimuli, and then, looking at a complex population of cells, such as human blood, which contains dozens, if not hundreds, of different cell subtypes, having the cells tell you exactly what happens to many targets in dozens of cell types simultaneously, under varying environmental stimuli.
What came out in the paper was the realization that you would find a drug that would act against a particular target, and then when you looked at similar cell types in similar targets, you would find a complete zoo of outcomes, where each drug showed a completely different profile that you never could have understood, and you might have found out about until say, a phase 2 clinical trial, with tens or hundreds of millions of dollars spent.
You could also miss an opportunity because you would see something that you could have stalled by some tinkering with the drug, or you could have used it against a totally different disease, once you realized what in fact the drug did, versus what you thought it did.  
Is yours the only group using the phosphoflow technique?    
GN: We developed the technique originally, then we licensed it to Becton Dickinson to promote fro research purposes and they have done a great job of establishing the antibody reagents for it. Some [undisclosed] pharmaceutical companies have begun to … use it on a limited basis in their pre-clinical trials.
I have also seen one pharmaceutical company that has done something close to what Peter did, because they realized the value of it.
We are very much at the forefront of making sure that we do not have a lock on this technique. If you go to our lab’s Web site, all of the protocols are there. We want competitors; that is the objective of this kind of publication.
I think what our work points out is that phosphoflow, reaching inside the cell in a multiparametric way, is one of several variations that other people could pick up on and take forward in ways that we have not.

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