Signaling pathways have traditionally been queried and analyzed at the protein level by bulk measurements using a Western blot, an ELISA, or perhaps a bead array. Yet these can mask the intracellular heterogeneity inherent in biological samples, making it impossible to distinguish between half the protein-of-interest in the cells being activated, say, and all the protein in half the cells being activated. Flow cytometry, on the other hand, by its very nature is the quintessential single-cell technique. Flow can phenotype the cell while at the same time provide multiplexed quantitation not only of which proteins are to be found, and in what form, but yields that information at high enough numbers and throughput to generate robust statistics.
From cytokines and small molecules interacting with a cell surface receptor, through phosphorylation, translocation, ubiquitination, cleavage, acetylation, methylation, and transcription factors binding to DNA to make new protein and start the process anew, there are a myriad of ways in which proteins participate in the cell’s information cascade. Yet the presence of a phosphate group (“or other post-translational modifications, to the extent that there are good antibodies,” adds Kjetil Tasken, center director of the Institute for Cancer Research at the University of Oslo) at a specific residue tends to present a more clear-cut, less ambiguous readout than does protein expression level—with the added bonus of comparatively rapid kinetics. Thus for many researchers “signaling cascade” has largely become synonymous with phosphorylation.
Phosphoflow
Examining cells’ phosphosites by flow cytometry has its own name: phosphoflow. When flow cytometry is used to phenotype cells, it’s typically run as a live cell assay in which fluorophore-conjugated antibodies query cell-surface antigens. “But nearly all phosphorylation and cleavage and other signaling-related events are happening within the cell. So that means that people need to fix and permeabilize the cells to allow the antibodies to get access,” explains Christopher Manning, flow cytometry group leader at Cell Signaling Technology (CST).
Because of the potential volatility of phosphorylation, researchers tend to use a fairly rapid time scale when looking at phosphorylation—“we’ll stimulate the cells and usually within 10 to 15 minutes we fix the cells and then start the staining,” says Garry Nolan, the Rachford and Carlota A. Harris Professor of Microbiology and Immunology at Stanford University School of Medicine. Similarly, notes Tasken, “you may want to put in phosphatase inhibitors in some cases, at least to validate the antibodies.”
“Fix and perm” is a fairly standard procedure, with many published protocols and commercial kits on the market. But the nature of antibodies is that “from clone to clone and from target to target there is not a universal protocol that works across the board,” says Manning. It is especially important to assure compatibility when designing multiplex experiments.
While a flow cytometer may in theory be capable of querying up to 50 different parameters, “in fact, people can barely get it to do 25, and even 10-15 colors is a heck of a lot of work,” Nolan says.
Manning finds that “most often our customers are doing lower-plex, where they’re just wanting to look at one protein phosphorylation event. And they might be combining that with a couple of extracellular markers.”
For example
“With flow analysis you don’t really get hard-core proteomics,” points out Tasken. With a limited number of readouts, you need to decide ahead of time what to look for.
Small molecule drug development in oncology largely looks to modulate the signaling strength in different cell-signaling cascades, explains David Draper, associate director of scientific development at MI Bioresearch, an oncology-focused CRO. And while it’s important to know how a test compound alters, for example, phosphorylation of STAT1, STAT2, STAT5, and STAT6, “these signaling cascades that these drugs affect can play both pro-tumor and anti-tumor roles, depending on what subtype you’re looking at.” Draper and his team developed a platform to establish the phospho-status of a variety of proteins involved in signaling on a cell-by-cell basis while simultaneously determining whether the cell is of tumor or immune origin.
Up the numbers
Not content to run just a single population per tube or well, several labs have found ways to barcode the samples by labeling them with different intensities of different dyes: “We can analyze 64 different cell populations at the same time by that type of technology,” extols Tasken. “That allows us to do experiments where we stimulate T cells to look at T cell signaling, we can do time courses, dose titrations, perturbations with inhibitors, for example, or we can have several patient samples—and analyze them at the same time with the same baseline in the flow cytometer.”
Sometimes researchers find the number of parameters they can query with flow cytometry to be too limiting. Just to properly phenotype blood, for example, “with 10 [colors] you can barely call out all the major cell subsets—T cells, B cells, macrophages, NK cells, and five other cell types, and all you’ve got is one marker to call them out for each,” laments Nolan. But with the 30 or more markers offered by the CyTOF mass cytometer—Nolan’s current go-to single cell protein platform—“now you can call out most of the major cell subsets and you can go inside with another 20 markers and look at the signaling proteins.” This has allowed insight into how the components of the immune system can affect each other as cogs in a machine, with expression of some proteins necessarily changing along with others, whereas otherwise it would have read out as so much statistical noise.
Phosphoflow and mass cytometry are already old technology, says Robert Balderas, vice president, biological Sciences at BD Biosciences. “Today, there is the advent of technologies that allow a conjugation of an antibody with an oligonucleotide … Ours is called Abseq, others are CITE-seq and REAP-seq.” The cells are subjected to single-cell RNA sequencing, with the oligos, like fluorophores (or heavy metal isotopes, as in the case of mass cytometry), being used as unique labels to indicate antibody binding to its target. The practical limit of how many parameters can be measured in a single cell comes down to how many good antibodies are available. But sequencing is still relatively expensive, and so Balderas recommends first using a flow sorter to subset the cells into a purer population “so that we don’t waste sequence.”
With its long history and easy availability of qualified reagents, flow cytometry is probably still the quickest and most entry-level way to get single-cell information about signaling known to be important in your system.