In my previous toolbox articles (sphere formation and xenotransplantation), I’ve talked about assays that are pretty useful in determining the existence of cancer stem cell (CSC) populations based on function. What these assays cannot do is provide us with a way to identify the specific cells. Well, guess what? There’s a tool for that too!
The idea of CSC identification is one that has guided CSC research almost from day one. After all, what good is it to prove the existence of a functional population, if you have no way to identify them? Enter the search for CSC markers.
These markers come in a few flavours. First, and most common, cell surface markers: proteins that are on the outside of a cell’s membrane. Second, internal markers: proteins that are found inside the cell. And last, but certainly not least, functional markers: a cellular process (i.e. the action of a specific protein) is used as the identifier. A short list of some common markers used for CSC identification, according to cancer type, is in the table at right (a more detailed list can be found here).
Flow cytometry is a powerful tool used to identify CSCs. It is dependent on fluorescence – the emission of light from a substance, after having absorbed light from another source. Researchers take advantage of this by attaching a fluorescent dye to an antibody (a protein that recognizes a very specific part of another protein) for their CSC marker of interest (Figure 1). This antibody-fluorescent dye conjugate (or conjugate antibody) is mixed with a cell sample, where it binds with its corresponding marker on the cells. Figure 2 outlines this process in a little more detail. The cells are then run through a flow cytometer (a super original name, I know; Figure 3).
How does the flow cytometer work? Remember, one of the important parts of fluorescence is that a substance needs to absorb light in order to give off light. This is part of what the flow cytometer does. First, cells are taken from a tube up into the flow cytometer, in single file – you can imagine the tubing in the machine must be pretty small to keep the cells in single file! Once in the machine, different colours of light are shone on the cells (again, one at a time!). Where does this light come from? LASERS!! Due to the bound conjugate antibody, cells with the marker of interest will emit light once they have absorbed light from the laser. This light is then sensed and recorded by a detector, and the flow cytometer sends this information to a computer. The amount of fluorescence detected is proportional to the amount of the marker on the cell (Figure 4A).
It is important to know that molecules with fluorescent properties don’t just give off light willy-nilly. Each type of fluorescent molecule has a very specific wavelength (colour) of light that they need to absorb before they emit their own light; therefore, a flow cytometer has multiple lasers, each a different colour. Similarly, each molecule will give off a very specific wavelength of light – the detectors in the flow cytometer are specific for a particular wavelength. This is quite useful, as it means researchers can look at multiple markers on the same cell (Figure 4B) using different fluorescent dye and antibody combinations.
This all seems pretty awesome (I mean, lasers!) and a sure-fire way to identify CSCs, but as you’ve probably realized by now, as with any kind of experimental tool, there must be limitations. And there are:
- Sample preparation can cut off surface markers of interest. Cancer cells are ‘sticky,’ so they need to be separated into single cells before going through the flow cytometer (single file, remember!). Sometimes the techniques used to do this can cut off surface proteins – including the marker of interest – so the cell may falsely appear negative for the marker.
- Different CSC markers for the same cancer don’t always identify the same cells. Take glioma CSC markers CD133 and CD15: each has been shown to identify cells with CSC properties, so you might expect cells assessed for both markers to be ‘double positive’ CSCs, and non-CSCs are negative. But often, this is not the case: there will be four populations of cells, each with different levels of CSC-ness. Results like these usually require more investigation, and can provide insight into CSC hierarchy.
- Surface markers, while easy to assess, don’t necessarily have a functional role in the cell – they are simply the way the cell looks. This is where functional markers come in. Here, a fluorescent dye that can enter the cell, and interact with a protein (usually enzyme) inside the cell, is used. Interestingly, these markers tend to be enzymes that have roles in drug metabolism and/or efflux (pumping out) – making the cells ‘resistant’ to chemotherapy. Drug-resistance is something researchers believe to be a property of CSCs, allowing for disease relapse.
Like any of the other tools found in the CSC researcher’s toolbox, flow cytometry can be useful on its own, but it is most powerful when used with other tools. And more importantly, the research question: different questions require different tool combinations.
Imagine an actual toolbox, filled with hammers, screwdrivers, wrenches, etc. Now imagine that a CSC research question is a lot like assembling a piece of Ikea furniture. You’re going to need several different tools in order to put it together for the finished product. And invariably, you will always have some leftover part that didn’t really fit in anywhere. And that is just like a research question: even once you’ve put an answer together, there’s always a piece of information left over with its own set of questions.
Sara M. Nolte
Latest posts by Sara M. Nolte (see all)
- Crazy for CRISPR! - December 5, 2016
- Made in Canada solutions for the scarcity of stem cell donors? - August 8, 2016
- From bench to bedside: clinical trials and cancer drug approval in Canada - September 28, 2015