Inside a cancer stem cell researcher’s tool box: Sphere formation

Author: Sara M. Nolte, 06/09/14

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I recently wrote about the dilemma many cancer stem cell (CSC) researchers face when trying to model their disease of interest in the lab. So assuming a researcher has successfully chosen their model – what happens next? Allow me to introduce to you some of the tools-of-the-trade found in any CSC researcher’s tool box. This post: Sphere formation.

Figure 1: Cancer cell cultures can grow in 3-D structures called tumorspheres. Images modified from originals: tumorsphere image photo credit “Sphere-ology” by Craig Aarts, McMaster University; golf ball from WikiCommons.

Figure 1: Cancer cell cultures can grow in 3-D structures called tumorspheres. Images modified from originals: tumorsphere image photo credit “Sphere-ology” by Craig Aarts, McMaster University; golf ball from WikiCommons.

While the phrase “sphere formation” itself could be considered self-explanatory, it represents something very specific for CSC researchers. It means that in a cell culture, cells from solid tumours are able to grow in suspension (i.e. floating) in these 3-D sphere-shaped structures (I always thought they looked a bit like golf balls: Figure 1).

As an assay (experimental test), sphere formation is used to identify the stem cell characteristic of self-renewal in vitro (in a dish). When applied to cancer, it can identify the presence CSCs in solid tumours, as these cells will have the ability to self-renew; and thus, form spheres (tumorspheres, in this case).

The usefulness of this assay hinges on the theory that only a self-renewing (i.e. stem cell or stem-like cell) can form a sphere in culture. Since sphere-forming cells are stem-like in nature, they also have the ability to differentiate into all of the non-stem-like cell types (i.e. cells that cannot self-renew) found in the initial cell culture/tumour. As a result, the structure of a tumorsphere is a mixture of CSCs and other, more ‘differentiated’, cell types (Figure 2).

Where does the actual ‘assay’ part of a sphere formation assay come in? By taking advantage of sphere-forming ability of CSCs, researchers can quantify the number of spheres in a specific sample, and thereby estimate the amount of CSCs in the sample. This can be further applied to assessing the effects of different drugs, genes, etc. on the CSCs’ ability to self-renew (which will hopefully translate into more effect treatments in the clinic!).

Figure 2: The process of a sphere formation assay. Spheres are composed of all cell types found in sample/tumor. Left: any initial spheres are dissociated to single cells, and the ability of these single cells to grow as spheres is observed. Some more differentiated cell types (yellow) are unable to grown as spheres; while stem-like cells (blue and yellow) can grown as spheres. Right: to distinguish between true stem-like cells (pink) and cells with residual self-renewing ability (blue), additional rounds of sphere dissociation and observation can be done.

Figure 2: The process of a sphere formation assay. Spheres are composed of all cell types found in sample/tumour. Left: any initial spheres are dissociated to single cells, and the ability of these single cells to grow as spheres is observed. Some more differentiated cell types (yellow) are unable to grown as spheres; while stem-like cells (blue and yellow) can grown as spheres. Right: to distinguish between true stem-like cells (pink) and cells with residual self-renewing ability (blue), additional rounds of sphere dissociation and observation can be done.

So how do we do it? First, we start with a single-cell suspension: the spheres of the cell culture are broken up into their individual cells (a process called ‘dissociation’), and it is now single cells floating in the media of choice (Figure 2). After this, there are several variations on how a sphere formation assay can be used, and how the data are presented. I have listed a couple below, and Figure 3 illustrates them in more detail.

  1. Limiting dilution assay: Looks at the ABSENCE of sphere formation over  a range of cell numbers; used to calculate the minimum number of cells needed in order to see one CSC.
  2. Clonal assay: Looks at the number of spheres formed by one cell; used to calculate sphere-forming efficiency, which can be used to estimate the percentage of CSCs in a sample.

Despite all its merits, there are a couple of important caveats to any sphere formation assay. First, sometimes a cell might not actually be a stem-like cell, but may be a differentiated cell that has temporarily regained or retains some residual self-renewal ability. This means that the cell may still form a sphere, causing us to overestimate the amount of CSCs. To address this issue, researchers will often repeat sphere formation assays in series (Figure 2, above), where it is thought that only the true self-renewing cells will continue to make spheres, while all other cells cannot.

Figure 3:  Several variations of a sphere formation assay exist, and use different methods to estimate the number of cancer stem cells (CSCs). An important piece of laboratory equipment is a “96-well plate.” This is a plastic plate about the size of your hand; each circle in the diagram represents one well. Top: Limiting dilution assay: each column in the plate contains a different number of cells (as written in the diagram). After a period of growth (typically 7-14 days) the researcher will count the percent of wells with NO spheres for each cell number, and plot this on a graph (right). Using mathematical models, the frequency of CSCs can be calculated. The solid line has fewer wells with no spheres at low cell numbers than the dotted line; indicating a higher CSC frequency. Bottom: Clonal assay: the entire plate contains a single cell in each well. After the growth period, the researcher counts the number of spheres in the entire plate, and divides this by the number of cells they started with (i.e. 96 cells), allowing them to calculate the sphere-forming efficiency. Sample ‘A’ (solid line) has greater efficiency than Sample ‘B’ (dotted line), suggesting there must be more CSCs present in ‘A.’

Figure 3: Several variations of a sphere formation assay exist, and use different methods to estimate the number of cancer stem cells (CSCs). An important piece of laboratory equipment is a “96-well plate.” This is a plastic plate about the size of your hand; each circle in the diagram represents one well. Top: Limiting dilution assay: each column in the plate contains a different number of cells (as written in the diagram). After a period of growth (typically 7-14 days) the researcher will count the percent of wells with NO spheres for each cell number, and plot this on a graph (right). Using mathematical models, the frequency of CSCs can be calculated. The solid line has fewer wells with no spheres at low cell numbers than the dotted line; indicating a higher CSC frequency. Bottom: Clonal assay: the entire plate contains a single cell in each well. After the growth period, the researcher counts the number of spheres in the entire plate, and divides this by the number of cells they started with (i.e. 96 cells), allowing them to calculate the sphere-forming efficiency. Sample ‘A’ (solid line) has greater efficiency than Sample ‘B’ (dotted line), suggesting there must be more CSCs present in ‘A.’

A second caveat: cancer cells can be thought of as being highly social creatures – they are constantly interacting with each other and their environment. These assays require extremely low numbers of cells; however, the fewer cells there are, the harder it is for them to interact, and much like a person, without any interaction, cancer cells may not be able to realize their full potential. This means that a cell might be a CSC and ready to form a sphere, but without a signal from a neighbouring cell, it doesn’t – leading a researcher to underestimate the number of CSCs. While this limitation may never truly be overcome (we are working in a dish, after all!), researchers will use specially-formulated growth media to ‘encourage’ cells to reach their full potential, in the absence of the interactions they need.

While sphere formation may not be the end-all-be-all, we CSC researchers often treat it as such – this assay can be found in almost any publication addressing cancer stem cells. This is definitely a staple in the CSC researchers toolbox, but as we’ll see in some upcoming posts, much like choosing a model system, the usefulness of this assay – or any assay, for that matter – is dependent on the research question being asked, and disease being studied.

Editor’s note: This post was modified shortly after initial publication to include a third Figure inadvertently omitted from the original post sent by the author -LW

Research cited:
Pastrana E., Silva-Vargas V. & Doetsch F. Eyes wide open: a critical review of sphere-formation as an assay for stem cells., Cell stem cell, PMID:
Tropepe V., Sibilia M., Ciruna B.G., Rossant J., Wagner E.F. & van der Kooy D. Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon., Developmental biology, PMID:

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Sara M. Nolte

Sara Nolte holds a Bachelor of Health Sciences and Masters of Science in Biochemistry & Biomedical Sciences from McMaster University. Her MSc research focused on developing of cancer stem model to study brain metastases from the lung. She then spent a year working on developing cell-based cancer immunotherapies. Throughout a highly productive graduate career, Sara became interested in scientific communication and education. She is now involved in developing undergraduate programs and courses in the health sciences at McMaster, and is looking for ways to improve scientific communication with the public. Outside of science, Sara enjoys participating in a variety of sports, and is a competitive Olympic weightlifter hoping to compete at the National level soon!
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