Monthly Archives: August 2012

Oh, trouble, stemness is thy name.

OK, just a few days ago, three major papers turned up that put a previously controversial idea about the way tumours are organised on a formidable footing, at least in cases of melanomas, gliomas and colorectal tumours in a mouse model.

We have been looking at how many cells are required to transmit a tumour from one mouse to another isogenic (genetically identical) mouse for a long time, and experiments in the fifties and early sixties led to the observation that you had to inject a certain number of cells to induce a tumour in half the injected animals, and this was way greater than one cell. However, there was also the fact that cancers were known to be clonal (originating from one cell) to contend with.

Putting the two ideas together led to the simple conclusion that not all transplanted tumour cells could induce the disease – you had to introduce so many tumour cells that one of them would be a cancer “stem cell” and could induce the tumour. This led to the formulation of the Cancer Stem Cell Hypothesis – that within a tumour there would be a small subpopulation of cells that could, if not eliminated completely, led to a return of the disease. This concept had become enshrined in the principles of radiotherapy but of course questions were raised in the molecular biology community, especially with respect to their presence in solid tumours. Skeptics took it upon themselves to point out that one could simply be looking at immune rejection or loss of viability as the reason why not all cells successfully transmitted the tumours.

The onus, then, was on proponents of the CSC hypothesis to show that there was such a thing as a resident subpopulation of cancer stem cells that led to recurrences. That evidence, it would appear, has finally come up, and in exquisite detail.

Cedric Blanplain and his group used a model of chemical carcinogenesis in mice where a chemical called DMBA is used to initiate a skin tumour and a substance called TPA is used to encourage the growth of the tumour in the site treated with DMBA. Eventually, that process results in a benign tumour called a papilloma which, after persistent TPA treatment, turns malignant. Papillomas are composed of a mass of terminally differentiated cells and an expanding population of undifferentiated, basal-epithelial cells. The number of the former stays fairly constant throughout but the latter expands.

Design of the TAM induced construct used to visualise clonal expansion. As you can see, the expression of the Yellow Fluorescent Protein is conditional to treatment with Tamoxifen, and individual basal cells can easily be identified. From Blanplain et al (See text for link)

To address whether there was a distinct CSC population that maintained the tumour, they used a very clever genetic engineering method to label individual cells – a construct that expressed Yellow Fluorescent Protein when the cells carrying them were exposed to Tamoxifen. They found that some of the labelled cells in the basal compartment expanded and also formed the non-basal differentiated structures in the tumour. In effect, they demonstrated that a subpopulation of cells could give rise to the entire tissue heterogeneity of tissue in the tumour – there was clear evidence for stemness in a population of tumour cells. They additionally found that the stem cell compartment in the tumours gave rise to progenitors capable of multiple fates and finally differentiated cells.
They were able to use microscopy to quantitatively evaluate what was happening and found that CSCs were dividing twice a day – extremely quickly compared to progenitors, which were dividing once in two days. Also, following Tamoxifen treatment, after three weeks only 20% of the originally YFP expressing cells were still expressing it (The basal ones were selectively labelled initially by using a basal specific promoter to drive gene expression)

Then of course we’ve had two more major studies. Parada and his group, again publishing in Nature showed that we had a similar thing going on with glioblastoma multforme, which has an abysmal prognosis. His group found that chemotherapy could wipe out most of the non-stem compartment but there was always a recurrence driven by a stem-like compartment of cells which escaped the effects of said chemotherapy. I find the work in question all the more intriguing because they started off with the hypothesis that these tumours were actually driven by modified versions of human adult neural stem cells in the Subventricular zone of the brain.

That hypothesis was of course well grounded in evidence – for they had identified what combination of mutations always resulted in tumours (using a conditional knock-out of genes that were known to be essential mutations in glioblastoma), and had been able to track those initiating cells to that location. Exploiting this, they used a transgenic construct encoding Green Fluorescent Protein and a Thymidylate Kinase (TK) protein driven by a Nestin promoter,which is active in adult neural stem cells (but not differentiated ones). TK expressing cells, in the presence of Ganciclovir, die if they are cycling, and this allows them to be ablated.

Telozolomide treatment kills proliferating cells, and this leads to subsequent repopulation, which is mediated by previously quiescent CSCs kicking off into division. (e) Reference – Parada et al, see text for link.

When they treated glioblastomas in these mice, they found that treatment with Telozolomide, an agent used to kill glioblastoma cells in clinical practise, was able to kill a mass of cells that was proliferating rapidly. Combining this with Ganciclovir resulted in enhanced cell kill by eliminating some of the stem cell compartment as well, but recurrence, they postulated, was inevitable because they found most of these cells to be quiescent (resting) and thus immune to drugs that hit proliferating cells (like TMZ). Having eliminated proliferating cells, though, the question was what would drive repopulation – would this be a random occurrence with any of the remaining cells kicking off? Or would the postulated CSCs be responsible? The reasoning they used to work this out was that the uptake of CldU and IdU, which are uracil analogues taken up only by proliferative cells, would be extraordinarily biased towards the GFP expressing CSC compartment if that were the source of repopulation following a pulse of TMZ treatment, and they promptly found it was indeed the case.

Treatment of Mice carrying the Nestin-TK-GFP constructs with Ganciclovir results in dramatically improved survival that is not seen in mice not carrying the construct when treated with the drug, or mice carrying the construct not treated with the drug. In some mice surviving after 10 weeks of treatment tumours have shrunk into low-grade lesions following the elimination of the CSC compartment.

And then came their piece de resistance’ – they showed that the only way to ensure long term survival in glioblastoma-afflicted mice was to eliminate the stem cell compartment altogether with ganciclovir treatment, and this led to massive survival benefits in GCV treated mice, and in some cases their brains only had low-grade lesions, benign vestiges of originally aggressive, malignant disease. A clincher if ever there was one for the CSC hypothesis.

The third paper, which I will not bore you with now, can be read here and features Hans Clevers and his group’s work showing that colorectal adenomas also depend on a CSC population that phenocopies normal intestinal crypt stem cells in terms of known surface markers. The great similarity of these CSCs with their normal adult counterparts worries me greatly, for it opens up the possibility that CSCs may be normal adult stem cells going rogue following a series of hits. Any therapy that is designed to hit these must also take care not to hit the normal stem cell compartments in tissues that are exposed.

Of course, there are still other solid tumours for which the CSC model has to be verified, but given that we have exquisite approaches like those described above to tease them out evidence either way shouldn’t be long coming.

What this means for therapy…
We will clearly have to make eliminating cancer stem cells a priority in dealing with cancer, while radiotherapy has the inherent potential to eliminate these most chemotherapy probably does not, and even with the advent of targeted therapies we will need to ensure that we don’t leave stem cells behind.
We have had some thinking heading this way already with the understanding that stem-like cells, as CSCs are otherwise known, may be sensitive to inhibitors of the Sonic hedgehog pathway which is hyperactivated in them, and there is some degree of preclinical evidence showing that this may be worthwhile investigating…

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2610257/

That is all from me this time round.

Cheers.
Ankur.