Cancer stem cells: new data

The existence of cancer stem cells has been proven

Vyacheslav Kalinin, "Elements"Stem cells are special populations of cells in a healthy body that are capable of dividing.

They divide, and some of them remain stem cells, and the other part turns into daughter cells that continue to divide and differentiate into cells of various tissues. In this way, the tissues are updated (more about stem cells can be found in the news Nobel Prize in Physiology and Medicine – 2012, "Elements", 10.10.2012; the hemato-testicular barrier is not torn, but updated, "Elements", 09.10.2012). However, sometimes something goes wrong, and a focus of uncontrollable (and, as a rule, very often) dividing cells appears in the body. This is how a cancerous tumor develops.

How does this tumor work? There are two main theories.

According to the first of them, all tumor cells are more or less equivalent and equally responsible for tumor growth.

According to the second theory, the tumor is organized like a healthy tissue: it has a limited number of stem cells (they are, respectively, called "cancer stem cells", or RSCs). Some of the split cells remain stem cells, while the other part continues to divide and gradually differentiates. They form the bulk of the tumor.

This second theory looks extremely attractive: after all, if RSCs really exist, then it is enough to destroy them, and the patient will not have any relapses of the tumor.

Possible cancer treatment regimens. The RSC theory assumes that the tumor consists of cancer stem cells (yellow) and differentiated cells (blue). If the cancer is treated with standard chemotherapy (the lower part of the scheme), then the stem cells can survive, and the tumor will grow again. If the therapy is aimed at the destruction of stem cells (the upper part of the scheme), then the tumors will not relapse. Image from the website en.wikipedia.orgHowever, so far there are more questions than answers in the RSC theory.

Where do these mysterious RSCs come from, from which cells do they develop – from stem or differentiated? What is the proportion of RSCs in the tumor – after all, if this proportion is large, then it makes no sense to concentrate on RSCs as such. Are they evenly distributed throughout the tumor or are they collected (like normal stem cells in healthy tissue) in one place? Can a differentiated tumor cell turn into an RSC again? And so on and so forth.

There are quite a few facts in favor of the RSK theory.

Firstly, the poor ability of cancer cells to transplant. One of the main methods in cancer research is the transplantation of tumors to animals (usually mice). In order for the tumor to "take root", it is necessary to transplant from several thousand to several tens of thousands of cells. Why so many? Is it because only very few of these cells are stem cells and are able to give offspring in a new organism?

Secondly, the phenotypic heterogeneity of tumor cells - in other words, the presence of differentiated and undifferentiated cells in the same tumor. Are these very undifferentiated cells the desired RSCs?

Finally, thirdly, it has recently been shown that metastases do not develop from any cell of a cancerous tumor, but only from some, at the early stages of tumor growth (see Malignant tumors and their metastases not only grow, but also actively change independently of each other, "Elements", 04/25/2012). Perhaps the "metastatic" cancer cells are essentially the same RSCs or their closest descendants, who "got lost", having broken away from the primary tumor, "took root" in a new place and thus gave rise to a secondary tumor.

In addition, there have been several direct proofs of the existence of RSC in certain types of cancer – ranging from leukemia to melanoma. However, these proofs were obtained by "dirty methods" – that is, those that can seriously distort the processes taking place in a real organism: for example, work on cell cultures or transplants of tumors to immunodeficient mice.

In a word, more substantial evidence of the existence of the RSC was needed. And finally they were received. Three independent groups of researchers were able to show that at least three types of tumors – glioblastoma, papilloma and carcinoma of the skin and intestinal adenoma – develop at the expense of stem cells.

Let's start with glioblastoma, an extremely aggressive malignant brain tumor. Previous studies in mice have shown that glioblastomas – malignant astrocytoma gliomas – develop from neural stem cells. This suggested that the progenitors of the tumor were neural stem cells that, as a result of some changes, turned into RSCs.

To test this hypothesis, it was necessary to examine "natural", not transplanted, tumors, and also to mark neural stem cells in them. To do this, the researchers bred a special line of mice, which, firstly, had a one hundred percent probability of developing malignant gliomas, and secondly, there was a complex genetic construct of Nes-deltaTK-GFP. This design had three remarkable features. Firstly, her genes were expressed only in neural stem cells and nowhere else. Secondly, it included the gene of the luminous protein GFP, which causes the cells expressing it to glow green when irradiated with ultraviolet light. Finally, thirdly, it carried the herpes virus thymidine kinase gene. The cell expressing this gene dies under the action of the herpes drug ganciclovir, which is completely harmless to cells in which this gene does not exist.

Thus, thanks to this design, researchers could easily distinguish neural stem cells from all others, just by illuminating them with light of the desired wavelength, and also quickly selectively kill these cells without touching any others.

At first, the researchers simply looked at which cells glow green in the gliomas that developed in mice. It turned out that, on the one hand, there really are such cells in tumors, and on the other hand, there are relatively few of them (Fig. 1). That is, the initial idea that the tumor originates from stem cells and that there are a limited number of these stem cells in the tumor had a right to exist.

Fig. 1. Stem cells (green) among other cells (whose nuclei are colored blue) in two different malignant gliomas. It can be seen that the number of stem cells varies in different tumors.

Image from the discussed article by Jian Chen et al in NatureThen the researchers decided to find out how the tumor resumes growth after chemotherapy.

As chemotherapy, the drug temozolomide was used, selectively killing only dividing cells.

Since the tumor grows anew after treatment with temozolomide anyway, it is obvious that the cells that survived during treatment, after the end of chemotherapy, begin to actively divide and give rise to a new tumor. The whole question was what kind of cells they were, and the experimental results clearly showed that these cells were stem cells (Fig. 2).

Fig. 2. Above: dynamics of cell divisions in the tumor. It can be seen that after chemotherapy, new tumor cells appear due to the division of cancer stem cells, and the same cell can undergo division in different periods of time. The GFP protein marks stem cells; synthetic nucleosides CldU (chlordeoxyuridine) and IdU (ioddeoxyuridine) mark cells divided in period 1 and period 2, respectively. The arrows show the same cells in different pictures. Image from the article under discussion by Jian Chen et al. in Nature.
Below: the timeline of the experiment. The next day and three days after the five-day chemotherapy, respectively, mice were injected with CldU and IdU, which are "eaten" by cells only during division. A drawing based on the description from the article under discussion by Jian Chen et al. in NatureNow it was time to apply another part of the genetic construct used, which allowed selectively killing neural stem cells.

If these cells are the originators of the tumor, then their destruction will not allow tumors to develop and will allow poor mice to live a little longer. And so it turned out (Fig. 3): although mice developed many disorders due to the absence of neural stem cells, but tumors in the subventricular zone, if they appeared, were much less pronounced than in control mice.

Fig. 3. Mice whose neural stem cells were killed by ganciclovir (green graph) showed better survival than control mice (black graph), which quickly died from developing tumors. On the horizontal axis – the time in days after the start of the action of ganciclovir. Image from the discussed article by Jian Chen et al in NatureTo summarize.

In this study, it was shown that glioblastomas originate from a limited population of RSCs, which, in turn, originate from neural stem cells, and that the early destruction of these neural stem cells helps prevent the development of a tumor.

Now let's see what was discovered by another group of researchers who studied papillomas and squamous cell carcinomas developing from them.

These scientists also worked on mutant mice whose cells contained the K14CREER/Rosa-YFP genetic construct, which caused skin stem cells and their descendants to express the yellow-glowing YFP protein if tamoxifen was injected into mice. (This technique is called fate mapping and is achieved due to the CreER-Lox system, which is described, for example, in the news To prevent Rett syndrome, it is necessary to constantly maintain the expression of the MeCP2 protein, "Elements", 13.07.2011). Having picked up a sufficiently low concentration of tamoxifen, it was possible to make only a few stem cells glow, because if too many cells glow at the same time, the image becomes "littered" and it becomes difficult to understand what is happening there at all.

Now it was necessary to cause the development of skin tumors in animals. To do this, scientists treated poor mice with two strong mutagens that cause the development of benign tumors on the skin – papillomas, some of which then turned into malignant squamous cell carcinomas.

After studying the resulting papillomas, the researchers found that the tumor originates from cells located in the basal layer of these tumors, just where the depot of normal stem cells is located in healthy skin. Some of the resulting daughter cells continue to divide and differentiate, forming a tumor, while the other remains in place and retains the "stem" properties (Fig. 4). In short, the organization of papilloma is extremely similar to that for healthy skin – all its cells seem to "remember" their role: they divide, differentiate. Due to this, all the layers that exist in normal skin are present in the papilloma. However, the ratio of cells in these layers is broken, and the natural loss of cells is much less than the "cellular profit" – and therefore a tumor forms instead of healthy tissue.

Fig. 4. Two left pictures: divisions occurring with papilloma stem cells within 6 days after YFP began to be expressed in them. The protein beta4-integrin (beta4) marks the basal layer of the epidermis, YFP marks some stem cells and their descendants, and the thymidine analogue EdU marks cells that have undergone division. It can be seen that only cells located in the basal layer divide in the papilloma, including some of the labeled stem cells (shown by white triangles). Since not all stem cells were labeled in this technique, but only a small part of them, some of the divided cells do not express YFP, however, apparently, they are still stem cells.
Two right-hand pictures: differentiation and migration, which the descendants of stem cells undergo within 14 days after YFP began to be expressed in them. Protein keratin 10 (K10) marks differentiated cells. It can be seen that many descendants of the stem papilloma cell leave the basal layer and go to the upper layers of the epidermis, gradually differentiating.
The length of the scale ruler is 50 micrometers. Image from the article under discussion by Gregory Driessens et al. in NatureWhat happens to papillomas next?

The researchers found that within a few weeks, most of the stem papilloma cells gradually differentiate and lose their stem properties. However, those cells that still retain the ability to divide begin to behave more and more "unbridled": they often divide, sometimes giving up to several thousand descendants; sometimes the descendants of only one stem cell form the entire tumor. In addition, in this work, as in the previous one, it was shown that the same cells undergo division in papillomas – which is another confirmation that it is the stem cells that are the originators of these tumors.

In malignant squamous cell carcinomas that develop from papillomas, clones of individual cells were also traced. The difference from papillomas was that, firstly, there were more dividing cells in carcinomas and they divided more actively, and secondly, that the differentiation processes in these tumors were more disrupted, and terminally differentiated cells had much less similarity with normal differentiated cells of healthy skin.

Let's summarize the results for this work as well. It was shown here that stem cells play an important role in the formation of papillomas and squamous cell carcinomas, which (at least initially) are localized in the same area where normal stem cells of healthy tissue are located.

Finally, let's see what results the third group of researchers managed to achieve.

For their work, this group used a mutant line of mice predisposed to the development of benign intestinal tumors – adenomas. To study the development of these adenomas, the researchers used an elegant modification of the fate mapping technique described above, known as "confetti". According to this technique, tamoxifen is injected into mice twice: the first injection causes intestinal stem cells to glow in one of four colors (red, blue, green or yellow), and the second in another. Moreover, if the cell glowed red after the first injection, then after the second it begins to glow blue and vice versa, and the same applies to yellow and green.

Thus, this technique allows us to study the dynamics of the development of stem cells and their descendants. Let's say, with the help of the first injection of tamoxifen, we "painted" a certain stem cell of the mouse intestine in, say, red. After waiting enough time for this cell to go through cell division many times and give many offspring, we inject tamoxifen into the mouse again. Those descendants of our red cell that have differentiated and lost their stem properties will remain red. However, those who have retained their "stemness" will change their color to blue and will pass the same color to all their descendants. Thus, we can see not only all the descendants of a given cell, but also trace the further fate of these descendants – which of them differentiated, which remained stem cells, where both are localized, and so on.

Using this technique, the researchers conducted a large series of experiments and found out that stem cells also play a crucial role in the development of adenomas. At the same time, some of the descendants of these stem cells differentiate, forming the bulk of the tumor, and the other, relatively small, part, localized just in the area that is characteristic of normal intestinal stem cells, retains stem properties and continues to divide and nourish the tumor (Fig. 5). Thus, the results of these studies, despite on a different technique and a different type of tumor, the results of the two previous groups are extremely similar.

Fig. 5. Study of the dynamics of the development of adenomas using the "confetti" technique. After the first injection of tamoxifen, the stem cells were "painted" in one of four colors. Then these cells divided within 24 days and gave rise to a tumor. After that, tamoxifen was injected into the mice for the second time, as a result of which those of the descendants of "multicolored" cells that retained stem properties changed color and turned from red to blue (and vice versa), and from yellow to green (and vice versa). These cells transmitted this new color to all their descendants. It can be seen that stem cells are localized only in a certain area of the adenoma; some of their descendants migrate and lose their ability to divide due to differentiation. Image from the discussed article by Arnout G. Schepers et al in ScienceSo, in these three works, the existence of stem cells in three types of tumors has been proven.

In addition, it has been shown that pathological stem cells originate from normal stem cells of the corresponding tissues. Of course, I immediately want to extrapolate these results to the whole oncogenesis in general and start developing means that selectively destroy the RSC as soon as possible. However, it is quite possible that everything is not so clear. Maybe RSCs are critical only for some types of cancer. In addition, it may also turn out that the results obtained in the works under discussion illustrate only a special case of oncogenesis, and the real picture looks much more confusing. For example, it is possible that differentiated cancer cells can so easily return back to the stem state that it makes no sense to focus specifically on the destruction of RSCs. Or, for example, it may turn out that not only normal stem cells, but also normal differentiated cells can turn into RSCs – just for stem cells this path is shorter. In addition, even the methods used in these studies, although they are much "cleaner" than those used to prove the existence of stem cells earlier, can still somewhat distort the real picture of cancer. However, all the same, the data obtained is extremely inspiring. It is quite clear that the RSCs will undergo intensive research in the near future and that the results of these studies will be extremely interesting.

Sources:
1) Chen et al., A restricted cell population propagates glioblastoma growth after chemotherapy // Nature. V. 488. P. 522-526.
2) Driessens et al., Defining the mode of tumour growth by clonal analysis // Nature. V. 488. P. 527-530.
3) Schepers et al., Lineage Tracing Reveals Lgr5+ Stem Cell Activity in Mouse Intestinal Adenomas // Science. V. 337. P. 730-735.

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