The roots of cancer

The destruction of cancer stem cells stops the growth of the tumor, but does not kill it

Vyacheslav Kalinin, "Elements"

American scientists have for the first time investigated the role of cancer "roots" – cancer stem cells – in the development of primary tumors and metastases in colon and rectal cancer. Their experiments showed that the elimination of these cells blocks the growth of the tumor, although it does not lead to its degradation. In addition, it turned out that the presence of cancer stem cells is critically important for the development and maintenance of metastases. The data obtained indicate a potential new method of therapy for both primary cancers and currently incurable metastases.

It is now obvious that a complete and final victory over cancer is impossible. The fact is that the very nature of a living cell has the ability to turn into a malignant one. The causes of this transformation are, as a rule, mutations in genes, the products of which provide control over the growth of the cell and prevent its excessive reproduction. Quite a lot of different cancers are already known, and the sets of genes in which mutations cause each specific type of disease are usually not the same. Therefore, different means are used to treat various cancers. Researchers are constantly developing new approaches and methods that make it possible to more effectively and selectively suppress the growth of cancer cells without affecting normal ones.

Colon and rectal cancer (colorectal cancer) – one of the most common and deadly types of cancer. Its cells are characterized by inactivating mutations in the genes of tumor suppressors APC, TP53 and SMAD4 and activating mutations in KRAS oncogenes.

Cancer cells differ in their functions and abilities, and only a small part of them have oncogenic potential, that is, they are able to give rise to new tumors. These are the so-called cancer stem cells (CSCs) responsible for the progression, that is, the development of tumors and the formation of metastases. Despite the fact that CSCs are found in many malignant tumors and, obviously, could be attractive targets for cancer therapy, for a long time it remained unclear whether it was possible to influence them in practice for clinical purposes. No experimental systems for selective elimination of CSCs have been developed so far.

In order to selectively destroy cancer stem cells, it was necessary to determine which marker molecules were on their surface, which were not present in noticeable quantities on other cells. It helped that stem cells (including cancer cells) have their own specific markers. One of them is a G–protein coupled receptor 5 containing leucine-rich repeats (leucine-rich repeat-containing G-protein coupled receptor 5, Lgr5). Working in the USA oncologist Felipe de Felipe de Sousa e Melo and his colleagues conducted a study to find out how the loss of Lgr5-bearing CSCs affects the development of cancerous tumors.

Earlier, the same team of authors, using genetic engineering methods, obtained lines of laboratory mice in which the genes of the diphtheria toxin receptor (diphtheria toxin, DT) and fluorescent protein were introduced into the Lgr5+ stem cells of the intestinal epithelium. Diphtheria toxin injected into such mice selectively destroyed Lgr5-bearing cells (H. Tian et al., 2011. A reserve stem cell population in small intestine renders Lgr5-positive cells disposable).

In the new work, the authors obtained in vitro cultures of organoids from the epithelium of the large intestine – three-dimensional multicellular structures resembling miniature organs. Using genetic engineering methods, mutations that are most common in colorectal cancer were introduced into the cells of organoids (Fig. 2). Mutant organoids were implanted into mice under the skin.

Fig. 2. At the top: introduction of mutations into organoids using CRISPR/Cas technology. The letters indicate the genes into which the mutations were introduced: A – Apcmin/+, K – KrasLSL-G12D/+, V – Vil1Cre, L – Lgr5DTR/eGFP, P – Trp53, S– Smad4. Green indicates Lgr5-GFP+ cells among single cells and in organoids (organoids). In the middle:
the state of cultures of mutant cells and organoids from which the latter were selected (the length of the scale segment is 500 microns). Bottom left: the growth rate of subcutaneous tumors caused by organoids with different sets of mutations, the vertical axis is the average tumor volume (in mm3), the horizontal axis is time (in days).  Bottom right:
 the introduction of diphtheria toxin (DT, gray graph) significantly inhibits the growth of subcutaneous AKVPL tumors compared to mice in the control group who were injected with saline (saline, black graph); average tumor volumes (in mm3) are shown depending on time (in days). It can be seen that after the cessation of DT injections (DT off), the tumors began to grow rapidly. Images from the discussed article in Nature.

The more different mutations peculiar to colorectal cancer were introduced into the organoids, the more actively the tumors caused by them grew. After tumors developed, animals were injected with DT to cause the destruction of Lgr5+ tumor cells. Although the tumors stopped growing as a result, they did not degrade completely. Consequently, some other cells that do not express Lgr5 and have not yet been identified could compensate for the loss of Lgr5+ cells and maintain the size of the tumor. Quantitative determination of the spectrum of matrix RNA transcribed in the cells of a tumor suppressed by diphtheria toxin showed that the signaling pathway of Mus, known as a growth stimulator, was activated in such cells. If the administration of DT was stopped, Lgr5+ cells reappeared and tumor growth resumed at the same rate.

It has long been suggested that CSCs may be involved not only in the formation of cancerous tumors, but also in the formation of metastases. But so far this assumption has not been tested in practice. Now the experiments of the authors of the work in question have shown that tumors that developed from organoids injected under the skin did not metastasize. To study the problem more deeply, the researchers performed orthotopic transplantation (tissue transplantation into the recipient's body area, homologous to the donor's body area) of mutant organoids into the rectal tissue of mice. In the case of AKVPL organoids, tumors arose only at the implantation site (the letters denote the genes in which mutations were introduced, see Figure 2). On the contrary, AKVPSL organoids not only induced tumors at the implantation site, but also metastasized to the liver for three weeks. If in the case of AKVPL transplantation, mice practically did not die, then rodents with AKVPSL died within 60 days.

To study the role of Lgr5+-CSCs in the formation of metastases, DT injections were started two weeks after implantation, before the appearance of metastases. A check carried out three weeks later showed that the liver metastases were much smaller than without the introduction of DT.

In conclusion, the authors studied the role of Lgr5+-CSCs in maintaining already developed liver metastases. This is a very important part of the work, since for patients with such metastases, the prognosis of the course of the disease is negative, and there are no effective remedies, that is, the probability of their death is high. When organoids were injected into the portal vein of mice, primary tumors were not observed, but liver metastases developed (Fig. 3). At the same time, the "treatment" of DT dramatically suppressed the growth of metastases. Even two weeks after the final injection of DT, only isolated small metastases were observed in the liver.

Fig. 3. a – injection of organoids into the portal vein of a mouse in order to cause the formation of liver metastases without the formation of a primary tumor.  b – scheme of this liver study: after the injection of organoids (Graft), scientists waited for the appearance of liver metastases (Liver mets), 3 weeks after the injection, they began to inject diphtheria toxin (DT on) for a week; then a week and two weeks later, liver tissue was analyzed.
  c–e – bioluminescence and type of liver a week after the end of injections in experimental mice (right) and in the control group, in which saline was injected instead of diphtheria toxin.
Images from the discussed article in Nature.

The work carried out is, in fact, a detailed analysis of the participation of Lgr5+–CSCs in the initiation and development of malignant tumors and metastases. It represents the first case when, in search of approaches to cancer treatment, the authors reached the "roots" of cancer – cancer stem cells.

Contrary to expectations, elimination of Lgr5+-CSCs in primary tumors does not lead to their degradation. Tumors show plasticity, and some other cells support their existence in the absence of Lgr5+-CSCs, although they do not provide the opportunity to grow. If the action of the suppressing agent ceases, Lgr5+-CSCs reappear, and the tumor again increases in size. This situation is similar to the previously obtained data on the maintenance of intestinal homeostasis during the elimination of Lgr5+ cells (J. Beumer, H. Clevers, 2016. Regulation and plasticity of intestinal stem cells during homeostasis and regeneration). It is possible that tumor degradation can be caused by a combined effect on Lgr5+-CSCs and the Myc signaling pathway, which is activated during the elimination of Lgr5+-CSCs.

The most interesting and important are the data on the key role of Lgr5+-CSCs in the initiation, development and maintenance of liver metastases in colorectal cancer. The data obtained indicate a potential new way of treating currently incurable metastases. In the clinic, for example, specific antibodies directed against Lgr5+ cells can be used for this. The results of the work of Melo and co-authors raise a number of important new questions. For example, to what extent is the role of Lgr5+-CSCs in the development of tumors and metastases of colorectal cancer characteristic of other cancers? It has yet to be answered.

Sources:
1) F. S. Melo et al. A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer // Nature. 2017.
2) F. R. Greten. Cancer: Tumour stem-cell surprises // Nature. 2017 (popular synopsis to the article under discussion).

Portal "Eternal youth" http://vechnayamolodost.ru  02.06.2017