01 September 2017

How melanoma metastasizes

The midkin protein secreted by melanoma forms niches for metastases

Vyacheslav Kalinin, "Elements"


Fig. 1. The general scheme of the pathway by which melanoma forms metastases in distant lymph nodes and organs. Contrary to the previously accepted point of view, this process does not include the formation of lymphatic vessels in and around the primary tumor. The midkin cell growth factor (MDK) produced by the tumor is secreted in free form and as part of exosomes. It reaches distant pre-metastatic lymph nodes and organs, in which metastases will then form. Whether midkin circulates in the lymph or/and in the blood remains unknown. Midkin induces lymphangiogenesis in these areas, which helps prepare them for the arrival of cancer cells. A drawing from the popular synopsis to the article under discussion in Nature

Lines of laboratory mice have been constructed to trace the formation of new lymphatic vessels throughout the body. It has been shown that the cell growth factor midkin, secreted by melanoma, stimulates this process and prepares niches for the formation of metastases far from the tumor. The results obtained may allow the development of new markers for determining the risk of metastasis, as well as new approaches for the prevention and treatment of metastases.

Cancer as a cause of death is confidently coming out on top in the world, and in a number of developed countries has already taken this position. Primary cancer is not so dangerous: it can often be surgically removed. Much more dangerous are cancer cells that spread throughout the body and can develop into secondary tumors (metastases), sometimes even in organs and tissues very far from the primary tumor. Active research is underway to determine the ways of migration of cancer cells through the body and block the formation of metastases. These problems have not been solved yet, and any noticeable step in their resolution is very important both from the point of view of theoretical and practical oncology.

Melanoma of the skin is a frequently occurring aggressive disease with very early colonization of the lymph nodes by cancer cells, which is preceded by the active formation of new lymphatic vessels (lymphangiogenesis). Removal of the primary tumor and the lymph nodes adjacent to it does not always prolong the patient's life. Moreover, during the progression of melanoma, the lymphatic vessels of the tumor collapse and lose their function. Therefore, it remained unclear whether lymphangiogenesis was associated with melanoma metastasis, and if so, how. It has been suggested that proteins secreted by the tumor and/or its stroma are associated with the formation of niches for metastatic melanoma cells. The study of the role of lymphangiogenesis in melanoma and other malignant tumors was complicated by the fact that there were no suitable experimental animals for visualization of distant pre-metastatic niches.

Scientists led by Maria Soengas (María S. Soengas) from the Spanish National Center for Cancer Research, together with colleagues from several other medical and scientific institutions in Spain and the United States, focused their efforts on tracing lymphangiogenesis throughout the body. To do this, they used the well-known property of vascular endothelial growth factor receptor 3 (vascular endothelial growth factor receptor 3, VEGFR3). Its expression in the cells of lymphatic vessels is normally strongly suppressed, but it is sharply activated in pathological conditions, in inflammation and in cancer. Using genetic engineering methods, the authors constructed lines of model mice in which the fluorescent luciferase protein gene was placed under the control of the Vegfr3 promoter.

Using a sensor that registers fluorescence, they observed the growth of lymphatic vessels throughout the mouse body after transplanting cultured melanoma cells under their skin, depending on the progression of metastasis (Fig. 2).


Fig. 2. Luciferase emission after transplantation of melanoma cells to mice. On the left, mice after transplantation of melanoma cell lines with a lower potential for metastasis are shown, on the right – with a large one. The numbers in the photo are the days after implantation. Primary tumors are outlined with a red dotted line. A drawing from the discussed article in Nature

It turned out that the growth of blood vessels in various areas of the body remote from the place of transplantation occurs independently of their growth around the primary tumor. Consequently, lymphangiogenesis around the primary tumor does not determine the metastatic colonization of cancer cells in other organs and tissues. Moreover, the level of expression of vascular endothelial growth factor in the tumor C (VEGFC), which binds to VEGFR3 and activates it, did not correlate with the formation of distant metastases. That is, lymphangiogenesis in remote areas of the body is not associated with the production of VEGF by the tumor.

It has already been shown that a tumor induces the formation of niches for metastases in distant organs before metastatic cells arrive there. If the tumor is removed, metastases can still form in these niches. The authors showed that even poorly developed tumors induced luciferase expression in the lymph nodes of the liver, spleen and lungs. After a while, metastases formed there. Removal of the tumor was accompanied by a significant decrease in the level of luciferase in the pre-metastatic areas, but later it could increase again, and subsequently metastases formed there (Fig. 3).


Fig. 3. Quantitative assessment of luciferase emission after transplantation of melanoma cells to mice and removal of the primary tumor (blue numbers indicate the moments: 1 – implantation of the tumor, 2 – before removal of the primary tumor, 3 – after removal, 4 – after detection of metastases). Data for metastases in lymph nodes, lungs and skin are shown. A drawing from the discussed article in Nature

These data show that activation of distal lymphangiogenesis induced by tumor cells is an indicator of the formation of a pre-metastatic niche.

Since it turned out that VEGF is not the cause of distant lymphangiogenesis and the formation of metastases, the authors investigated other factors secreted by the tumor that could be associated with these processes. Using liquid chromatography and mass spectrometry, they compared sets of peptides obtained by cleavage of proteins secreted by cultures of melanoma cells with different ability to form metastases. Comparison with the catalogs of "peptide signatures" showed that the most likely candidate for the role of an inducer of lymphangiogenesis is midkin (MDK). This small protein is known as Cell growth factor, in particular, stimulating the growth of blood vessels, but its involvement in lymphangiogenesis was not previously known.

Immunohistological analysis showed that midkin is actively produced by melanoma cells with a high potential for metastasis formation, and very weakly in cells with a low potential (Fig. 4).


Fig. 4. From left to right, histological preparations of cells transplanted into mice with melanomas with an increasing potential for metastasis are shown. Midkin is visible in the form of red veins and spots. A drawing from the discussed article in Nature

Suppression of expression and secretion of midkin using specially selected "hairpin" RNA (shRNA) did not affect the expression of VEGFC or VEGFD. Moreover, it did not affect the growth of blood vessels in xenograft (caused by human melanoma cells) tumors. But the formation of lymphatic vessels in them was suppressed. The most striking effect of midkin suppression is the cessation of luminescence and the formation of metastases in distant lymph nodes and organs (Fig. 5). In mice with suppressed midkin expression, after removal of the primary tumor, metastases did not form at all. At the same time, an increase in midkin expression induced by the introduction of a lentiviral vector into cells carrying the midkin coding gene turned cells with a weak potential for metastasis into actively metastasizing cells.


Fig. 5. On the left: luciferase emission (color indicates intensity) after mouse transplantation of melanoma cells. The use of special "hairpin" RNAs that turn off the gene encoding midkin inhibits lymphangiogenesis (the results of the use of two of the five such RNAs used in the work are shown). A drawing from the discussed article in Nature

The culture medium of cells actively secreting midkin stimulated the proliferation of the endothelium of lymphatic vessels. And in the mouse body, midkin accumulated in the emerging lymphatic vessels (Fig. 6). This led to the activation of the proliferation-stimulating protein mTOR in the endothelium. In addition, midkin not only stimulated lymphangiogenesis, but also as such promoted the adhesion of cancer cells in newly formed lymphatic vessels.


Fig. 6. Midkin (green) accumulates at the sites of formation of new lymphatic vessels and their entry into lymph nodes (shown by arrows). Asterisks mark stroma cells. The lymphatic vessels are contrasted with red. A drawing from the discussed article in Nature

Thus, the conducted studies revealed the mechanisms by which the primary melanoma tumor secreting midkin causes the growth of lymphatic vessels. This, in turn, leads to the formation of pre-metastatic niches through hitherto unknown processes.

A retrospective study of benign moles and human melanoma also showed a link between midkin expression and the clinical picture of the disease. For the operated patients who had a high level of midkin in the lymph nodes free of tumor cells, the prognosis of life expectancy without relapses was significantly worse than for patients with low midkin levels.

The results of the work under discussion are of great importance for both theoretical and practical oncology. But a number of important questions have remained unanswered so far. What are the ways in which midkin gets to distant parts of the body through the blood and/or lymphatic vessels? Which of the receptors of lymphatic vascular endothelial cells is associated with lymphangiogenesis? To what extent can the results obtained on melanoma be extended to other cancers?

From a practical point of view, midkin and lymphangiogenesis can serve as good molecular and functional markers of the risk of metastasis. Lymphangiogenesis and its drivers (midkin, mTOR and others) may become targets for new methods and means of preventing metastasis.

1) D. Olmeda et al. Whole-body imaging of lymphovascular niches identifies pre-metastatic roles of midkine // Nature. 2017. V. 546. P. 676–680.
2) A. Hoshino, D. Lyden. Metastasis: Lymphatic detours for cancer // Nature. 2017. V. 546. P. 609-610. (Popular synopsis to the article under discussion.)

Portal "Eternal youth" http://vechnayamolodost.ru  01.09.2017

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