At the mercy of the tumor

Why are metastases so difficult to stop?

Maria Rafaeva, "First-hand Science"

When at least one of the 100 trillion normal cells of the body degenerates into cancer and is not destroyed, the trigger is triggered and tumor growth is triggered. With the development of the disease, some cancer cells form metastases – secondary foci of tumor growth. And often even timely removal of the primary tumor and postoperative therapy are not able to cause remission. It turns out that the primary tumor, already at the early stages of its growth, is able to "train" the microenvironment in the foci of the development of future metastases, and the metastatic cancer cells themselves "adjust" the work of their genes so as to better take root in a new place. If we know how to prevent the growth and development of metastases, we can save up to 90% of people dying from major cancers.

In recent decades, the efforts of many oncologists have been aimed at establishing detailed mechanisms of the origin of a cancerous tumor, the regulation of its active growth and the formation of a favorable microenvironment from stroma cells, the connective tissue framework of the organ. Over time, it became clear that the main threat of cancer lies in its ability to spread throughout the body.

Some cancer cells of the primary tumor separate from it and enter the bloodstream or lymphstream, through which they travel to the main arteries of the vascular system. Cells that have overcome this pathway exit into the stroma of the organ due to the delay in narrow capillaries and adhesion ("sticking") to the inner vascular wall. Only a small part of these cancer cells survive in the new environment, but they are the originator of new tumor growth foci.

The process of scattering of cancer cells throughout the body, called metastasis (from other-Greek. "change, transfer"), was first described back in 1889 by the British surgeon and pathologist S. Paget, but its mechanism remained a mystery to the scientific community for a long time.

Paget drew an analogy between metastasis and germination of seeds, which also survive only in a suitable "soil"-microenvironment. At that time, it was impossible to give experimental confirmation to this idea, therefore, for a long time the theory of the American pathologist J. J. was prevailing. According to Ewing, the main role in the spread of metastases in the body is played by the features of the dynamics of blood flow and the structure of the vascular system.

Some cells of the primary tumor enter the bloodstream or lymph flow and travel through the vascular system. Lingering in the narrow capillaries of organs and "sticking" to their inner wall, they pass from the lumen of the vessel into the stroma of the organ. Only a few cancer cells manage to survive at the same time, but they form new foci of tumor growth – metastases. By: (Massagu, Obenauf, 2016).

Finally, in the 1970s, thanks to experiments on laboratory mice that were injected with cancer cells labeled with radioactive isotopes, the American researcher I. Fiedler was able to prove that the nature of cancer cells affects the result of metastasis: melanoma cells metastasized to the lungs, but not to the liver, in the vessels of which they did not survive.

Later, other facts were established confirming that cancer cells of different nature metastasize mainly to certain organs, sometimes even in a certain sequence. For example, breast cancer cells form metastases first in the bones, liver, lymph nodes and lungs, and only then in the brain. The phenomenon of a specific distribution of metastases in the body was called organotropy of metastasis.

And today there are still many unanswered questions in oncology. For example, are metastatic cancer cells different from other primary tumor cells? What is the basis of organotropy? And most importantly: how do metastases manage to survive after removal of the primary tumor and chemotherapy?

Metastases: from genetics to epigenetics

The behavior of any cell is genetically determined. The malignant transformation of normal cells into cancerous cells is associated with mutations in the driver genes that lead to uncontrolled cell division. These beneficial mutations for tumor cells are accompanied by mutations in other genes that initially do not affect tumor growth – passenger genes. With each generation of cells, each new clone, these genetic changes accumulate. Some cell clones succeed more than others, which allows us to talk about "evolutionary" changes inside the tumor.

According to one hypothesis, some tumor cells acquire the ability to metastasize as a result of a similar accumulation of mutations in the driver genes that trigger this process. However, there is no experimental confirmation of this: today scientists agree that most cells of the primary tumor can metastasize. Thus, a genetic study of pancreatic cancer cells that disperse into lymph nodes, liver and lungs revealed that the founder cells of metastases have the same "profile" of driver mutations as the primary tumor: their genetic similarity is even higher than that of randomly taken cells of normal tissue (Alderton, 2017).

But what happens later with the genomes of metastatic cells? When comparing the cells of primary mammary tumors and their local (in the nearest lymph nodes) and distant metastases, it was found that in new foci, the genomes of cancer cells continue to evolve independently of the primary tumor (Yates et al., 2017). At the same time, the "profile" of mutations in metastatic foci in one organ turns out to be similar, but different in different organs.

This means that cancer cells adapt to the new microenvironment depending on the niche they occupy. Consequently, although initially the cells of metastases and primary tumors had similar mutations, the process of adaptation to the new environment initiated the emergence of new genetic changes. And these mutations are mostly associated not with driver genes, but with passenger genes.

Normal tissue cells have predictable behavior: the rate of proliferation, the life span, the nature of interaction with other cells. With malignant transformation, mutations accumulate, and the tumor gradually becomes genetically heterogeneous, i.e. consisting of generations of several clones of cancer cells. As a result of chemotherapy, cancer cells that are sensitive to the drug die, while others survive and multiply. Chemotherapeutic drugs themselves are mutagens and can provoke changes in the genome of cancer cells, as well as give rise to new tumor clones. At this stage, the behavior of cancer cells is already unpredictable, since their genetic and epigenetic changes lead to the acquisition of new properties by cells. By: (Baranova, 2017).

Interestingly, at least one gene has been discovered – FBXW7, mutations in which counteract metastasis (Mlecnik et al., 2016). This happens due to an increase in the pro-inflammatory reaction, an increase in the number and activity of T-lymphocytes, which stimulates the formation of immune reactions against tumor cells. Thus, the appearance of mutations in the FBXW7 gene of cancer cells prevents the development of immunosuppression characteristic of tumors.

So far, we have been talking about direct changes in the DNA structure itself. The next step in the regulation of gene expression is epigenetic changes associated with the methylation of DNA and histones (DNA–binding proteins). By their nature, such changes are more plastic compared to genetic ones: they are influenced by cellular signal transmission cascades that adapt to the action of external factors. It turned out that in metastatic cells, the "epigenetic code" is significantly different from the "code" of the primary tumor. Thus, in the case of pancreatic cancer, metastases show a significant weakening of histone and DNA methylation. As a result, areas of inactive chromatin (substances of chromosomes) become active, accessible to transcription factors that control the reading of information from DNA to matrix RNA. This mechanism leads to increased expression of oncogenesis genes in metastatic cells (Alderton, 2017).

At the fork of metabolic pathways

Epigenetic regulation is one of the most important mechanisms of programming cellular metabolism. With the growth of the tumor, areas are formed inside it where blood vessels do not reach and where oxygen deficiency (hypoxia) occurs. As a result of DNA demethylation, a gene encoding the HIF-1-alpha protein, which is called hypoxia–induced factor, begins to work actively in cancer cells. The enhanced formation of this protein, in turn, affects the level of gene expression of many metabolic enzymes and transporter proteins, which leads to complex changes in the metabolism of cancer cells.

As is known, cellular metabolism includes the processes of splitting compounds with the release of energy (catabolism) and their formation using energy (anabolism). The energy metabolism of the cell is provided by cellular respiration, in which glycolysis (enzymatic breakdown of glucose), the cycle of tricarboxylic acids (oxidative transformations of intermediate products of the breakdown and synthesis of proteins, fats and carbohydrates) and oxidative phosphorylation (energy storage as a result of oxidation of organic molecules) are isolated.

Cancer cells adapt their energy metabolism to conditions of lack of oxygen in their own way. In the primary tumor, they mainly use anaerobic glycolysis, rather than oxidative phosphorylation, as in normal cells. This enhanced absorption and breakdown of glucose to lactic acid, which is secreted by cancer cells, is called the Warburg effect. This adaptation allows cancer cells to successfully survive and actively multiply with a lack of oxygen. But all this applies to the primary tumor, and for metastatic cells, the features of energy generation are still poorly understood.

Nevertheless, using breast cancer cells that have a wide organotropy in metastasis, the scientists found differences in the metabolism of the primary tumor and metastases. Cancer cells that colonized bones and lungs used oxidative phosphorylation more actively, and those that colonized the liver used glycolysis. When cancer cells colonized all possible target organs, both metabolic pathways were activated in them (Rosen, Jordan, 2009).

It seems that this metabolic plasticity helps cancer cells to explore new niches. Why is it more profitable for metastases in one case to use mainly one type of metabolism, and in the other – another? The answer to this question, as well as to the question of the role of various factors in the regulation of metabolic plasticity of cancer cells, has yet to be found out.

How the "soil" for metastasis is prepared

Despite all their adaptive plasticity, metastatic cancer cells cannot cope alone with such a difficult task – the development of a completely unfamiliar habitat.

In 2005, in the experiments of D. Liden's group, it was shown for the first time that a primary tumor stimulates the formation of so-called premetastatic niches in various organs.

This occurs, firstly, due to stimulation of the vascular endothelial growth factor receptor (VEGFR‑1) on myeloid progenitor cells of blood cells (erythrocytes, granulocytes, monocytes and platelets) in the bone marrow, which stimulates their migration to the foci of metastasis.

Secondly, fibroblasts (connective tissue cells) located in these foci begin to intensively produce fibronectin, one of the components of the extracellular matrix. Myeloid progenitor cells have cell adhesion receptors to this protein, so they literally "fall for the bait" and populate premetastatic niches, where inflammatory cytokines, growth factors and proangiogenic factors that stimulate the formation of blood vessels begin to secrete. All this contributes to the modification of the stroma of the organ and its colonization by metastases.

There are several stages in the formation of metastases. Already at an early stage of primary tumor growth, priming takes place – "training" of the future secondary focus with the help of growth factors secreted by the tumor and exosome – membrane structures that deliver proteins and nucleic acids to cells. At the licensing stage, the primary tumor is immunosuppressed and a favorable microenvironment is created in the premetastatic niches; at the initiation stage, the development of the premetastatic niche by tumor cells occurs; the progression stage is the final in the formation of a secondary tumor. By: (Liu,Cao, 2016).

Although the scheme described above is typical for most organs, it has features depending on the place (organ) where metastases are formed. For example, in the liver and lungs, another blood cell, neutrophil granulocytes, is actively involved in the process of creating a premetastase niche. It is known that leukocytes of this type help cancer cells integrate into a new niche by secreting proteases and cytokines and directly contacting cancer cells when exiting capillaries.

Bone metastases are one of the most insidious, and their premetastatic niche has pronounced features. For example, in breast cancer, only those cancer cells that do not have receptors for the hormone estrogen metastasize to the bone. Such primary tumor cells actively secrete the enzyme lysyl oxidase. In bones, this enzyme stimulates the formation of osteoclasts – giant macrophage cells that "eat" bone tissue (Cox et al., 2015). Later, these cavities remaining in the bone as a result of the destructive work of osteoclasts occupy metastases.

The main function of lysyl oxidase is to form cross-links between the collagen fibers of connective tissue. With the development of hypoxia, breast cancer cells increase the synthesis of this enzyme, which contributes to the remodeling of the extracellular matrix in the preparation of another premetastatic niche – in the lungs. In some types of cancer (for example, melanoma), for successful metastasis to lymph nodes and organs, it is necessary that new lymphatic vessels form in their premetastatic niche.

Recently, it was found that such lymphangiogenesis begins at the early stages of primary tumor growth, and the mediator of its launch is the protein growth factor midkin secreted by melanoma cells (Olmeda et al., 2017).

The described differences in the specific mechanisms of niche formation can serve as one of the explanations for organotropic metastasis. But on what basis do cancer cells, which in principle can metastasize to different organs, choose one of them? The key mechanism currently known is the "training" of future foci with the help of specific exosomes – microscopic extracellular vesicles secreted by cells. Receptors are located on the lipid membrane of exosomes, and RNA and proteins are located in the inner cavity.

Scientists conducted an interesting experiment: having isolated exosomes from cancer cells of different origin (breast, pancreatic cancer, etc.), they injected them into the bloodstream of laboratory mice that had been vaccinated with tumors of a different type (Hoshino et al., 2015). It turned out that with the help of exosomes, it is possible to reprogram the distribution of metastases across organs.

Cancer cells (colored green) leaving the blood vessel (colored red) and colonizing the stroma of the lung. Multiphoton fluorescence microscopy. Micrograph of A.M. Guiliani (Denmark).

Due to what is this happening? Exosomes of different types of cancer carry on their surface receptors for a certain extracellular matrix protein, playing the role of "intercellular mail": they are delivered specifically to the organ whose stroma contains a lot of such a specific protein. Merging with the membranes of stroma cells, exosomes are released from the contents. A premetastase preparation program is launched in the cells: in lung fibroblasts – through the activation of some genes of the S100 group, in Kupfer cells in the liver – other genes of the same group. As a result, cellular signal transmission cascades and inflammatory reactions are stimulated, thanks to which the premetastase niche is "trained" (Hoshino et al., 2015).

Let's summarize. All the described mechanisms of metastasis, of course, complicate the picture of the course of cancer and the tactics of their treatment. The heterogeneity of the primary tumor and metastases requires special attention for a number of very different signs, which implies the need to use combined and targeted (targeted) treatment at different stages of the disease.

This is confirmed by the results of one of the latest studies based on the treatment of an oncological patient with relapses between courses of long–term immunotherapy (Jiménez-Sánchez et al., 2017). Comparison of populations of T-lymphocytes from the microenvironment of different metastases showed that they are heterogeneous. Consequently, primary, secondary and subsequent tumors respond differently to treatment.

Today we also know that under the action of cytostatics, cancer cell subclones increase the secretion of growth factors and trigger cellular signaling cascades that prevent cell death. In addition, their survival is helped by the support of stroma cells, which, under the influence of chemotherapy drugs, change their behavior to "defensive". As a result, after chemotherapy, subclones of cancer cells with mutations that have proved useful in the new environment often survive, i.e. with resistance to used medicines.

The discovery of premetastatic niches and the understanding of their structure allowed us to take a fresh look at the problems of cancer therapy. After all, if ways were found to prevent such preparation of the "soil" for metastasis, this would greatly increase the likelihood of remission.

Literature

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