Updates on mechanistic insights and targeting of tumour metastasis

Abstract Malignant tumours are one of the major diseases that seriously endanger human health. The characteristics of their invasion and metastasis are one of the main causes of death in cancer patients, and these features cannot be separated from the participation of various molecules‐related cells living in the tumour microenvironment and specific structures. Tumour invasion can approximately be divided into several specific steps according to the movement of tumour cells. In each step, there are different actions in the tumour microenvironment that mediate the interactions among substances. Researchers are attempting to clarify every mechanism of the tumour dissemination. However, there is still a long way to the final determination. Here, we review these interactions in tumour invasion and metastasis at the structural, molecular and cellular levels. We also discuss the ongoing studies and the promise of targeting metastasis in tumour therapy.


| INTRODUC TI ON
At present, research on tumours is in full swing. Primary tumours can be treated by surgical resection, but cancer mortality increases significantly once the tumour metastasizes. Tumour metastasis, as an important signal of cancer staging, has become a hot topic in cancer treatment. Therefore, research on tumour invasion and metastasis is particularly important. The metastasis caused by carcinomas is formed following the completion of a complex succession of cell-biological events, collectively termed the invasion-metastasis cascade. 1 In this process, there are not only related oncogenes, tumour suppressor genes, tumour metastasis-associated This review describes recent findings on the mechanisms of how these associated components convert their roles and the different activities occurring afterwards according to the chronological sequence of invasion.

| S TAG E OF TUMOUR PROG RE SS I ON
At present, the TNM staging system is the most widely used staging system in the world. 4 The TNM staging system is based on the local growth (T), lymph node metastasis (N) and distant metastasis (M) of the tumour. A tumour has four T stages, three N stages and two M stages, with a total of 24 TNM combinations. There are multiple classification methods for each site: clinical classification is represented by cTNM or TNM, pathological classification (pTNM), recurrence classification (rTNM) and autopsy classification (aTNM). cTNM system is essential for the selection and evaluation of initial treatment options. This system is determined before treatment without any subsequent information changes.
When patients are no longer treated, clinical staging must be stopped. Pathological staging provides more accurate information on the basis of pretreatment data, and other evidence obtained from surgery (especially pathological diagnosis). In fact, the clinical and pathological classification are combined to make the final judgment. Histological grade divides tumour differentiation into four levels, expressed by the degree of similarity between tumour and normal tissue at the site of invasion. G1 to G4, respectively, represent highly differentiated, medium-differentiated, low-differentiated and undifferentiated tumours. There are also specialized abbreviation for other identifiers including lymphatic invasion (L), venous invasion (V) and residual tumour (R). 5

| S TRUC TUR AL BA S IS OF TUMOUR M E TA S TA S I S
As mentioned previously, there are several steps in the invasionmetastasis cascade: local invasion through the surrounding extracellular matrix (ECM) and stromal cell layers, intravasation into the lamina of blood vessels, surviving the rigours of transport through the vasculature, arresting at distant organ sites, extravasation, surviving the foreign microenvironments to form micro-metastasis and finally, reviving the proliferative programmes at metastatic sites, thereby generating macroscopic and clinically detectable neoplastic growths. 1

| Local invasion
The so-called local invasion is that the cancer cells located in the primary tumour enter the surrounding matrix and then migrate into the adjacent normal tissue parenchyma, 1 which is closely re- In addition, the connection between epithelial cells is a great obstacle to the local invasion programme. To overcome this barrier, cancer cells dissolute adhesions, eliminate cell polarity, breakdown the cells into individual forms, assisting in epithelial-mesenchymal metastasis. 8 Moreover, there is a two-way interaction between tumour cells and the nearby matrix. Cancer cells stimulate the formation of the inflammatory matrix, thus establishing a positive feedback of self-amplification. 1

| Intravasation and survival in circulation
Lymphatic and blood diffusion are two ways of infiltration, and the latter seems to be the main mechanism of metastasis. 9 All solid tumours require a vascular supply in order to progress. 10 Tumour blood vessels have three characteristics as the structural basis of intravasation as described below ( Figure 1B). Firstly, these vessels cause tumour cells to grow rapidly. Unlike blood vessels in normal tissues, the neovascularization produced by cancer cells is tortuous and spirally extended. The spiral vessels are much longer in length, the blood flow volume is larger and the blood pressure is much higher, which triples the normal levels. The above features result in the rush of nutrient absorption. Secondly, too many tumour blood vessels could destroy tissue. The normal growth period of vessels is one year, while the growth cycle of tumour blood vessels is only a few days. Because of the rapid growth, the number of tumour blood vessels can reach hundreds quickly, pulling down the original tissue. Thirdly, there are holes in tumour blood vessels causing pleural effusion and ascites later on. Normal human blood vessels have three layers, as blood vessels of the tumour only have the intima with tiny holes. 11 The weak interaction between adjacent endothelial cells and the lack of pericyte coverage may promote intravascular dilation and enhance the capability of crossing the barrier between pericytes and endothelial cells, forming microvascular walls. 12 In addition, tumour endothelial cells (TECs) differ from normal endothelial cells. TECs, grow faster with F I G U R E 1 Tumour metastasis cascade. Tumour cells transfer from the primary organ through the vasculature to the target tissue. This process has been divided into four stages in human, that these stages are actually continuous in vivo, without any hesitation or pause. A, Tumour cells invade locally through the surrounding extracellular matrix (ECM) and stromal cell layers where growth factors participate in the degradation of the basement membrane and other ECM components. B, Tumour cells intravasate into the lamina of blood vessels. During their residence in vessels, these cells fight against the primary cells in the vessels and hide from immune substances to survive the rigours of transport through the vasculature. C, Tumour cells can be arrested at distant organ sites by special surface makers and membrane ligands subsequently. This specialty may not be one hundred per cent. D, Tumour cells begin to extravasate into the parenchyma of distant tissues and initially survive in these foreign microenvironments to form micro-metastasis that collect useful molecules and finally reinitiate their proliferative programmes at metastatic sites, thereby completing the tumour metastasis lower serum requirements, respond to growth factors better, express special genes, have cytogenetic abnormalities and are more resistant to chemotherapeutic drugs. Lately, a new process related to tumour metastasis termed vessel co-option has been found in human tumours growing in the brain, liver, lungs and lymph nodes.
Vessel co-option is a non-angiogenic process through which tumour cells utilize pre-existing tissue blood vessels to support itself, but its adhesion pathways are unknown.

| Being arrested at a distant organ site and extravasation
Although CTCs can theoretically spread to a variety of secondary sites, clinicians have noticed that a single cancer type forms metastatic organ in a limited number of target organs ( Figure 1C).
There are two assumptions regarding this formative mechanism: one belief claims that it is a passive physical process due to structure. According to this view, the tissue orientation of cancer cells is only a passive process. However, some CTCs may not have this rapid capture due to their unusual plasticity or the chance of shunting through arteriovenous channels, thus enabling these cells to remain in further organs. 15 The other expects predefined preferences for hosting in some organizations. In fact, some cancer cells can form specific adhesions in particular tissues, which is more conducive to the entrapment of these cancer cells. It has been suggested, for example, that the expression of metadherin in breast cancer cells leads to lung metastasis by promoting the binding of metadherin to its pulmonary vessels. 16 Alternatively, CTCs can metastasize to specific organs through the interaction of these cells with ligand receptors of the microvascular lumen, whose mechanisms require further study.
After the tumour cells are captured, the next step is to infiltrate into the substance of the distant tissue. Although most models of metastasis include an extravasation step early in the process, study shows that metastasis is mainly induced by the proliferation of tumour cells attached to the vascular endothelium while the extravasation of tumour cells is rare. 17 In addition, as discussed earlier, the neovascularization of primary tumours is zigzag and leaky. However, microvessels in normal tissues at a distance are normal-like, resulting in reduced permeability. For example, disseminated cancer cells that attempt to reach the brain's parenchyma must cross the blood-brain barrier. 18 Similarly, endothelial cells lining the pulmonary microvessels typically form a large degree of the air-blood barrier. To overcome the physical barrier of low permeability microvessels in distant normal tissues to the exosmosis of cancer cells, primary tumours can secrete protein angiopoietin and MMPs to interfere with these distant tissues. These factors destroy vascular endothelial cell-cell connections and promote the overflow of breast cancer cells in the lungs. 19 In contrast, cancer cells that reach the bone or liver encounter highly permeable sinuses, even under normal conditions, which means little to no tumour cell spills. 20 In other words, the characteristics of special microenvironments may have a profound impact on the fate of disseminated cancer cells.

| Micro-metastasis formation and metastatic colonization
The microenvironment of the metastatic site usually differs from that of the primary site, which means that cancer cells, at least in the beginning, are not fully adapted to their newly discovered home. The diversity may include the types of stromal cells, the components of the extracellular matrix, the effective cytokines or even the microstructure of the tissue itself ( Figure 1D). Some researchers speculate that cancer cells can solve the problem of an incompatible microenvironment by establishing a pre-metastatic niche. 21 According to this model, primary tumours release systemic signals, induce fibroblasts in tissues to specifically upregulate the secretion of fibronectin and mobilize VEGFR1 positive hematopoietic progenitor cells from bone marrow through homing interactions to these future metastatic sites. These hematopoietic progenitor cells then secrete MMP-9 to alter the local microenvironment of these loci. 22,23 Importantly, all these events are thought to occur before the cancer cells reach the metastatic site. In addition, cancer cells can also initiate cellular autonomy, regulating signal transduction pathways. 21 In the end, the remote microenvironment better suits tumour cells.
If only disseminated cancer cells are first exposed to the microenvironment of foreign tissues and successfully survive, they still do not guarantee proliferation. Conversely, most disseminated tumour cells appear to have experienced slow depletion for several weeks or even months, otherwise to remain dormant as microcolonies for long periods of time. 24 Disseminated tumour cells are static to a large extent, and their proliferation in metastatic sites is greatly inhibited because of their incompatibility with the surrounding microenvironment. 24 To solve this problem, these cells activate a cellular nonautonomous mechanism. For example, the outgrowth of otherwise indolent disseminated tumour cells may depend on the activation and mobilization into the circulation of bone marrow-derived cells and the subsequent recruitment of these cells to a metastatic site. 25 Structural basis is a prerequisite for tumour metastasis, but so far, thorough research is still lacking.

| MOLECULE S IN THE PRO CE SS OF TUMOUR ME TA S TA S IS
Some kinds of endothelial adhesion molecules, such as integrin and selectin, can regulate the infiltration of T cells in the tumour microenvironment as long as desmosomes and tight junctions are present.
However, little is known about the genetic mechanism of these molecules when they affect immune cells in cancer patients.
The expression of the Ras gene can be used as a G protein to participate in the signal transduction process of tumour cells. 26 The ErbB

| Cadherins
The classic cadherin is a multifunctional cell adhesion receptor. It is a multidomain, trans-membrane protein in which the extracellular domain forms the homotypic, adhesive interaction while the intracellular domain interacts with the actin cytoskeleton through the catenin family of adaptor proteins. 30 There are many subtypes of the cadherin family, including E-cadherin, P-cadherin and N-cadherin.
Taking E-cadherin as an example, it has been shown that E-cadherin is an inhibitor of tumour invasion and metastasis in various human tumour tissues. 31

| Selectin
Selectin is a surface lectin that regulates the adhesion of leuco-

| Growth factors
The  teristic of CTCs is that they undergo EMT reversible transformation ( Figure 2B) and the same is true for CSCs. EMT is an activity based on the CTC plasticity that tumour cells divert between the two phenotypes: an epithelial type and a mesenchymal type, which can promote the selective distal implantation of tumour. 50 The decrease in the expression of E-cadherin induces the falling off of tumour cells.

| REL ATED CELL S IN THE PRO CE SS OF TUMOUR ME TA S TA S IS
It also activates the metastasis process together with the infiltration of cells into the blood vessels. 51 In turn, when CTC is reversed in distal organs, the adhesion between cells increases, benefiting the plantation of tumour cells in distal organs and the establishment of metastatic foci. 52 According to the self-implantation theory, CTCs can also release signals before tumour invasion to stimulate the tumour cells to return to the primary site for self-implantation depending on the SCUB3 pathway. 53 CSCs are defined as cancer cells with the stem-cell-like ability.
The original CSCs may participate in metastasis or a new type of CSC derived from the first one or another cell in the tumour that acquires metastatic traits could do so. The formation of such a 'metastatic CSC' might be observed as EMT. 54 Once CSCs have metastasized, they must contribute to neoplastic growth in the new location where a smaller percentage of CSCs is contained than the primary tumour. 55  Under the action of many tumour-derived factors, medullary tumour cells proliferate and mobilize into the blood to act on distal target organs. MDSCs in the pre-metastatic microenvironment can promote further amplification through the S1PR1-STATS signalling pathway and help tumour cells penetrate into the circulatory system ( Figure 2C). S1PR1 is a G protein-coupled receptor of lysophosphatidyl 1-sphingosine, and its expression is increased in STAT3 + tumour cells. The upregulation of the S1pr1 gene can activate STAT3 and promote tumour growth and metastasis. Some studies have shown that S1PR1 leads to the sustained activation of STAT3 and that STAT3 can induce the expression of the S1pr1 gene. Both activities promote the expression of STAT3 in tumour cells and play an important role in tumour progression. [60][61][62] Subsequently, MDSCs can also mediate changes in inflammatory and immunosuppressive components in the microenvironment before metastasis. Inflammation has its own place in the cancer process and provides the necessary conditions for tumour development, which means that MDSCs indirectly affect the de-

| THE PRE S ENT S ITUATI ON AND THE PROMIS E OF TARG E TING ME TA S TA S IS IN EMERG ING TUMOUR THER APY
The current treatments for tumour limited mainly in surgery, chemotherapies and radiotherapies. The interdisciplinary collaboration among nanotechnology, biology, chemistry, pharmacology, oncology and other disciplines has led great changes in these three primary therapy. 73 Targeted drugs are imperative in the treatment of cancer. As molecules or their preparations endowed with targeting ability, they form a relatively high concentration of drugs in the target area, so as to improve the efficacy and inhibit the toxic and side effects to normal tissues and cells. 74  Photothermal therapy (PTT) is another emerging tumour therapy by thermal ablation of tumour cells. 81 In PTT process, a photosensitizer converts the light energy to heat energy, using the temperature evolution to kill tumour cells and avoid significant side effects on normal cells because tumour cells have a lower heat tolerance than normal ones. 82 Additionally, genetically engineered T cell with the addition of genetic material, including cytokines and cytokine receptors, enhances receptor affinity and functional avidity of the genetically engineered T cells and has got promising results in current clinical trials. 83 Oncolytic virus also works, and oncolytic vectors are designed for improved tumour specificity, intratumoural spread, therapeutic gene delivery and especially as targeted cancer immunotherapeutics. 84 The field of cancer immunotherapy has recently received a significant attention. The treatment of targeted tumour metastasis prevents or slows down tumour metastasis, which prolongs the life of tumour patients and improves the clinical survival rate.
However, it is still difficult for some of the targeted drugs to focus on tumour tissues and then affect other healthy tissues, resulting in adverse reactions due to the lack of specificity. More research is needed on the safety, specificity and stability of targeted therapy for tumour metastasis. In addition, the combined targeted delivery of treatments is an important method to enhance the therapeutic efficiency and reduce adverse side effects for current therapies. 85 More attention should be paid to the design of optimal combination tumour microenvironment targeting therapy in the future.

| CON CLUS I ON AND PER S PEC TIVE S
In this paper, we discussed the mechanisms of promoting tumour metastasis from the genetic, molecular, cellular and structural When we are looking at an old tree, we are still doing research on its broomy branches and looking forward to digging into its main root. Generally, the research of tumour metastasis is developing continuously, but it is still not enough to support antitumour therapy. We hope that the study of tumour metastasis will not only broaden the number of molecules but also deepen the research on each related substance and determine the ultimate key point of tumour metastasis.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest to this work.