Tumor metastasis: Mechanistic insights and therapeutic interventions

Abstract Cancer metastasis is responsible for the vast majority of cancer‐related deaths worldwide. In contrast to numerous discoveries that reveal the detailed mechanisms leading to the formation of the primary tumor, the biological underpinnings of the metastatic disease remain poorly understood. Cancer metastasis is a complex process in which cancer cells escape from the primary tumor, settle, and grow at other parts of the body. Epithelial‐mesenchymal transition and anoikis resistance of tumor cells are the main forces to promote metastasis, and multiple components in the tumor microenvironment and their complicated crosstalk with cancer cells are closely involved in distant metastasis. In addition to the three cornerstones of tumor treatment, surgery, chemotherapy, and radiotherapy, novel treatment approaches including targeted therapy and immunotherapy have been established in patients with metastatic cancer. Although the cancer survival rate has been greatly improved over the years, it is still far from satisfactory. In this review, we provided an overview of the metastasis process, summarized the cellular and molecular mechanisms involved in the dissemination and distant metastasis of cancer cells, and reviewed the important advances in interventions for cancer metastasis.


INTRODUCTION
methods for preventing or treating metastasis, it is inevitable to understand the cellular and molecular mechanisms of metastasis.
In contrast to numerous discoveries that reveal the detailed mechanisms leading to the formation of the primary tumor, research on metastatic cancer is still lagging behind. Stephen Paget and James Ewing proposed the "seed and soils" and the "mechanical metastasis" hypothesis, respectively, which laid the foundation for the study of tumor metastasis. [4][5][6] Cancer metastasis is a complicated process, which is regulated by various signaling pathways and modulated by the surrounding extracellular matrix (ECM). Tumor cells spread from the primary tumor mass to distant organs through blood vessels, lymphatic vessels, and transcoelomic routes. 7,8 In order to successfully metastasize from the prior site, tumor cells need to go through the following stages: local invasion, intravasation, survival in circulation, extravasation, and colonization. 9 Furthermore, multiple components in the tumor microenvironment (TME), including immune cells, stromal cells, chemokines, and cytokines are involved in a complex crosstalk with tumor cells that affects tumor growth and metastasis. [10][11][12] Surgery, chemotherapy, and radiotherapy are the three cornerstones of cancer treatment. With the deepening understanding of mechanisms of tumorigenesis and metastasis in recent years, a plethora of treatment approaches, including targeted therapy and immunotherapy have been established. [13][14][15] However, the underlying mechanisms of cancer metastasis are not yet fully understood, and the strategies for preventing and inhibiting cancer metastasis are also limited.
Here, we provided an overview of the metastasis process, summarized the mechanisms underlying the dissemination and distant metastasis of cancer cells, and reviewed the important advances in interventions targeting cancer metastasis.

COMPONENTS AND MECHANISMS INVOLVED IN METASTASIS
Tumor metastasis is one of the hallmarks of tumor malignancy and one of the causes of tumor-related death. 16 The malignant behavior of tumor metastasis is mainly related to the malignant degree of the primary tumor, but one of the common features is that all metastases need to go through a cascade called "invasion-metastasis cascade." 17 The metastasis process begins when the tumor cells gain invasiveness and lose their adhesion to the surrounding matrix including basement membrane (BM) and ECM; tumor cells migrate out of the primary tumor and invade surrounding tissues. 18 Subsequently, disseminated tumor cells penetrate blood vessels or lymph vessels into the circulation and respond to various resistance conditions such as shear force, anoikis, and immune surveillance in the circulation. 19 Only a small percentage of tumor cells can survive in the harsh conditions of circulation. After successfully entering the secondary site, tumor cells adhere to the endothelium of the target organ, and exudate and migrate into the organ parenchyma, which is the "pre-metastatic niche." [20][21][22] They either enter a long-term dormant state in the form of a single cell, or enter micrometastasis in the form of multiple cells, and finally begin to grow continuously to form clinical metastases ( Figure 1). 23,24 At the beginning of the whole metastasis process, the most important thing is that there are a group of invasive and plastic tumor cells in the cancer nest, which are collectively referred to as metastasis initiating cells. 25 Cancer stem cells (CSCs) have been proved to have the above characteristics and play a crucial role in tumor metastasis. 26,27 In addition to the role of the tumor itself, metastasis is induced by various factors, including genetic alation, abnormal epigenetic modifications, immune escape, and changes in the growth environment. 9 Here, we will describe in detail the major factors and mechanisms involved in the metastasis cascade.
The EMT is the transition of epithelial cells to mesenchymal cells under certain physiological and pathological conditions, which was proposed by Greenberg and Hay in 1982. [28][29][30] Studies in the last decades have shown that EMT is an important molecular event for epithelial tumor cells to gain invasiveness and plays an important role in the development and metastatic dissemination of malignant tumor cells. 31 Tumor cells can obtain a higher mesenchymal phenotype through EMT, which manifests as reduced contact with surrounding cells and matrix, and enhanced cell migration and motility at the beginning of tumor cell invasion and metastasis. 30 Therefore, interrupting or reversing EMT can inhibit the invasion of malignant tumor cells and reduce the rate of tumor metastasis. 32 The process of EMT is shown in Figure 2.
Cancer cells undergoing and molecular EMT exhibit morphological changes, such as decreased expression of epithelial markers (e.g., E-cadherin, zonula occludens-1, and occluding) and increased expression of mesenchymal markers (e.g., N-cadherin protein, fibroblast-specific protein 1, and fibronectin). 33,34 Cell adhesion molecules, such as E-cadherin, N-cadherin, and β-catenin, are closely F I G U R E 1 Overview of the metastatic cascade. Carcinoma cells escaping from the primary tumor migrate and invade through the basement membrane and extracellular matrix, enter the blood or lymphatic vessels, intravasate into the circulation, penetrate the blood or lymphatic vessels (extravasation), and adhere and grow in secondary sites. A variety of stromal cells, immune cells, and other molecular components surrounding the tumor provide signals that enhance the metastatic potential of cancer cells. Platelets and neutrophils can protect tumor cells by providing physical protection against shear stress, secreting mediators (such as transforming growth factor-beta (TGF-β)), neutralizing the cytotoxicity of NK cells and favoring immune escape. Abbreviations: BM, basement membrane; CAF, cancer-associated fibroblast; CTC, circulating tumor cell; ECM, extracellular matrix; EMT, epithelial-to-mesenchymal transition; MSC, mesenchymal stem cell; NK cell, natural killer cell; RBC, red blood cell; TAM, tumor-associated macrophage related to EMT and are regulated by EMT-related transcription factors (EMT-TFs), such as zinc finger E-boxbinding homeobox 1/2 (ZEB1/2), snai1/2 and twist. [35][36][37] The down-regulation of E-cadherin is accompanied by the upregulation of N-cadherin, which reduces the adhesion of cancer cells to epithelial cells and increases their adhesion to stromal cells, leading to subsequent invasion of tumor cells into matrix. 38 β-catenin has also been found to promote tumor metastasis, mainly driven by the ectopic expression of β-catenin. 39 Vimentin has been found to promote cell invasion by regulating the E-cadherin/β-catenin complex. 40 EMT-TFs can be regulated by signaling pathways such as Wnt/β-catenin, transforming growth factorbeta (TGF-β), and Notch; these factors work together to give tumor cells the characteristics required for metastasis under different conditions, thereby promoting the occurrence of metastasis. [41][42][43][44][45] EMT is also involved in the process of tumor invasion and colonization in metastasis, which mainly depends on circulating tumor cells (CTCs) and CSCs. 41,46,47 CTCs are divided into three groups, including epithelial CTCs, hybrid epithelial/mesenchymal phenotype CTCs, and mesenchymal CTCs. 48 With the development of modern medical technology, tumor metastasis can be monitored throughout the process. Compared with epithelial CTCs, mesenchymal CTCs may be easier to metastasize because they are resistant to anoikis and chemotherapy and have a stronger ability to migrate to distant organs. 49,50 Previous studies reported that up-regulating the expression of EMT-TFs in breast cancer cells increased the expression of CSC-specific cell markers, improved the ability to form spheroids, and accelerated tumor formation of breast cancer cells in mice. 51 In non-CSCs, the activation of EMT promotes their conversion to CSCs. 51-54 Tumor formation and metastasis rely on the tumor-forming ability of tumor cells, which suggests that CSCs with tumor-initiating ability are the crucial prerequisite for disseminated cancer cells to establish metastatic colonies. 55 Another mechanism of EMT promoting metastasis is to protect tumor cells from immune cell-mediated killing. 56-58 Recent researches on breast cancer found that, compared with epithelial cancer cell lines, mesenchymal breast cancer cell lines can recruit more immunosuppressive regulatory T cells (Tregs) and M2 macrophages. 57 Compared with epithelial cancer cells, mesenchymal cancer cells are less responsive to anti-cytotoxic lymphocyteassociated protein 4 (CTLA4) immunotherapy. 59 In addition, the expressions of programmed death ligand-1 (PD-L1), PD-L2, and B7 homolog 3 (B7-H3) are up-regulated in tumor cells that have undergone EMT, which can facilitate the immune escape of cancer cells. 58,60 During dormancy and colonization, the process of mesenchymal cells to transform to epithelial cells (MET) is required to help tumor cells survive in the foreign microenvironment. 31 Indeed, the role of MET in promoting tumor cell colonization at a secondary site during metastatic growth has been confirmed in numerous studies. 31,61 It is worth noting that the tightly interconnected transfer network of EMT and MET has been proved to be subject to epigenetic regulation mediated by DNA F I G U R E 2 Cancer cells undergo EMT and invade into circulation. (A) A single transformed epithelial cell remains quiescent for a period of time. (B, C). The transformed cells proliferate and generate a small intraepithelial colony, accompanied by the formation of cancer stem cells. Cancer cells destroy the basement membrane, undergo EMT, and migrate and invade through the basement membrane and extracellular matrix. Normal extracellular matrix undergoes cancer-associated remodel. Meanwhile, cells and molecular components in tumor microenvironment (TME; CAFs, TAMs, neutrophils, MSCs. . . ) surrounding the primary tumor enhance cancer cell survival, proliferation and metastasis. (D) Cancer cells escaping from primary tumors can invade into the circulation as single CTCs or multicellular CTC clusters. Abbreviations: BM, basement membrane; CAF, cancer-associated fibroblast; CTC, circulating tumor cell; EC, endothelial cell; ECM, extracellular matrix; EMT, epithelial-to-mesenchymal transition; MSC, mesenchymal stem cell; TAM, tumor-associated macrophage methylation and histone modification. 62-64 For example, in breast cancer, histone methyltransferase nuclear receptor-binding SET domain 3 (NSD3) cooperates with enhancer of zeste homolog 2 (EZH2) and RNA polymerase II to stimulate the Notch pathway-mediated Ecadherin transcriptional inhibition to promote EMT and trigger tumor invasion and metastasis. 65 During the initiation of EMT, histone demethylase lysine (K) -specific demethylase 6A (KDM6A) is inhibited, leading to the transcriptional inhibition of epithelial genes, whereas during the MET process, the expression of KDM6A is restored, thereby reactivating the epithelial gene by the trimethylation of histone 3 lysine 27 (H3K27me3) and reversing the phenotype. 66,67 Therefore, in general, a series of epigenetic regulation of tumor cells makes EMT dynamically change, giving tumor cells the ability to survive in different microenvironments.

2.2
The most important soil factor: TME In addition to the characteristics of tumor cells, numerous studies have shown that TME is the soil for tumor cells and provides the necessary energy for tumor growth ( Figure 3). 68 In the past two decades, many achievements have been made regarding the impact of TME on cancer metastasis. 10,11,69,70 TME can promote the occurrence and development of tumor by affecting metabolism, secretion, immunity, structure, and function of tumor cells, thereby playing an essential role in the overall F I G U R E 3 TME involved in the processes of invasion-metastasis cascade. The cellular components in TME can be classified into cancer cells, stromal cells, and immune cells. These cells interact with each other through ligand-receptor interactions, and the secretion of cytokines, chemokines, exosomes, and extracellular vesicles, forming an evolving microenvironment. Cancer cells that are good at recruiting and establishing a supportive metastatic niche may be able to survive and initiate the process of proliferation and metastasis. The formation of the metastatic niche may occur before the arrival of cancer cells, also known as pre-metastatic niches. Here, we summarized the role of important cellular components in TME in tumor metastasis cascade of tumor metastasis. 12,71,72 The members in TME are generally classified into two categories: cellular components and extracellular components. Cellular components mainly include immune cells, cancer-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), and endothelial cells. 73-75 Extracellular components, such as tumor-secreted extracellular vesicles (EVs), growth factors, chemokines, and cytokines are also closely involved in tumor metastasis. 76 Cellular components and extracellular components restrict and influence each other, which form a mutual feedback network.

Immune cells
Under normal circumstances, the immune system will recognize foreign antigens to initiate autoimmunity and passive immunity to eliminate harmful pathogens. For this reason, tumor immunity has become a hot spot in the field of tumor research. Studies have pointed out that some types of immune cells are closely related to tumor occurrence and development and play an essential role in tumor metastasis. 72,77 Among the immune cells in TME, tumorassociated macrophages (TAMs), Tregs, and neutrophils are noteworthy for their roles in tumor development and metastasis.

TAMs
According to the expression of specific markers, differentiation status, and functional role in the immune system, macrophages are conventionally classified into two major phenotypes, M1 and M2, which can be converted to each other. 78 M1 macrophages promote inflammation responses against tumor cells, whereas M2 macrophages tend to exert immune suppressive effects. M2 macrophages (M2-TAMs) express abundant arginase-1, mannose receptor, and scavenger receptors, and secrete a large number of anti-inflammatory cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10), and interleukin-13 (IL-13). 79,80 In general, researchers tend to consider TAMs as M2-like phenotype-acquired macrophages. 81 The cytokines and chemokines secreted by tumor cells or fibroblasts could recruit more M2-TAMs in TME. [82][83][84] TAMs are elevated in TME and are associated with poor clinical prognoses. 85 The role of TAMs in promoting metastasis involves multiple mechanisms. First, TAMs participate in the regulation of the EMT process by enhancing the expression of Ncadherin and snail, secreting various soluble factors such as IL-1β, IL-8, tumor necrosis factor-α (TNF-α), and TGFβ, and secreting a number of proteolytic enzymes. [86][87][88] Second, TAMs promote vascularization of tumor cells through stimulating the formation of new tumor vessels and the remodeling of the established vascular system into a more tortuous and leaky form. 89,90 Third, TAMs promote intravasation of tumor cell, favor tumor cell survival in the circulation, and promote extravasation of tumor cells. 91 Last, TAMs are important determinants for the formation of pre-metastatic niches. Notably, TAMs contribute to the immune escape of tumor cells throughout the metastasis process. 81

Tregs
There are mounting studies that convincingly demonstrated that Tregs play a prominent role in promoting metastasis. Tregs have a strong immunosuppressive function in TME by inhibiting adaptive and innate immune responses. [92][93][94][95][96] Here, we summarized the main mechanisms. 97 First, Tregs secrete inhibitory cytokines, including IL-10, TGF-β, and IL-35. [98][99][100] Tregs can inhibit the function of CD8+ T cells and dendritic cells (DCs) through membrane-bound TGF-β. For example, Tregs impede CD8+T cell-mediated anti-tumor immune responses by inhibiting interleukin-2 (IL-2) production and activating the TGF-β signaling pathway. 92 It has been reported that TGF-β1 secreted by Tregs in breast cancer can induce the up-regulation of IL-17Rb and promote tumor lymph node metastasis. 101 Second, Tregs can kill effector cells by granzymes and perforin and diminish the function of T cells and natural killer (NK) cells through inhibitory receptors, such as CTLA-4, lymphocyte activation gene 3 (LAG-3), and programmed cell death 1 (PD-1). [102][103][104] Tregs can also suppress CD8+ T cells and NK cells secretion of interferon-γ (IFN-γ). 105 Olkhanud et al. found that chemokine receptor (CCR) 4+Tregs are mainly recruited at the lung metastasis site in breast cancer, and the Tregs secret β-galactoside-binding proteins to induce the apoptosis of NK cells, thereby promoting tumor metastasis. 95 Third, Tregs affect effector cell functions by interfering with cell metabolism mainly through the following three ways: depriving IL-2, promoting the production of adenosine, and transferring a large number of cyclic adenosine monophosphate to effector T cells. [106][107][108][109] Fourth, Treg induces DCs tolerance through the expression of inhibitory receptors CTLA-4 and LAG-3, with the latter further inhibiting T-cell capacity through indoleamine 2,3-dioxygenase. 110,111 Finally, factors pro-duced by myeloid-derived suppressor cells (MDSCs) and Tregs form positive feedback loops to reinforce the suppressive microenvironment. [112][113][114] In addition, it has been found that Tregs could secrete vascular endothelial growth factor A (VEGF-A) in metastatic ovarian cancer, thereby promoting angiogenesis and tumor cell dissemination. 115 TGF-β1 secreted by Tregs can promote tumor metastasis by enhancing EMT. 116 Some preclinical studies have shown that Tregs were closely related to lymph node metastasis and peritoneal metastasis and suggested that Treg may be involved in pre-metastasis niche reprogramming to promote metastasis. 101,117 Therefore, eliminating or alleviating the immunosuppressive state of Tregs may suppress the progression of tumor metastasis.

Neutrophils
Neutrophils, as innate immune cells, protect the body from infection by foreign microorganisms and promote inflammatory responses under normal conditions. 118 However, it is worth noting that neutrophils have been found to promote tumor metastasis in a variety of ways, including inhibiting anti-tumor T cells and influencing tumor cell invasion. 119 In a mouse model of sarcoma, pleomorphic neutrophils were found to reduce the expression of intercellular adhesion molecule 1 in tumor cells, thereby increasing tumor motility and facilitating the initiation of metastasis. 120 In addition, pro-metastatic neutrophils activate various signaling pathways to promote EMT by secreting pro-EMT-related cytokines, including IL-8 and IL-17A. 121,122 In the process of metastasis, neutrophils bind with CTCs in circulation to protect CTCs from immune killing, increase CTCs invasiveness, and promote metastasis. 123 A large number of neutrophils were found in the secondary sites. 124,125 In terms of mechanism, recruited neutrophils promote the formation of pre-metastatic niche and support angiogenesis mainly by forming an immunosuppressive microenvironment and secreting neutrophil elastase. 126

2.2.2
Cancer-associated fibroblasts (CAFs) CAFs are one of the most common fibroblasts in tumor tissues. CAFs are derived from a variety of different fibroblasts, including normal fibroblasts stimulated by exosomes, bone marrow MSCs induced by TGF-β, and epithelial cells transformed by EMT. [127][128][129] Since CAFs originate from many kinds of cells and have apparent heterogeneity, there is no clear marker that can distinguish CAFs from normal fibroblasts. CAFs are the primary source of various factors and enzymes of TME and are one of the critical members involved in tumor metastasis.
In previous studies, it was believed that the role of CAFs in tumor metastasis is mainly manifested in the reconstruction of ECM structure. 130 However, current studies have found that CAFs promote EMT-mediated tumor metastasis in ovarian cancer, bladder cancer, and breast cancer cells in various ways, including the secretion of TGF-β and exosomes. 131 The increased autocrine and paracrine TGF-β signaling in the mesenchymal transcription factor forkhead box F2 (FOXF2) deficient base-like breast cancer cells induces EMT to mediate tumor metastasis. 132 In turn, TGF-β silences FOXF2 expression by up-regulating the post-transcriptional regulator (miR-182-5p) of FOXF2 and promotes breast cancer metastasis. 133 In addition, chemokine (C-X-C motif) ligand 11 (CXCL11) secreted by CAFs promotes the metastasis of liver cancer by up-regulating the expression of circUBAP2. 134 In order to gain a deeper understanding of the role of CAFs in the tumor, Wang et al. analyzed CAFs by single-cell sequencing combined with RNA-sequence and divided CAFs into four types. 136 One of the subtypes has high glycolytic activity and is termed MeCAFs. It was found that pancreatic ductal adenocarcinoma patients with abundant MeCAFs had a higher risk of metastasis but had a significantly better response to immunotherapy. 136 These results indicate that abnormal glycolysis directly or indirectly affects tumor metastasis. CAFs up-regulate carnitine palmitoyltransferase 1A (CPT1A) to cause reduction of fatty acid oxidation, leading to peritoneal metastasis of colorectal cancer. 137 Fibroblasts play an important role in both the primary tumor site and the secondary tumor site. Fibroblasts distributed in the metastatic site are called metastasisassociated fibroblasts (MAFs), which promote angiogenesis and the formation of immunosuppressive microenvironment in pre-metastatic niche. 138 The main difference between MAFs and primary tumor-related fibroblasts is that MAFs have a stronger ability to inhibit the antitumor effect of immune cells, which is reflected explicitly in the higher levels of CC motif chemokines ligand 2 (CCL2), CXCL12, and interferon-related genes secreted by MAFs. 139 Additionally, in metastatic liver cancer and gastric cancer, it was found that the decrease of CD3+ infiltrating lymphocytes and the increase of TAM cells were related to the high levels of MAFs, partly due to the chemokine CXC receptor 4 (CXCR4) signal transduction in α-smooth muscle actin (α-SMA)+ MAFs. 140 In another study conducted by Costa et al., fibroblasts that inhibit tumor immunity were discovered, named cancerassociated fibroblasts subset 1 (CAF-S1). 141 Furthermore, it was found that the presence of CAF-S1 in breast cancer promoted bone metastasis of cancer cells. 142

MSCs
More and more studies have recognized the importance of MSCs in regulating tumor metastasis at the initial tumor site and distant metastasis sites. At the beginning of tumor metastasis, MSCs support the generation of the cancer-promoting microenvironment by transforming into CAFs and increase the motility of cancer cells through secreting growth factors and chemokines in autocrine and paracrine manners. 128 Pendergast et al. co-cultured MSCs and lung cancer cells and found that MSCs can mediate EMT to promote lung cancer metastasis by activating the abelson-matrix metalloprotein 9 (ABL-MMP9) signaling pathway. 143 In the late stage of tumor metastasis, MSCs participate in the colonization and metastatic growth of tumor cell. For example, in ovarian cancer, MET of cancer-associated MSCs mediated by Wilms' tumor 1 (WT1) and EZH2 promote metastatic tumors growth in distant organs. 135 MSCs can also promote tumor metastasis by regulating anti-tumor immunity. As an immune checkpoint, PD-L1 is one of the crucial members involved in tumor immune escape and treatment resistance. 144 Studies have found that PD-L1 is highly expressed on tumor-associated MSCs, and MSCs can up-regulate the expression of PD-L1 in a variety of tumor cells. Hamidreza et al. reported that MSCs promoted the up-regulation of PD-L1 expression in breast cancer cells by secreting CCL5. 145 Sun et al. found that IL-8 secreted by MSCs can increase the expression of PD-L1 in gastric cancer cells. 146 Further experiments suggested that MSCs can increase the binding of PD-L1 to the transcription factor CCCTC-binding factor, thereby affecting the stemness of gastric cancer cells and leading to tumorigenesis. 147

Tumor-associated endothelial cells (TECs)
The role of TECs in metastasis is complex. In the process of tumor metastasis, TECs play an important role in obtaining anti-anoikis properties of CTCs, which is one of the mechanisms by which TECs promote tumor cell metastasis to secondary organs. 148 In addition, studies have found that biglycan in TECs is significantly higher than that in normal endothelial cells. 149 TECs-biglycan activates nuclear factor-κB (NF-kB) and extracellular signalregulated kinase (ERK) signal transduction to stimulate the metastasis of tumor cells expressing toll-like receptors (TLR) and promote tumor angiogenesis of tumors by activating the TLR signaling pathway. 150 IL-6 secreted by tumor cells can activate the signal transducers and activators of transcription 3 (STAT3)-VEGF pathway of lymphatic endothelial cells and encourage lymphatic metastasis of the tumor. 151

Extracellular matrix (ECM)
ECM is a network of various proteins represented by collagen and macromolecules, which maintain tissue structure outside tumor cells. 18 A hallmark of metastasis is ECM degradation. 152 In the process of metastasis, it is necessary to enhance the invasion ability of tumor cells and reconstruct the ECM structure. 153 ECM reconstruction is a relatively complex process, which requires the participation of multiple chemokines. 154,155 Matrix metalloproteinases (MMPs) are a group of enzymes that can directly degrade collagen and multiple connexins in the process of ECM degradation. Among matrix MMPs family, MMP-2 and MMP-9 are the two important enzymes involved in the EMT process, which specifically degrade gelatin, collagen, elastin, and fibronectin, thereby inducing invasion and metastasis of tumor cells. 156 Furthermore, Musashi-1 can promote the degradation of ECM by upregulating Timp3 to encourage the expression of MMP-9 in breast cancer, and thus regulating cell-ECM adhesion. 157

Cytokines and chemokines
The involvement of various cytokines/chemokines, along with their receptors and signaling axis in the promotion of tumor metastasis has been well-studied. The convoluted cross-talk between various cell types and these secreted factors helps drive the sequence of events that lead to tumor metastasis. 158,159 These cytokines/chemokines are involved in ECM remodeling, tumor invasion, EMT, angiogenesis, pre-metastatic niche reprogramming, extravasation, and modulating stromal cells and immune cells. 115,154,[160][161][162] The cytokines/chemokines including TNF-α, IL-8, CCL20, CXCL5, CXCL12. 163-166 CXCR2, CXCR3, and CXCR4, up-regulate EMT-TFs (snail and ZEB1), promote MMP-2 expression, and accelerate EMT of cancer cells, thereby promoting tumor metastasis. 160,165,166 Cytokine IL-8 mainly down-regulates E-cadherin through activating the Wnt/β-catenin pathway in ovarian cancer, thereby promoting tumor invasion. 163 Some cytokines can promote the expression of chemokines and mesenchymal transformation. For example, TNF-α has been reported to promote the up-regulation of CXCL10 and activate the phosphoinositide3-kinase/protein kinase B (PI3K/AKT) pathway to inhibit the phosphorylation of GSK-3β, lead-ing to the up-regulation of snail and promoting tumor metastasis. 160 Cytokines/chemokines, such as VEGF, TGF-β, IL-1, CXCL8, and CCL21 are involved in promoting angiogenesis and lymphatic formation. [167][168][169][170][171] Among the known proangiogenic factors, VEGF is the most effective cytokine in promoting angiogenesis. By directly binding to receptors on endothelial cells, VEGF induces endothelial cell proliferation and promotes tumor metastasis. 167 VEGF-C and VEGF-D in the VEGF family can activate the generation of lymphatic vessels, recruit chemokines, such as CCL21, CCL27, and CCL28, to lymphatic vessels and promote the occurrence of lymph node metastasis. 172,173 Cytokines/chemokines also play an indispensable role in promoting the recruitment of immune cells in the tumor microenvironment. For example, the recruitment of Treg requires the participation of CCL20 and CCL22, while the recruitment of TAMs requires the participation of CCL2. [174][175][176][177] Single-cell sequencing analysis revealed that chemokine CCL5 is an important mediator of CTC immune escape. 162 CCL5 can promote immune escape and metastasis of CTCs by recruiting Tregs, which further suggests that chemokines can promote immune escape. 162 In addition, MDSCs secrete TGF-β in esophageal cancer, which can increase the expression of PD-1 on tumor-infiltrating CD8+ T cells, leading to immunotherapy resistance. 178,179 Cytokines and chemokines are involved in the organotropism of tumor metastasis. Lung metastasis is associated with the high levels of CXCL1, CXCL9, and CXCL10 secreted by human pulmonary artery endothelial cells or lung fibroblasts. 179,180 Ricardo et. Al. reported that blocking CXCL5/CXCR2 signaling can inhibit the bone colonization of breast cancer cells. 181 Blocking CXCL12/CXCR4 signaling significantly impair the metastasis of breast cancer cells to regional lymph nodes and lung, which indicate that chemokines and their receptors play a key role in determining the destination of metastasis. 182,183 Overall, the potential of cytokines and chemokines to induce metastasis is mainly achieved by promoting EMT, angiogenesis, immunosuppression, and pre-metastatic niche reprogramming.

2.2.7
Tumor-secreted extracellular vesicles EVs secreted by tumors are the key mediators of cell-tocell contact in the local and remote microenvironments. 184 EVs are exceptional cargo for various nucleic acid, proteins, and lipids in TME, and play a prominent role in the metastasis of the tumor to distant organs. [183][184][185][186][187][188][189][190] Tumor-associated EVs in breast cancer can promote breast cancer colonization by changing the composition and structure of lung tissue fibroblasts. 185 It was found that EVs secreted by bladder cancer cells mediate their intercellular communication with human lymphatic endothelial cells through long non-coding RNA (lncRNA) ELNAT1 and promote lymph node metastasis in a SUMOylation-dependent manner. 186 In nasopharyngeal carcinoma (NPC), EVs migrate from highly metastatic to poorly metastatic NPC cells, mediate intercellular communication, and enhance the metastatic potential of poorly metastatic NPC cells by inducing the up-regulation of epidermal growth factor receptor (EGFR) and down-regulation of reactive oxygen species (ROS). 191 Mechanistically, overexpression of EGFR mediated by EGFR-rich EVs down-regulates intracellular ROS levels through regulating PI3K/AKT pathway, thereby promoting the metastatic potential of NPC with poorly metastatic ability. 191 The role of EVs in tumor metastasis depends on their components; the function of EVs may be different for different tumor metastasis sites. For example, in brain metastasis, EVs mainly mediate pre-metastasis regulation by destroying the blood-brain barrier or by distinct increasing the expression of cell migration-inducing and hyaluronan-binding protein in brain-tropic EVs. 187,188 In hepatocellular carcinoma, nidogen 1 (NID1) in EVs promote tumor cell colonization and extrahepatic metastasis by enhancing angiogenesis and lung endothelial permeability and promoting the formation of pre-metastasis niches in the lung. 189 EVs-NID1 also activates fibroblasts that secrete TNF receptor 1 (TNFR1), promote lung colonization of tumor cells, and augment the growth and motility of cancer cells. 189 EVs in bone metastasis mainly mediate cancer-induced osteolysis to produce a suitable microenvironment for tumor cells. 190 In addition, EVs can also promote the formation of pre-metastasis niches by stimulating CAFs, inducing MSC differentiation, and regulating tumor immunity. 183

2.2.8
Neutrophil extracellular traps (NETs) NETs are net-like structures composed of DNA histone complexes, and proteins released by neutrophils in response to infection or inflammatory cytokines were discovered by Brinkmann et al. in 2004. 192 NETs are initially discovered to be one of the host defense mechanisms of neutrophils against pathogens, and they are also involved in the progression of sterile inflammation-related diseases, such as autoimmune diseases, diabetes, and carcinoma. 193 In the absence of infection, NETs can also be stimulated by cancer cells and CAFs, and hijack anti-tumor immune system to promote tumor cell proliferation and metastasis. 193 The levels of plasma NETs in patients with lung cancer, cervical cancer, and pancreatic cancer are significantly higher than those in healthy controls and are related to tumor recurrence and metastasis. 194 In tumor metastasis, NETs are mainly involved in the spread of cancer cells and awakening dormant tumor cells. NETs can induce EMT of primary tumor cells in the early stage of metastasis and lead to the activation of inflammatory signaling pathways such as NF-kB and STAT3, thereby enhancing the mobility and invasiveness of cancer cells and promoting metastatic growth. 195,196 In the circulation, NETs wrap CTCs to avoid contact between CTCs and immune cells and can also form a physical barrier for immune escape. 197,198 The molecular mechanisms by which NETs can trap CTCs have been investigated, and it has been found that expression of β1-integrin on both cancer cells and NETs is important for the adhesion of CTCs to NETs. 197,198 After spreading to other sites, cancer cells from primary tumors often do not start growing immediately but enter a dormant state. Disseminated cancer cells can remain dormant for years or even decades before they relapse or "wake up" as metastatic cancer. 199 Studies have found that the formation of NETs induced by sustained inflammation is required for awakening dormant cancer. 199 Two NET-associated proteases (neutrophil elastase and MMP9) sequentially cleave laminin, and then NET-remodeled laminin facilitates the proliferation of dormant cancer cells by activating integrin α3β1 signaling and focaladhesion kinase (FAK)/ERK/myosin light chain kinase (MLCK)/Yes associated protein (YAP) signaling. 199

A double-edged knife in tumor metastasis: Autophagy
Autophagy is generally defined as a lysosome-dependent mechanism of intracellular degradation and recycling process, which is highly conserved in all eukaryotes. [200][201][202] Several forms of autophagy have been discovered. In all types of autophagy, autophagosomes are the hallmark morphological characteristics, and the formation of autophagosomes is the core process of this dynamic process. 200,203 Autophagy is a dynamic physiological process that maintains cell homeostasis when the body encounters stress, including nutrient deprivation, hypoxia, and infection. 204,205 It has been reported that autophagy plays a key role in the prevention of cancer and other diseases by promoting cellular senescence and antigen presentation and preventing genomic instability and necrosis. 206 However, autophagy also plays a vital role in the occurrence and progression of tumor. 207,208 In many types of tumors, including melanoma, breast cancer, and liver cancer, the expressions of autophagy-related proteins light chain 3 and beclin-1 in tumor tissues of patients with distant metastasis are higher than in tumor tissues of patients with non-metastatic tumor, suggesting that autophagy is closely related to tumor metastasis. [209][210][211] Partly due to the heterogeneity of tumor cells, autophagy is a double-edged sword in the process of tumor metastasis. 212 In the early stage of metastasis, autophagy mainly suppresses the occurrence of metastasis by regulating the antitumor immune response. [213][214][215] Autophagy-related protein 5(ATG5) is considered to be closely related to autophagy. In pancreatic ductal adenocarcinoma, mice with ATG5 knockout developed more tumors and metastases than control mice. Moreover, compared with the control mice, the expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophages was up-regulated in the tumors of mice with ATG5 knockout. Meanwhile, the number and activity of M2-TAMs were substantially higher in ATG5 knockout mice, indicating that autophagy can inhibit TAMs infiltration and limit tumor metastasis. 214 In addition, studies have found that autophagy can stimulate tumor-related spontaneous inflammation by releasing high mobility group box 1 and enhance the antitumor immune response of DCs to limit metastasis. 215 In the later stage of tumor metastasis, autophagy reduces the adhesion between tumor cells and ECM by regulating the activity of the Rho family so as to promote tumor migration and invasion. 216 In addition, autophagy can enhance tumor invasion and promote the progress of tumor metastasis by inhibiting anoikis of CTCs, maintaining the stemness of CSCs, and reawakening dormant tumor cells. 210,217,218 Another significant aspect of autophagy in metastasis is involved in the regulation of EMT. P53, an oncogene, also plays an important role in regulating the relationship between EMT and autophagy. 219,220 Under normal circumstances, P53 exists in the cytoplasm and inhibits autophagy. However, under cellular stress, P53 translocates from the cytoplasm to the nucleus and induces autophagy. 221,222 In addition to autophagy, recent studies have found that P53 inhibits EMT and metastasis by affecting the TFs involved in the EMT process. 223 It is well known that AKT/ mammalian target of rapamycin (mTOR) signaling pathway participates in modulating the EMT process. 224 A large number of studies have found that oncogenes activate mTOR-related pathways to promote tumor metastasis by inhibiting autophagy. 225 Suppressor of cytokine signaling-5 (SOCS5), a member of the SOCS protein family, has also been found to promote tumor cell invasion and metastasis in hepatocellular tumors by up-regulating PI3K/Akt/mTORmediated autophagy pathway. 226 Interestingly, opposite findings have been reported in NPC. Annexin A1 (ANXA1) inhibits autophagy and promotes EMT by activating PI3K/Akt/mTOR pathway. 227 ANXA1 inhibits autophagy by activating PI3K/Akt/mTOR pathway, leading to the upregulation of tumor cell migration and invasion capabilities, EMT-like changes, and metastasis in vivo. 227 During this process, autophagy inhibited by ANXA1 induces EMTlike alterations, possibly by inhibiting autophagy-mediated Snail degradation. 227 Autophagy can also be induced by activating the adenosine monophosphate activated protein kinase (AMPK) pathway that promotes tumor metastasis. 228 For example, tight junction protein 1 (CLDN1) promotes EMT and metastasis of esophageal cancer cells by triggering autophagy in vivo and in vitro. 229 Mechanically, CLDN1 induces autophagy by up-regulating the expression of Unc-51-like autophagy activating kinase 1 (ULK1) through AMPK/signal transducer and activator of transcription 1 (STAT1)/ULK1 signaling pathway. 229,230 In general, autophagy is a double-edged sword in the process of tumor metastasis, and the mechanisms involved are very intricate and require further research.

Lipid metabolism
Under hypoxic conditions, tumor cells mainly rely on glycolysis to obtain sufficient energy. 231 This metabolic mode different from normal cells is also called tumorrelated metabolic rearrangement. 232 Glycolysis is essential to maintain the malignant proliferation behavior of the tumor, but there are relatively few studies on the effect of lipid metabolism on tumor progression. Obesity has been recognized as a risk factor for cancer, so investigating lipid metabolism in the tumor is necessary. 233,234 The results of researches in the past 10 years have shown that abnormal lipid metabolism in the TME is one of the key steps involved in tumor metastasis. 235 Therefore, correcting abnormal lipid metabolism may prevent the occurrence of tumor metastasis. It has been found that a variety of enzymes involved in lipid anabolism and catabolism are related to tumor metastasis, including adenosine triphosphate (ATP) citrate lyase (ACLY), fatty-acid synthase (FASN), stearoyl-CoA desaturases (SCD), and monoacylglycerol lipases. 236 ACLY and SCD-1 are associated with facilitated colon cancer metastasis, and FASN can promote retroperitoneal metastasis of ovarian cancer. 237,238 Identifying tumor metastasis initiating cells is considered to be one of the most challenging problems in the field of tumor metastasis research. In recent years, fatty acid receptor CD36 has been shown to play an essential role in the metastasis of oral cancer because CD36 is positively correlated with lymph node metastasis and can be used as a marker of metastasis initiating cells. 239 Palmitic acid or a high-fat diet can enhance the metastatic ability of CD36+ tumor cells. Subsequent studies have found that CD36+ cells are associated with a poor prognosis of ovarian cancer, and blocking CD36 can attenuate tumor metastasis. 240 These findings suggest that the initiating cells of tumor metastasis may depend on lipids, and blocking CD36 can reverse tumor metastasis.
With the development of proteomics and metabolomics, new discoveries have been made about cancer metabolic disorder and its correlation with metastasis. In breast cancer, it was found that the levels of phospholipid are different between mammary epithelial cells and breast cancer cells, as well as between breast cancer cells with different levels of aggressiveness. 241 In short, lipid metabolic reprogramming is associated with breast cancer carcinogenesis and metastasis. 241 In pancreatic cancer, fatty acid synthesis was found to maintain the stemness of pancreatic cancer cells, indicating that abnormal lipid metabolism can make cancer cells more invasive and promote the spread of tumor cells. 242 Promoting cholesterol biosynthesis may promote tumor metastasis.
More and more molecules regulating lipid metabolism have been proved to promote tumor metastasis by regulating EMT, including apolipoprotein C, sterol regulatory element-binding transcription protein 1, stromal-interaction molecule 1, human hydroxysteroid dehydrogenase-like 2, and cytosolic phospholipase A2α. [243][244][245][246] In triple-negative breast cancer, nicotinamide adenine dinucleotide phosphate (NADP) steroid dehydrogenase-like (NSDHL), a cholesterol metabolic enzyme, has been reported to be a potential metastatic driver. 247 The functions of NSDHL rely on its enzyme activity in the biosynthesis of cholesterol and is mediated by the NSDHL-TGFβR2 signaling pathway. 247 TME has a unique lipid structure, which is characterized by being rich in sphingolipids and cholesterol and is called lipid rafts. 248 The role of lipid rafts in tumor metastasis is different from other lipid metabolism-related regulatory molecules. Rina et al. reported that palmitoylated CD44 is wrapped in lipid rafts, and its binding to promigration binding partners (such as Ezlin) is restricted, thereby inhibiting cancer metastasis and spread. 249 Moreover, another study reported that squalene synthase could promote lung cancer metastasis by activating TNFR1, NF-Κb, and matrix metallopeptidase 1, and destroying the lipid raft structure. 250 Therefore, lipid rafts mainly play an inhibitory role in tumorigenesis and metastasis. For cells in TME, abnormal lipid metabolism may be a potential mechanism to promote tumor metastasis. For example, lipid accumulation in TME can lead to CD8+T cell dysfunction and promote TAMs differentiation. 251,252 In addition, the abnormal elevation of fatty acid synthase in CAFs may enhance the aggressiveness of tumor cells. 253 There-fore, lipid metabolism reprogramming plays an important role in regulating the formation of the pro-metastatic TME.

Long non-coding RNA
LncRNA is a standard non-coding RNA with a length of more than 200. 254 Researches in recent years have shown that lncRNA plays an important role in tumorigenesis. 254 In fact, lncRNA plays a double-edged role in tumor metastasis. LncRNA may be involved in all the processes of metastasis, including EMT, tumor invasion and migration, and tumor cell colonization in secondary sites. We briefly summarized the representative lncRNA that have been shown to be relevant with metastasis in vivo and in vitro ( Table 1).  As mentioned above, EMT is an essential step in the initiation of tumor metastasis. It has been reported that LncRNA H19 can mediate the transformation of tumor cells into mesenchyme and is significantly elevated in bladder cancer, breast cancer, and colorectal cancer. [255][256][257] H19 directly binds to EZH2 in bladder cancer, resulting in a reduction in EMT epithelial marker E-cadherin. 255 In colorectal cancer, H19 acts as a competing endogenous RNA, leading to the up-regulation of ZEB1/2 protein expression. 257 However, in prostate cancer, the opposite phenomenon has been observed, that is, H19 inhibits tumor metastasis, mainly by encoding miR-675 to mediate the down-regulation of TGFBI. 258 Similar to H19, lncRNA-PNUTS serves as a competitive sponge for miR-205 and miR-200, leading to the up-regulation of EMT in breast cancer. 259 LncRNA LINC00460 is increased in colon cancer, and it induces EMT and promoteS tumor growth and metastasis by enhancing the expression of high-mobility group AT-hook 1 and decreasing the expression of Ecadherin. 260 The influence of lncRNA on tumor invasion and migration is also one of the mechanisms by which it affects tumor metastasis. For example, colon cancer-associated transcript 2 (CCAT2) and RNA associated with metastasis-11 (RAMS11) have been reported to participate in the process of tumor invasion and migration. CCAT2 is related to the stability of microsatellites; knockdown of CCAT2 inhibits tumor invasion and migration in mouse models. CCAT2 may interact with TCF7L2 to activate Myc transcription and Wnt signaling pathways and thus promote metastasis. 281,282 RAMS11 is overexpressed in colon cancer with liver metastasis. Knockout of RAMS11 gene using CRISPR-Cas9 technology can reduce the invasion and migration of colon cancer cells in vitro and reduce liver metastasis in mouse model. 283 Lnc01232 was found to promote metastasis by inhibiting ubiquitination, upregulating HNRNPA2B1, and activating the mitogen-activated TA B L E 1 Important metastasis-related long non-coding RNAs protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway in pancreatic cancer. 261 As the first lncRNA found to be associated with metastasis, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been shown to be associated with poor prognosis of lung cancer and breast cancer, and its expression is significantly increased in patients with metastatic cancer. 284,285 In the breast cancer mice model lacking the promoter of MALAT1 or MALAT1, tumor differentiation and E-cadherin are increased, whereas lung metastasis is significantly reduced, indicating that MALAT1 may serve as essential factors for tumor cell colonization at distant metastasis sites. 285 With the progress in understanding the functions of lncRNAs, increasing evidence has indicated that lncRNAs play a role in the physiological and pathological processes of malignancies. Although many findings need further verification, it is certain that lncRNAs can carry out diverse functions in carcinogenesis and metastasis. However, the specific role and mechanisms of different lncRNAs in cancer metastasis need to be further elucidated.

INTERVENTIONS FOR TUMOR METASTASIS
Metastatic disease is the major contributor to cancerrelated mortality. Therefore, there is an urgent need for effective cancer treatments that can eliminate large solid tumors and disseminated and metastatic nodules, while simultaneously preventing tumor recurrence. With the deepening understanding of the molecular mechanism of tumorigenesis and metastasis in recent years, a plethora of treatment approaches, including targeted therapy and immunotherapy have been established for antitumor treatments. 13,286,287 Although the emergence of new therapies, such as those based on immune checkpoint inhibitors (ICIs), is rapidly changing the treatment modality of metastatic patients in some cases, the current standard of cancer treatment for localized disease is still usually based on surgery, chemotherapy, and radiotherapy. 288,289 In addition, the combination therapies are receiving more attention and are being actively evaluated. 290 We summarize the interventions for metastatic diseases based on preclinical and clinical evidence.

Surgery, chemotherapy, and radiotherapy
Surgery, chemotherapy, and radiotherapy are the three cornerstones of tumor treatment. According to the purpose of treatment, surgical treatment can be divided into rad-ical surgery, local surgery, and palliative surgery. 291 For patients with early-stage tumor, radical surgery can prevent and reduce tumor recurrence and metastasis. However, for most patients, due to the insidious tumor-related symptoms, distant metastasis has already occurred at the time of diagnosis. In the past, it was considered that metastatic tumors cannot be treated surgically, but a number of recent studies and clinical practices have confirmed that local surgery can be performed on some patients with localized metastasis, which can reduce the tumor burden and may achieve the goal of radical cure. [292][293][294][295] For example, palliative surgery for patients with intraperitoneal metastases such as liver cancer and ovarian cancer can alleviate their symptoms and improve their quality of life. 296,297 For intervention and treatment of metastatic tumors, surgery alone is not enough. Dormant CTCs is the main cause of early postoperative metastasis. Therefore, many clinical studies have been carried out to reduce the probability of tumor metastasis. Neoadjuvant chemotherapy and adjuvant chemotherapy are currently widely used in the treatment of cancer because these interventions can kill CTCs and reduce the possibility of metastasis while reducing the size of the preoperative tumor and preserving some functional organs. [298][299][300][301] Except for a small number of patients, chemotherapy, such as 5-FU, Adriamycin, and platinum, is the first-line choice for most patients with advanced cancer. [301][302][303][304] It has been observed that diverse malignancy patients with metastatic lesions benefit from chemotherapy, especially patients with lymphoma and leukemia, and some patients can even obtain a durable response. 305,306 However, conventional maximum-dose chemotherapy cannot be tolerated by many patients with advanced cancer due to severe side effects and ultimately leads to treatment failure. In addition, it has been found that chemotherapy may induce the production of pro-metastatic cytokines and chemokines, thereby inducing tumor metastasis. 307 Therefore, many studies have been carried out to optimize chemotherapy regimens, and some progress has been made. Recent studies have shown that metronomic chemotherapy can inhibit angiogenesis and reduce the expression of pro-metastatic cytokines and chemotaxis, thereby reducing tumor recurrence and the distant spread of tumor cells. 308,309 These results indicate that low-dose metronomic chemotherapy may be effective in the prevention and treatment of tumor metastasis.
Radiotherapy has been recognized as a radical treatment for some early-stage cancer, such as NPC, and progress has also been made in the treatment of patients with metastatic cancer. 295,310 Compared with radiotherapy alone, the combination of radiotherapy and other therapies has better therapeutic effects on metastatic cancer, especially when combined with chemotherapy. For locally advanced NPC, concurrent chemoradiotherapy has been approved as a standard regimen by the National Comprehensive Cancer Network Guidelines. 310 Recently, two Phase III clinical trials from China proposed that induction chemotherapy before concurrent chemoradiotherapy can significantly improve the prognosis of patients with NPC. 311,312 It can be reflected that multidisciplinary combination therapy will be the main means to improve the prognosis of patients with metastatic cancer.

Targeted therapy for metastasis
For most patients with advanced-stage cancer, surgery and radiotherapy are difficult to achieve satisfactory therapeutic effects, which is also the reason for the high tumorrelated mortality. In the process of tumor metastasis from initiation to colonization, the occurrence of each step is the result of the joint action of some specific genes and signaling pathways. Blocking one of these steps may block the formation of metastases. Therefore, the development of drugs targeting these targets may provide an alternative for patients with advanced tumors.

Targeting EMT and cell motility
Blocking EMT is one of the key strategies to prevent tumor cells from spreading from the primary tumor site. As a marker of mesenchymal cells, N-cadherin is elevated in metastatic tumors and is associated with a poor prognosis. 33,38 ADH-1 is the first humanized antibody selectively targeting N-cadherin, which has been proved to improve the prognosis of patients with tumor-expressing N-cadherin. 313 Recently, a study showed that blocking Ncadherin by ADH-1 can also inhibit the expression of PD-L1 and the recruitment of Tregs in TME and augment the cytotoxicity of tumor-infiltrating lymphocytes (TILs) against tumor cells. 314 The steroid receptor coactivator (SRC) tyrosine kinase family is one of the central members that mediate EMT. 45 Drugs targeting SRC, including dasatinib, bosutinib, and saracatinib, have been proved to inhibit tumor growth and prolong patient survival. 315 When combined with other treatment modalities, the antitumor effect is stronger. In a Phase III prospective clinical trial (NCT01584648), the 3-year progression-free survival rate of patients receiving dabrafenib plus trametinib was 22% and 12% for patients receiving dabrafenib monotherapy, and the 3-year overall survival rate was 44% and 32%, respectively. Furthermore, in the subgroup with normal lactate dehydrogenase and less than three metastatic sites, the 3-year overall survival rate of patients receiving combination therapy reached 62%. 316 The motility of tumor cells is one of the critical factors that determine whether the metastasis can proceed smoothly, and the integrin family can affect cell motility by regulating cell adhesion. 37 Therefore, targeting the integrin family can weaken the motility of tumor cells. Currently, drugs targeting the integrin family mainly include cilengitide, intetumumab, 264RAD, and MK-0429, which have been proven to prolong the survival of cancer patients in preclinical studies and prospective clinical studies. [317][318][319][320] The allosterisms of actin and myosin in ECM promote structural remodeling and tumor cell polarization during tumor cell migration; Ras homolog gene family, member A (RhoA), MMP, and non-muscle Myosin-II are involved in the allosterisms of these two proteins. 130 Small molecule inhibitors of molecules regulating actin and myosin have been shown to attenuate tumor cell motility and inhibit tumor metastasis in vivo and in vitro, but their effectiveness in humans needs to be confirmed by further clinical studies. 156,321 After separation from ECM, only a small percentage of cells can survive and achieve subsequent metastasis. The main reason is that this small part of tumor cells has acquired the properties of anti-anoikis. 19,322 Therefore, tumor metastasis can be further inhibited by promoting tumor cell anoikis and eliminating anoikis resistance. T0070907 inhibits PPAR-γ and leads to cell death by reducing adhesion and inducing anoikis. 323 In addition, galectin-3 inhibitors have been shown to be effective in the treatment of thyroid cancer. The mechanism is mainly attributed to the inhibition of anoikis resistance, which is expected to provide a new strategy for inhibiting tumor metastasis. 324

Targeting CAFs
CAFs have also been proven to be important members involved in promoting tumor metastasis, and targeting CAFs have gradually attracted more attention in the treatment of metastatic tumor. 130 The activity of CAFs promoting tumor metastasis is mediated by a variety of signaling pathways, especially TGF-β and EGFR signaling pathways, which may provide targets for anti-metastasis. [325][326][327] In a variety of malignancies, including bladder cancer, breast cancer, colorectal cancer, and pancreatic cancer, TGF-β1 can induce the transformation of resident normal fibroblasts into CAFs and lead to the differential expression of α-SMA and fibroblast activation protein (FAP) genes (specific markers of CAFs) through the typical TGF-β signaling pathway. 161,[328][329][330] These data suggest that TGF-β1 plays a role in promoting CAFs production.
As the first oral small-molecule selective inhibitor of TGF-β1, galunisertib has been proved to have anti-tumor effects in clinical trials of liver cancer, pancreatic cancer, and glioma. [331][332][333] In addition, the therapeutic effect of combination gemcitabine with galunisertib is significantly better than gemcitabine monotherapy in advanced pancreatic cancer (combination group 8.9 months vs. monotherapy group 7.1 months). 332 In a Phase II trial, the median overall survival of galunisertib combined with the multitarget tyrosine kinase inhibitor (TKI) sorafenib in the treatment of metastatic liver cancer was 18.8 months. 331 Researchers have also developed a fusion protein, M7824, which blocks both PD-L1 and TGF-β signaling pathways; M7824 has shown potent anti-tumor activity in preclinical studies. 334 Subsequently, a Phase I clinical study was conducted in advanced lung cancer patients, and M7824 showed tolerable toxicities and preliminary anti-tumor effects. 335 In addition, a variety of cytokines secreted by CAFs act as ligands on the janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway, participate in the activation of this pathway, and promote tumor metastasis. [336][337][338] For example, CAFs-derived IL-6 can activate the STAT3 pathway to promote EMT of tumor cells, thereby facilitating metastasis. 62,339 Therefore, antagonizing those cytokines or blocking JAK/STAT signaling can inhibit the occurrence of tumor metastasis. The JAK inhibitor ruxolitinib has been shown to inhibit the invasion and migration of breast cancer, lung cancer, and colorectal cancer cells in vivo. 336,340,341 Two Phase II clinical trials have shown that ruxolitinib combined with gemcitabine improved overall survival in patients with advanced HER2negative cancer or advanced pancreatic cancer. 342 The IL-6 receptor inhibitor tocilizumab and IL-6 antagonist siltuximab have been shown to augment the anti-tumor activity of chemotherapeutic agents in preclinical trials, and further clinical studies are expected. 343,344 Blocking the activity of CAFs directly can also play an anti-tumor metastasis role. FAP is one of the markers of activated CAFs, and targeting FAP could transfer activated CAFs into a quiescent state. The monoclonal antibody targeting FAP (sibrutuzumab) and the small molecule inhibitors of FAP (F19 and PT100) were well-tolerated, but no obvious clinical anti-tumor effect was observed in Phase I clinical trials, and subsequent structural adjustments may be needed. 345-347

3.2.3
Targeting angiogenesis Angiogenesis is considered to be one of the components involved in tumor metastasis. A variety of smallmolecule tyrosine kinase inhibitors and monoclonal anti-body against angiogenesis have been approved as firstline or second-line therapy in a variety of advanced malignancies, including pazopanib targeting vascular endothelial growth factor receptor (VEGFR), imatinib targeting platelet-derived growth factor receptor (PDGFR), bevacizumab targeting VEGF, and multi-targeted receptor TKI anlotinib. [348][349][350][351] In addition, the combined application of existing drugs and the development of new drugs inhibiting angiogenesis are being explored in pre-clinical and clinical trials. 352 Several clinical trials of advanced lung cancer have shown that the combination of targeted drugs such as gefitinib, erlotinib, and anlotinib with radiotherapy can enhance the efficacy of anti-brain metastasis and are expected to be applied in clinical practice. [354][355][356] There has been a large amount of literature reviewing the therapies targeting angiogenesis, so we will not describe them in detail.

Targeting TAMs and Tregs
Regulating tumor immune microenvironment is another crucial therapeutic strategy to interfere with tumor metastasis. There is increasing evidence that TAMs and Tregs in the microenvironment promote tumor development and metastasis. 93,353 Targeting TAM and Tregs is a promising strategy to modify the immunosuppressive TME and prevent metastasis. A large number of studies have shown that CC chemokine receptors are essential mediators involved in tumor metastasis. 357 Among them, CCR1, CCR2, CCR3, and CCR5 promote the recruitment of TAMs in the TME, and CCR4 mainly mediates the recruitment of Tregs, indicating that CC chemokines can be used as potential anti-tumor pharmacological targets. 82,175 In recent years, CCR inhibitors have shown strong anti-tumor effects in preclinical and clinical studies. 358 For example, in a Phase I clinical study, CCR5 antagonists have demonstrated their ability to inhibit liver metastasis in colon cancer. 359 A preclinical experiment has shown that mogamulizumab, which antagonizes CCR4, can reduce tumor lung metastasis, and a Phase III clinical trial has demonstrated that antibodies targeting CCR4 are effective in treating patients with cutaneous T lymphoma. 360 Furthermore, mogamulizuma combined with the immune checkpoint inhibitor nivolumab may provide a new strategy for the treatment of advanced or metastatic solid tumors. 361 Another monoclonal antibody targeting CCR, AMG 820, also showed preliminary anti-tumor ability. 362 Colony-stimulating factor 1 (CSF1) can enhance macrophage recruitment and activation into a pro-tumoral TAM phenotype and plays a role in promoting metastasis. 363,364 The pleiotropic signaling of CSF1 receptor (CSF1R) supports multiple functions of macrophage, including proliferation, differentiation, and migration. 365 The CSF1/CSF1R axis has received much attention, and approaches targeting the ligands or receptors are currently being clinically developed. Pexidartinib, which target KIT, CSF1R, and Fms-like tyrosine kinase 3 (FLT3), has been approved by the US Food and Drug Administration (FDA) for the treatment of symptomatic tenosynovial giant cell tumor in adult patients. 366 Emactuzumab, a humanized monoclonal antibody targeting CSF1/CSF1R, inhibit tumor cell proliferation and metastasis by blocking the activity of CSF1R-dependent TAMs, suppressing the recruitment of TAMs to the microenvironment, and enhancing the T-cell infiltration. 367 However, the antitumor ability of emactuzumab is not evident in a Phase I clinical trial, and further studies are needed. 367 The CD4+ Treg cell population in the TME is the main cell group leading to the inactivation of effector T cells and is also one of the factors that promote tumor progression and metastasis. Therefore, targeting CD4+ Treg cells directly is a promising strategy to effectively prevent tumor metastasis and progression. IT1208, a defucosylated humanized anti-CD4 monoclonal antibody, demonstrated tolerable toxicities and encouraging preliminary efficacy in a Phase I clinical trial involving 11 patients with advanced solid tumors. 368 In addition, a microsatellite-stable colon cancer patient receiving IT1208 treatment showed increased infiltration of both CD4+ and CD8+ T cells in the tumor and achieved a durable partial response. 368 These results suggest that Tregs-targeted drugs, such as IT1208, are expected to provide a new idea for immunotherapy, but further researches are needed.

ICIs
A major hallmark of T-cell exhaustion is the increased expression of multiple immune checkpoints, such as PD-1, CTLA-4, LAG-3, T-cell immunoglobulin-3 (TIM-3), and T-cell immunoglobulin and ITIM domain (TIGIT). [369][370][371] Immune checkpoints have distinct ligands and inhibit anti-tumor activity of T cell through multiple mechanisms; T cell function decreases with the increase of immune checkpoint expression. 373 Recent advances in cancer immunotherapy have shown that ICIs, including inhibitors of PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, and TIGIT, can improve the clinical response and survival of patients with a broad spectrum of metastatic cancers, such as melanoma, non-small lung cancer, and renal cell cancer (Table 2). 13,369,371,[374][375][376] ICIs limit the inhibitory pathway of T cells and stimulate the activation of effector T cells to enhance the anti-tumor immune response. So far, at least eight different ICIs have been approved by the US FDA for the treatment of more than a dozen different cancers, including melanoma, kidney cancer, lymphoma, colorectal cancer, lung cancer, head and neck carcinoma, liver cancer, and sarcoma. 377 Among them, immune checkpoint therapy-targeting PD-1/PD-L1 pathway has achieved remarkable success in various types of tumors. For example, the most dramatic effects have been observed in metastatic melanoma, a malignancy that only slightly responds to conventional chemotherapy, and the historical average survival time of metastatic melanoma patients is less than 1 year. 378 Surprisingly, when combined with anti-CTLA4 and anti-PD-1 therapy, nearly 60% of the patients can achieve radiographic responses, with a median survival time of more than 3 years. 379,380 In a Phase II trial, 42 patients with brain metastases from non-small-cell lung cancer (NSCLC) were recruited to receive PD-1 targeting inhibitor pembrolizumab, 11 of whom responded, and the median followup time was 8.3 months. 381 In addition, clinical studies have shown that the PD-L1 inhibitor atezolizumab can significantly improve the prognosis of patients with advanced or metastatic solid tumors. 382 Pembrolizumab and atezolizumab have been approved as first-line or second-line therapies for some patients with advanced or metastatic solid tumors, such as NSCLC, kidney cancer, breast cancer, and melanoma. [381][382][383] However, only a limited number of patients can benefit from immunotherapy, and some patients who initially respond to immunotherapy may eventually relapse and progress. 384 Therefore, a large number of pre-clinical and clinical studies have investigated the combination of immunotherapy with other therapies, including chemotherapy, radiotherapy, or targeted therapy, as well as the combination of ICIs targeting different immune checkpoints to overcome the dilemma. To date, many therapies combined with immunotherapy and chemotherapy or anti-angiogenic drugs have been recommended as first-line treatments for multiple solid tumors. 385,386 For example, in patients with squamous NSCLC, the addition of pembrolizumab to chemotherapy (carboplatin plus paclitaxel or nab-paclitaxel) result in significantly better overall survival and progression-free survival than chemotherapy alone. 387 As a result, the combination of pembrolizumab with chemotherapy is recommended as the first-line treatment for patients with advanced squamous NSCLC. 388 In advanced renal cell carcinoma, the FDA approved pembrolizumab combined with lenvatinib or axitinib as the first-line treatment. 389,391 Among them, pembrolizumab combined with axitinib is the first anti-PD-1 antibody plus targeted drug combination therapy approved by the FDA. 390  that radiotherapy can improve the response of patients with metastatic NSCLC to immunotherapy. 392 Overall, a broad range of immunotherapy-based combination therapies have been approved for the treatment of cancer, and a large number of preclinical studies and clinical trials are ongoing to explore more possibilities for combination therapies in the treatment of metastatic cancer.

Others
In recent years, due to the abundance of natural herbal compounds and the diversity of their chemical compositions, they have received more and more attention as antitumor drugs. Triptolide, anthocyanidins, and gigantol have been confirmed to inhibit the proliferation and invasion of NSCLC cells, glioblastoma cells, and bladder cancer cells in vivo and in vitro. [393][394][395] Studies have found that the antitumor effects of certain compounds are mainly achieved by inhibiting EMT. 394,395 In addition, the chemical compound DZ-50 and the natural product curcumol can inhibit tumor metastasis by weakening the resistance of tumor cells to anoikis. 396,397 However, their anti-tumor effects need to be confirmed by further clinical studies. The failure of metastatic cancer treatment may be partly due to the inability of drugs to persist in the blood or lymph and the inability of drugs to cross certain natural barriers. For example, for patients with metastatic brain cancer, the difficulty in treatment is that most anti-tumor drugs cannot cross the blood-brain barrier. The emergence of nanomedicine has brought revolutionary updates to tumor treatment. Due to the small molecular weight of nanometers and the ability to carry specific pharmacophores to distant metastatic regions, nano-related chemotherapy and nano-related immunotherapy have demonstrated outstanding potential in tackling metastatic cancer. 398,399 The combination of different nano-polymer materials (such as hydrogels, micelles, and nanoparticles) and chemotherapeutic drugs, small molecule inhibitors, or immunotherapy drugs, has successfully improved the control rate of metastatic tumor. [400][401][402][403] For example, losartan-loaded injectable peptide hydrogel can inhibit the formation of CAFs collagen, thereby increasing the concentration of adriamycin at the metastatic sites and killing tumor cells. 400 Succinobucol is a small molecule inhibitor targeting vascular cell adhesion molecule-1, which is believed to be able to reverse lung metastasis, but the results in in vivo experiments are not satisfactory. Then, He et al. successfully designed succinobucol on ph-responsive wormlike micelles as a novel nanomedicine. Both in vivo and in vitro experiments demonstrated that succinobucol could be successfully delivered to the site of metastasis through ph-responsive wormlike micelles, thus improving treatment efficiency. 401 In addition, nanotechnology can enhance the anti-tumor effect of immunotherapy by rebuilding the tumor immune microenvironment, enhancing the anti-tumor effect of immune cells, enhancing the effectiveness of anti-tumor cytokines, and awakening tumor cells to release antigens. 398,399 Moreover, nanotechnology may reduce the side effects of immunotherapy.

CONCLUSIONS AND FUTURE PERSPECTIVES
Metastasis accounts for the great majority of cancerrelated deaths, which is the result of the interaction between tumor cells and TME components. In contrast to numerous discoveries that have revealed the detailed mechanisms leading to the formation of primary tumor, the biological underpinnings of metastatic disease are still poorly understood. However, some progress has been made in elucidating the cellular and molecular mechanisms that drive cancer metastasis over the past decades.
Despite the diversity of tumor types, the mechanisms of metastasis have some commonalities. The invasionmetastasis cascade involves angiogenesis, detachment of metastatic cells from the primary tumor, invasion through the BM and ECM rounding tumor, intravasation of the metastatic cells into the blood and lymphatic vessels and survival during hematogenous transit, adhesion of these cells to the endothelium of capillaries, extravasation through vascular walls into the parenchyma of distant tissues, and colonization at the target organ site. 17,23,404 The EMT and anoikis resistance of tumor cells are the main forces to promote the metastasis cascade, and multiple components in TME are inducers to ensure the success of metastasis by reprogramming the pre-metastasis niche and promoting metastatic cell growth. 21,35,325 EMT modulates cell-to-cell or cell-to-matrix adhesion and allows cells to increase their migration and invasion capabilities by forming invasive protrusions. 405 Metastatic cells develop an ability to resist anoikis, enabling them to adapt and survive when detached from the ECM or in the absence of the ECM. 19 The interactions between tumor and ECM components are essential for EMT and acquisition of tumor invasive abilities, and TME is also closely involved with all the processes of metastasis. 69 As presented earlier, immune cells (e.g., TAMs, Treg, and neutrophil), stromal cells (e.g., CAFs, MSCs, and TECs), chemokines, cytokines, and growth factors in TME, are involved in a complex crosstalk with tumor cells that affects tumor growth and metastasis. 69,79,138,406 Furthermore, recent studies have emphasized the role of autophagy, lipid metabolism, and lncRNA in tumor metastasis. 212,240,255 Those discoveries have brought new insight into our understanding of cancer metastasis. For most patients, due to the insidious tumor-related symptoms, distant metastasis has already appeared at the time of diagnosis. 1 In addition to the three cornerstones of tumor treatment, surgery, chemotherapy, and radiotherapy, novel treatment approaches, including targeted therapy and immunotherapy, have been established in patients with metastatic cancer. According to the chronological order and biological characteristics of metastasis, intervention strategies can be roughly classified into four types: early prevention, blocking metastasis-specific pathways, reducing angiogenesis, and enhancing anti-tumor immunity. For low-grade early malignancies, preoperative neoadjuvant chemotherapy and postoperative adjuvant chemotherapy can prevent and reduce the probability of recurrence and metastasis to a certain extent. 298,301 For advanced or metastatic tumors, targeted therapies, especially drugs targeting metastasis-specific pathways and angiogenesis have been approved for the treatment of most types of tumors. 15 What is more exciting is that immunotherapy, especially ICIs, is revolutionizing the treatment of malignancies and has demonstrated their potential as "cancer terminators." The advent of immunotherapy and combination therapies based on immunotherapy has provided more options for patients with metastatic cancer. Despite many challenges, we hold an optimistic view of the future prospect of immunotherapy.
In summary, this review provided our current understanding of the mechanisms that underlie the dissemination and metastatic outgrowth of cancer cells. As presented above, although many critical obstacles are still lying ahead, tumor metastasis-related pathways and TME represent novel and attractive directions, which may change the landscape of cancer therapy in the future. However, further researches and more endeavors in this area are needed.

A C K N O W L E D G M E N T S
This work was supported by the National Natural Science Foundation of China (No. 81772863 and 82072958) and the China Postdoctoral Science Foundation-funded project (No. 2020M683119). This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

C O N F L I C T O F I N T E R E S T
We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

E T H I C S A P P R O VA L
Not applicable.

A U T H O R C O N T R I B U T I O N S
ML and YJ: data curation, formal analysis, and writingoriginal draft. BX: data curation and formal analysis. XZ: conceptualization, supervision, validation, and writingreview editing. All authors read and approved the final manuscript.