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beyond reason
암세포가 전이되기 위해서는 암세포가 이동해야 하고 tumor invasion되어야 함
그것에 관한 메카니즘
기전을 알고 해결방안 찾기!!
Traditionally, the metastasis has been detected in the late stage of the cancer, which mostly leads to death. The classical opinion about tumor metastasis is that tumor cell migration begins with the single tumor cell and goes through a series of complicated procedures, and lastly arrives and survives at distant tissues and organs. However, emerging studies have found a new migration mechanism called collective cell migration in many cancers. The collective cell migration could move as clusters with the tight cell-cell junction in the tumor microenvironments, toward the traction established by the leader cells. In addition, the collective cell migration has been shown to have higher invasive capacity and higher resistance to the clinical treatments than the single tumor cell migration. Interestingly, the collective clusters of tumor cells have been detected in the early stage of the cancer patient, which has led to the understanding of the significance of early cancer screenings. Here, we reviewed the major principles and guidance of the collective cell migration mechanisms, and the specific manifestations in the different tumors such as breast cancer and lung cancer.
Metastases are responsible for advanced cancers, which are usually connected with the worse clinical outcomes. The whole process is complex, multi-step but inefficient, including the escape of primary cancer cells into the circulatory system, and finally colonization and proliferation in the distant organs [1]. The traditional opinions demonstrated that the cancer invasion began with the single colonial growth tumor cell from the primary tumor, which has comprised the foundation of the tumor transmission models, such as epithelial-mesenchymal transition (EMT) and migratory cancer stem cells [2] (Figure 1). However, there are emerging evidences found in many types of cancers, like breast cancer, lung cancer, and mesenchymal tumors, whose metastases can also be seeded as large, cohesive cohorts of cells clustered into adjacent tissues. The first report of collective cell clusters was in 1950s, which found that the blood sample of cancer patient contained both individual and collective tumor cells [3]. Some studies have also been reported that the tumor clusters could travel more efficiently [1], and circulating tumor cells (CTC) clusters had significance of clinical outcomes [1]. Recent studies have suggested that collective cell migration revealed worse clinical outcomes than single cells.
The invasion mechanism of single cancer cell and collective cancer cell. The single cell invasion begins with a single tumor cell from the primary tumor site and undertakes the EMT program to lose some epithelial characteristics, and then comes into the circulatory system, lastly arrives at the distant tissues and organs after experiencing the MET program which gains the epithelial characteristics again. The collective cell invasion begins with a clusters of cells of the primary tumor while does not need completed EMT program that remains some epithelial characteristics and locates at the secondary organs as a whole units.
Collective cell migration is a fundamental process which is a coordinated movement of group cells that maintain connected via cell-cell junctions [4-6]. This process has often been observed in the epithelial regeneration and the formation, and reshaping of large tissue structures during the embryonic development period, such as angiogenic sprouting and neural crest cell streaming [4,7,8]. Nowadays collective cell migration has been reported to connect with cancer migration, and even metastases [9,10]. Collective cell migration is a comprehensive achievement of a variety of processes, such as collective polarization, mechanical coupling, and cytoskeletal kinetics. It majorly followed the guidance of cell-intrinsic multicellular organization, the leader-follower cell behavior, and integration of extracellular signal guidance cues. The knowledge of collective cell migration in tumors has had great significance on the treatment of cancer. In this review we illuminated some common equipments and ways for studying collective cell migration, and discussed the emerging principles and guidance mechanisms of collective cell migration, and how it particularly performed in common tumors.
Collective cell migration can move as a unit with the help of the cell-cell junction coupled to dynamic actin cytoskeleton. Cell-cell junction keeps cellular adhesion, polarization, and senses and integrates external guidance signals, then further passes mechanical signal processing and forces transmission within the migrating clusters in the whole movement [11,13]. Like in embryonic development, cell-cell junction may be the production of the complementary adhesion systems [14]. The ingredients of cell-cell junction in the collectively migrating cells involve adhesion receptor and cytoskeletal adaptor systems, including desmosomal proteins, gap junctions, tight junction constituents and integration between immunoglobulin family members (Table 2) [8]. Most of them participate in the entire process of signal transmission mediated by cell-cell junction, such as PI3K/AKT, focal adhesion kinase (FAK) and Rho GTPases [10,15,16]. The adhesions take alterations in surface expression and cytoskeletal coupling under the control of up-streaming signaling, then generate various cell-cell junction [17]. And adhesion receptors can help to stabilize the junction between cells. Thus, the adhesion-positive cell-cell junction can be seen as a strong marker of the collective invasion [18]. Besides, the mechano-transducing bridge was made by these cell junctions in the nearby cells, which underlies organizations on the actin cytoskeleton [19]. Cell-cell junctions connect the actin cytoskeleton of multiple cell bodies, which establish the basis for conformity of the forces of individual cells and the leader-follower polarity in the whole group [20-22]. Cell-cell junctions can stabilize mechanical connections, however, cytoskeletal connections and adhesion sites are dynamic and constantly changing to adapt to the microenvironment during the migration [23]. What’s more, the dynamic collective cell function deeply depends on different period cell-cell junctions, from hours to days or even weeks [24]. The critical cell-cell junction medicating the direction and speed of collective cell movements is in few minutes via unstable and short-lived adhesions sensing [25]. There are a series of cell-cell junction mechanisms that have been involved in tumor collective cell invasion. Epithelial tumors metastasis majorly referred to E-cadherin and β-catenin positive cell-cell junctions which can mediate AJs and cell-cell interactions in tumor cells [26]. EMT can reprogramme cell-cell junctions to enhance the ability of invasion and metastasis of epithelial cancers, which includes weakening or even dissolving cell-cell adhesions within the tumor cells, cleaving cadherins via up-regulateing the expression of stromal proteases, deregulating integrin adhesion systems, and turning Rho-mediated actomyosin contractility from cell-cell junctions toward cell-matrix interactions [27-29]. Thus, the adaptability of cell-cell coping program strategies makes cancer invasion and metastasis suitable in different tissue environments. The specific transmission to the different environments during the collective invasion process still remains to be further explored.
Adhesion systems and their roles in cell-cell junction
Adhesion systems | Location and roles | Relevant regulation proteins and signaling pathways | References |
---|---|---|---|
Adherens junctions (AJs). | Cell-cell junctions of epithelial and endothelial tissues | Rho family GTPases: Cdc42 in Par6/aPKC and CIP4 (Cdc42-interacting protein 4); | [86-88] |
Connect the actin cytoskeleton of adjacent cells | activator of AJs: Rac guanine nucleotide exchange factor (GEF) TIAM1, and Nectin and Nectin-like proteins | ||
Tight junctions (TJs). | In plasma membranes of adjacent cells | Composed transmembrane proteins: claudin, occludin, tricellulin, marveld3, junctional adhesion molecules (JAMs), ZO1, ZO2, ZO3 | [89-92] |
Create a barrier as restricting diffusion as paracellular gates | Rho GTPase signaling: RhoA, Cdc42, Rac | ||
Gap junctions (GJs). | Intercellular membrane channels | Cx43, N-cadherin, cytoskeletal proteins such as microfilaments and microtubules | [93-95] |
Form a tight connection between adjacent cells contributing to cell-cell adhesion | |||
IgCAMs | Iimmunoglobulin-like cell-adhesion molecules | Proteins at the cell membrane: growth-factor receptors, integrins, cadherins and intracellular proteins: effectors of signal transduction pathways and cytoskeletal proteins | [96,97] |
Mediate adaptive cell-cell interactions | |||
Slit/Robo | Roundabout receptors (Robo) and their Slit ligand | Netrin, GTPase activating proteins (GAPs), | [36] |
Control actin cytoskeletal dynamics by interacting with different signaling molecules | RhoA, Cdc42, Rac | ||
Ephrin/Eph receptor. | Tyr kinase receptors and ephrins | Tyr kinase adaptor protein 1 (Nck1), Nck2, Vav2, Vav3, Src, ephexins | [98] |
Control actin cytoskeletal dynamics in short-distance cell-cell signaling | RhoA, Cdc42, Rac | ||
Integrins. | Transmembrane proteins that connect the cytoskeleton with ECM | F-actin, FAK, Src, ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase) | [99,100] |
Important transducers of mechanical forces |
The guidance for individual tumor cell invasion has been widely understood, including chemical and physical guidance [9], which also works in the collective cell migration. What’s more, the collective cell migration also needs to integrate guiding cellular signals for moving and maintaining the migration as a cohesive cell group. The process relates to several cell-intrinsic and extracellular mechanisms of guidance and polarity under the multicellular decisions [30]. The major mechanism refers to the leader-follower polarization, which is related to the coordination of two kinds of cells - leader and follower cells. In the front of the moving groups, there are usually localized leader cells which receive and integrate the signals, while the nearby cells with cell-cell junction, migrate to the direction given by the leader cells, calling follower cells [31,32]. The leader cells maintain their polarity and invasion into tissue structures by acquiring their leading edge to the substrate supported by AJs signaling, such as actin-based structures, signal processing and special gene expression [33].
Like in mammalian sprouting vessels [33], the leader cells integrate the guidance of the extracellular inputs and downstream intracellular signals in the specific cell-type and tissue-context, such as mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), FAK and Rho GTPases [6,34-38]. The early activation of these pathways contributes to the intrinsic bipolarity in leader cells, which causes leader cell selection in cell subsets and inhibits the leader cell selection in adjacent cells, known as followers. Leader cells can regulate actin cytoskeleton polymerization, the actomyosin contractility and force transmission and then pass the force in the whole units to stabilize the focal adhesions with the substrates with the help of the activation of Rho GTPases in a case [39,40]. Meanwhile the follower cells maintaining cell-cell junction could silence Rho/Rock signaling and reduce actomyosin contractility [41] (Table 3). The units of collective cell movements demonstrate leader cells that contact with nearby structures according to Rac-driven filopodal protrusions and substrate adhesions [36]. Moreover, the adhesion to substrates of the leader cells is mainly balanced by two kinds of forces: the integrin-based pulling force at the cell-cell junction of tumor cells, and the pushing force by the neighboring cells. The leader cells with mesenchymal characteristics grasp the direction and speed of migration by stretching actomyosin-mediated protrusions, degrading the surrounding microenvironments and resetting the extracellular matrix (ECM) [9]. The follower cells move with the cell-cell junction behind along the traction made by the leader cells and readjust the ECM [42].
The proteins and signaling pathways in collective cell migration
Cell guidance/environment | The guidance and functions | Proteins and signaling pathways | References |
---|---|---|---|
Cell-cell junction | Connect the actin cytoskeleton of multiple cells with adhesions | Rho GTPase signaling: RhoA, Cdc42, Rac | [10,15,16,88] |
Keep cellular polarization and force transmission within the whole clusters | PI3K/AKT/FAK/ERK | ||
Leader-follower polarization | Integrate signals of various environments and regulate the actomyosin contractility | MAPK, ERK, FAK, Rac | [6,34,41,46,50] |
Connect with substrates and cells around via actin cytoskeleton | Rho/Rock signaling | ||
N-WASP, LIM Kinases 1 and 2, Notch1-Dll4 | |||
CTC cells | Express a hybrid epithelial-mesenchymal (hybrid E/M) phenotype during the metastasis progression | OCT4, NANOG, SOX2, and SIN3A | [57] |
Na+/K+ATPase | |||
Breast cancer | Maintain the leader-follower polarization of K14+ cells in MMTV-PyMT and TNBC models | YAP signaling, ITGA3, ITGA5, Rho signaling, | [50,61,63,64] |
POSTIN and TNC, DOCK10, ITGA11, DAB2, PDFGRA, VASN and PPAP2B | |||
Lung cancer | Promote collective cell migration and metastasis in lung cancers | LPP, vimentin | [79,80] |
Consequently, collective cell migration mainly relies on the leader-follower cell behavior besides the integrated mechanocoupling and guidance in migration transversion also contributes a lot. The actomyosin contractility has been found to exert as the central hub disposing coordinating mechanical sensing and mechanical transduction responses [43]. While collective tumor cells migrate in the tissue structures, cell adhesion molecules sense the changes in substrate, cytoskeletal rearrangements promoted by ECM and environmental sensing promoted by actomyosin contractility together contribute to signal polar activation and changes of gene expression (Figure 2), which is crucial for the translation of cytoskeletal forces into migration motion [13,33,44,45].
The guidance of leader-follower cell polarization in collective cell clusters. The leader cells could exert pulling force to the follower cells by establishing the cell-ECM adhesion and impose a pushing force to the ECM, and receive pushing force from the mitosis of rear cells. The actomyosin cytoskeleton plays an important role in the whole movements as the core of the force transmission from cells to the matrix and between cells. The collective cells move as a unit with the cell-cell junction that the actomyosin contractility level is low at the junctions, which makes the intracellular pressure which is important to the ECM remodelling.
However, cadherin-mediated cell-cell junctions at the mechanosensitive adhesions develop an antagonistic relationship with integrin-based adhesions that induce cell polarization and determine the migration direction by promoting cell contractility of the rear of end cells [46-48]. And the cadherin-mediated cell-cell junction also suppresses the formation of protrusion in leader cells and the combination of the integrins with the ECM by transversal RhoA-mediated contractility. Cadherin-mediated mechanical sensitive adhesions interact with the actomyosin network by the catenins like p120- and α- and β-catenin, and this interaction is vital for the maintenance of the cell groups [34]. The balance between the force of cadherin and integrin integrates the leader and follower cells moving migration. Furthermore, the actomyosin cytoskeletal needs to be continuously remodelled by the changes in transcriptional programs to pass different tissue structures [49]. For example, the leader cell plays its role via the Notch1-Dll4 lateral inhibition, while the Rho signaling regulates the rear cells [46]. Also, the expression of N-WASP participates in the formation of cellular protrusions extended by leader cells. Besides, the cellular protrusions need the help of LIM Kinases 1 and 2 to increase actin filament stability [50,51]. Therefore, adhesion molecules regulations, dynamic changes of actomyosin cytoskeleton activities and specific gene expression programs in the leader-follower cells are fundamental in efficient collective cell migration.
The classic single tumor cell metastasis mechanism has been studied for some years, suggesting that cells need to undergo the EMT process to be invasive [52]. During the EMT, single tumor cells loose cell adhesion factors which are the characteristics of the epithelial cell and express classical mesenchymal markers induced by the tumor cell environment factors. When the single tumor cells reach the distant tissues, they will experience the MET program and reacquire the epithelial cell characteristics [53,54]. However, the collective cell migration found in CTC clusters did not need to undergo the complete EMT process to accomplish the metastasis [55]. Tumor cells in CTC clusters could maintain epithelial gene expression and furthermore express a hybrid epithelial-mesenchymal (hybrid E/M) phenotype to finish the proliferation and metastasis [1,56]. Moreover, in CTC clusters it has been found that stem-cell-related and proliferation-related genes are enriched and the transcription factors are hypomethylated in the binding site, such as OCT4, NANOG, SOX2, and SIN3A [57]. Interestingly, knockdown of the cell-cell junction or the Na+/K+ATPase inhibitors could turn CTC clusters into single CTC cells and lead to the DNA methylation remodelling at critical sites and further suppress the metastasis process, which provides potential clues to clinical treatments for the late stage of tumor diseases [57].
There has been some common mechanisms for breast cancer invasion, including epithelial-mesenchymal transition (EMT), collective invasion, and the macrophage-tumor cell feedback loop [58]. Collective invasion has been reviewed as the frequently mechanism of breast cancer invasion, in which the leader cells expressed a basal epithelial gene program, including intermediate filament cytokeratin-14 (K14) and the nuclear transcription factor p63, rather than molecular EMT [26], and guided the rear cells to migrate toward the way established by the leader cells that maintain the cell-cell junction and the connection with the substrates in microenvironments. The studies for tumor invasion in breast cancers usually used multicolor lineage-tracing strategy in a classic mouse model of breast cancer-MMTV-PyMT [59], by which we could observe the major stages of metastasis such as collective invasion, locally clusters dissemination, CTC clusters and distant metastasis [60]. The K14+ cells in MMTV-PyMT has been observed much more efficient than single seed cell in breast cancer invasion as they counted for 2% in the clusters but made >88% tumor collective invasion [26] and could enhance the survival of tumor clusters in distant tissues. K14+ cells were enriched in local dissemination and CTC clusters connected with tumor spread, while K14- cells were predominated in large colonies and large metastasis associated with proliferation [60]. Compared to the K14- breast cancer cells, K14+ cells in CTCs have been found to be enriched expression of 87 genes [61]. K14+ cells have been shown to be required by collective cell invasion rather than single seed dissemination as the increased genes expression of cell-cell junction and the adhesion of cell-ECM [60]. Furthermore, in MMTV-PyMT model, the leader cell may acquire K14 expression according to the contraction of the tumor cell-matrix border rather than existed as fixed [26]. K14+ cells could be induced by a stromal ECM which contains abundant fibrillar collagen and lacks collagen IV [26]. Meanwhile, collective cell invasion in basal-type breast cancer cells is limited because the tumor cells can only move through the paths patterned by fibroblasts in the ECM [62]. Therefore stromal fibroblasts play a role in breast tumor collective invasion as the leader cell by remodelling ECM, which determines the trails followed by the remaining tumor cells in the model of MMTV-PyMT and the activity of fibroblasts as regulated by YAP signaling [63]. The leading fibroblasts were regulated by ITGA3, ITGA5, and Rho signaling, while follower cells were dependent on Cdc42 and MRCK [64]. Some studies reported that the K14+ cells had some connections with the proteins required in metastasis transcribed by POSTIN and TNC encoding [61]. Similarly, collective migration could be eliminated by the hydrolysis of the MT1-MMP-mediated protein, which can express the leader cells creating the paths trailed by the follower tumor cells [65]. The intrinsic differences of tumor cell subpopulations could affect the migration movements of the collective invasion [65,66]. In triple-negative breast cancer (TNBC) an epigenetically distinct subpopulation of breast tumor cells has been identified called “trailblazer” cells, which may be expressed by a pattern of genes and could be more invasive than the K14+ breast tumor cells [50]. The cohort is comprised of 7 genes, including DOCK10, ITGA11, DAB2, PDFGRA, VASN and PPAP2B, which regulate the activation of collective invasion, and DOCK10 is required for tumor metastasis [50].
Cadherin has been found in most breast cancers as a crucial characteristic, and the level of membrane E-cadherin has played a dual role in promoting cancer invasion and metastasis [67]. A special collective migration mechanism has been explored in the 4T1 cells of a highly metastatic mouse breast carcinoma model, where tumor cells move expressing highly invasive phenotype remaining loosely inter-connected by tethers moderately mediated by E-cadherin [68] rather than extensive cell-cell junction similar to the other collective migration cells. Furthermore, knockdown of the E-cadherin would prevent the formation of tethers and switch the migration mode from collective to single-cell, and reduce the dissemination of 4T1 cells to the lung and others [69].
However, in the classic EMT program induced by the stromal cells, E-cadherin has usually been reported as down-regulated in the tumor migration cells with the loss of cell-cell adhesion and the phenotype of epithelial cells [70,71]. Inflammatory breast cancer (IBC) has been a good model to explore the principles of collective cell dissemination, and is a highly aggressive subtype of breast cancer that mainly invades via CTC clusters [72]. CTC cells highly express E-cadherin which is the hallmark of epithelial traits, rather than go through EMT program during the metastasis process, which may have connections with the worse clinical outcomes compared to the non-IBC patients [61,72,73]. Together, the interaction between collective cell migration and single tumor cell migration regarding cancer metastasis requires further research.
It has been reported that CTC clusters were detected in the blood of lung cancer patients, which have greater potential to metastasize and the clustered cells have more survival advantages than single CTC cells [74]. And the CTC clusters in SCLC (small cell lung cancer) has been shown a capability of being tumorospheres with loosely and irregular connection, which had resistance to radio chemotherapy and worse prognosis (Figure 3) [75,76]. Also, the CTC clusters have been identified to have higher expression of IL6, BCL2, ERCC1, Ki-67 and IL-17 than single CTC cells, indicating that CTC clusters were highly invasive and connected to the worse clinical outcomes [77,78]. Furthermore, during the early stage of the lung adenocarcinoma metastasis process, the tumor cells experience mostly epithelial-like collective invasion, and are surrounded by vim+þ/FSP1+ cancer-associated fibroblasts (CAF) which require vimentin to maintain heterotypic tumor cell-CAF interactions [79]. Additionally, LPP regulates the expression of matrix metalloproteinase 15 (MMP-15) by degrading N-cadherin in PC14PE6 cells to promote collective cell migration and metastasis in lung cancers [80]. Together, the emerging studies detailing the collective cell migration in lung cancers could contribute to clinical treatments.
The invasion and metastasis mechanism of CTC clusters in SCLC. CTCs originate from the lung tumor and invade into the circulation. Small clusters of CTCs may become to tumorospheres. Then the whole tumorosphere extravasates from circulation into the distant organs as liver, bone and brain. Tumorospheres keep loosely and irregular connection and showed resistances to radiochemotherapy and worse clinical prognosis.
The collective cell migration is an emerging mechanism distinguished from the classic single tumor cell metastasis, which does not need a complete EMT program and can move as a cohort unit in which cells maintain the cell-cell junction and express some epithelial characteristics. Also, collective cell migration has been reported to have greater potential for metastasis and dissemination and has higher therapeutic resistance compared to single cell migration. A summary of the instruments and typical methods used in collective cell migration, are shown in Table 1. There are some challenges remaining to be further studied. First, what are the intrinsic differences between different cell clusters in different cancer diseases? Second, what are the specific mechanisms that change the single-cell migration to the collective cell migration? Third, how do the clusters adapt to the complicated environments during the metastasis process and finally survive in the distant organs? The discovery of collective cell migration has brought a new perspective to solving tumor disease. And there have been some FDA-approved medicines targeting collective clusters, which bring emerging clues to the terminal cancer treatments. Furthermore, screening of the collective cell migration could contribute to the detection of the early stage of tumor patients.
Instruments and ways to research collective cell migration mechanism
Instruments or ways | Field | Function | References |
---|---|---|---|
Time-lapse microscopy | Intracellular mechanism | Group dynamics, molecular process evaluation, interaction between cells and cells and substrates and son on | [81] |
Intravital imaging | Intracellular mechanism | Monitor of speed, location and type of invasion | [82] |
Mouse models | Breast cancer and other cancers | Tumor development process, tumor microenviroments representation | [83-85] |
3D matrix-based cell culture | Epithelial cancers, melanoma and other cancers | Tumor development process, tumor microenviroments representation | [9,13] |
Lineage analysis | CTC clusters | To define CTC clusters metastasis arisen from polyclonal origin | [1] |
Gene ontology analysis | CTC clusters | To prove the survival advantage of clusters in circulation | [77] |
Histological studies | Collective cell invasion | The morphology of collective cells and the extent of the invasion | [4] |
The work was supported by the National Natural Sciences Foundations of China (No: 81773218, 81703009, 81802791, 81472773 and 81272566) and The Natural Sciences Foundations of Hunan Province (No: 2018JJ3858 and 2017JJ3457).