Cancer cells include epithelial cells and complex stromal cells (1,2). To maintain motility, cancer cells can modify their morphology and migration patterns according to the environmental conditions they are exposed to. The initial stage of tumour metastasis is invasion, during which cancer cells move away from their original location. Collective cell migration may confer a more powerful invasive capacity on cancer cells than individual cell migration (3). Collective invasion is accomplished by both leader and follower cells moving together in cooperation. The leader cell directs the group, while the follower cells follow closely behind (4-6). The morphology of leader and follower cells differs significantly, with leader cells being polarized and spindle-shaped, while follower cells are tightly packed and maintain epithelial sheet-like properties (7). In addition, epithelial tumours have a predominant retention of epithelial characteristics and intercellular adhesion. Conversely, mesenchymal tumours typically invade individually, with only certain subtypes displaying collective invasion tendencies through epithelial-mesenchymal transition (EMT) (8). As an example, histopathological analyses frequently reveal that squamous cell carcinoma (SCC) invades in cluster forms (9). In organotypic coculture invasion models, cancer-associated fibroblasts (CAFs) act as leader cells to coordinate cancer cell migration (10). Follower cells in lung cancer exhibit an epithelium-like morphology when cultured in two dimensions, whereas leader cells appear mesenchyme-like (11). Therefore, further study of epithelial tumours is essential to gain a comprehensive understanding of follower cells.

Numerous studies have been conducted recently on the characteristics of leader cells in vivo and in vitro, demonstrating their important role in collective invasion (11-13). Furthermore, follower cells, which comprise the majority of the cell groups involved in collective invasion, have received increasing attention. Optimized spatiotemporal genomic and cellular analysis (SaGA) can specifically target, extract, separate and amplify leader and follower cells from a 3D microenvironment (11). The separated follower cells proliferate rapidly and divide frequently but are not highly invasive, whereas the separated leader cells are highly invasive, and divide and proliferate slowly (12,13). At the leading edge, specialized leader cells exert traction force on the follower cells (14). In addition to being pulled along by leader cells, follower cells are actively involved in selecting a specific direction of travel by extending protrusions underneath leader cells; these protrusions are known as cryptic lamellipodia (c-lamellipodia) (15). Since they typically possess c-lamellipodia, which are typically smaller and exhibit fewer adhesions with the extracellular matrix (ECM), the follower cells have less interactions with the ECM and exert less traction (16). The biomechanics generated by follower cells through c-lamellipodia facilitates collective invasion, allowing follower cells to invade as a group. Death of the leader cell causes the follower cells to cease migrating in the same direction, leading to random, slow movement of cells and an end to collective migration (17). Additionally, follower cells help the leader cells initiate appropriate polarization, strengthen leadership of the cells and ensure that sufficient leader cells are available to lead the invasion. This is achieved through the utilization of a multitude of signalling molecules, including chemotactic factors and physical contacts, whereby follower cells migrate in the wake of the leader cells and constitute a significant portion of the multicellular cluster. A variety of strategies are employed by follower cells to increase their invasive capacity, including contact inhibition locomotion (CIL), biomechanics, matrix remodelling, chemotaxis, lateral inhibition and exhibiting genetic and metabolic heterogeneity (18-21) (Fig. 1). Elucidation of the role of follower cells in collective invasion may identify new molecular targets for cancer treatment and intervention.

The ECM, offering both a biochemical and biomechanical context, plays a crucial role in cancer progression by regulating the ability of cancer cells to cross its barrier. By secreting diverse profibrotic growth factors and inflammatory factors, follower cells are primarily responsible for recruiting and activating stromal cells within the tumour microenvironment (119). In response to tumour-derived activation factors, stromal cells differentiate into CAFs, and CAFs act as myofibroblasts to reconstruct the ECM to facilitate tumour invasion (120-122). Follower cells themselves have also been shown to express altered ECM components, such as collagen I and III, and ECM-modifying enzymes (123-125). Lysyl oxidases are secreted by leader and follower cells to crosslink collagen fibres (126). Crosslinked collagen stiffens the ECM, leading to integrin-dependent invasive behaviour. Due to increased ECM stiffness, force-loading rates at FAs change, resulting in the stretching of talin1 and vinculin recruitment (127). FA kinase (FAK), RhoA and Src are activated as a result of this, increasing the contractility of follower cells.

To regulate follower cell behaviour, integrins transduce signals from the ECM by assembling FAs, thereby stimulating follower cell cytoskeletal remodelling (128). Integrins act as major receptors for ECM molecules during cancer metastasis (129). The activation of integrins and downstream mechanotransduction adapters, such as p130CAS, occurs when mechanical tension is increased (130). As a result, there is an increase in FA and actin stress fibre formation in follower cells (130,131). In cancer, actin-rich protrusions attach to ECM molecules bound to integrins and to contractile structures within follower cells, causing the basement membrane to breach without protein hydrolysis (132). Moreover, follower cells can exert force on ECM networks, affect the ECM architecture reversibly or permanently and enhance stiffness and ligand density locally (132-134). A growing body of evidence indicates that the ECM is significantly stiffer in this state, compared with its normal state, indicating abnormalities in its composition and structure (135,136). The ECM is undoubtedly an important component of the tumour microenvironment, not only promoting malignancy but also regulating tumour invasion (20,135,136).

Follower cells may exhibit lower levels of durotaxis and matrix metalloproteinase (MMP) secretion than leader cells, but still play a critical role in the collective invasive process (137,138). Durotaxis is the directed migration of cells towards regions of high mechanical resistance. Moreover, cells migrate towards the 'optimal matrix stiffness' region, where they can generate the greatest traction, and when in a region above the optimal matrix stiffness, cells show an apparent tendency to migrate towards softer regions (138,139). By undergoing durotaxis, the follower cells are able to move towards regions of high mechanical resistance and utilize secreted MMPs to degrade the ECM and facilitate cell invasion. Follower cells are typically more dependent on the behaviour of leader cells and the signals they produce. Follower cells often follow the path carved out by the leader cells and can contribute to the spreading of the tumour (137,140-142). By traversing a larger space created by the leader cells, follower cells encounter less mechanical resistance and can spend less energy remodelling the ECM (143). As a result of increased crosslinking and force-mediated ECM remodelling, the tumour-surrounding interstitial matrix becomes linearized. For efficient cell migration, follower cells migrate along densely aligned collagen fibres (126). Membrane-type-1-MMP (MT1-MMP) is typically localized to invasive actin-rich cell structures (144). The delivery of MT1-MMP and other proteinases by exosomes outside the cell can also facilitate invasive lamellipodia maturation and degradation of the ECM (145). By inhibiting MMP activity in CAFs before adding SCC follower cells, the invasion of follower cells was effectively halted (10). MMP function is not required by follower cells once the matrix has been remodelled by CAFs (10). In addition, as follower cells tend to undergo glycolysis, increased lactate generation and excretion favour protease-mediated matrix remodelling and thus enhance the invasion of cancer cells (146-148) (Fig. 4A). Lactate-induced acidity has also been found to enhance the activity of certain proteases, namely MMP-2, MMP-9, cathepsin B and cathepsin L (149).

In glioblastoma, follower cells remodel the extracellular hyaluronic acid (HA) through a combination of synthesis, by hyaluronan synthases (HASs), and degradation, by hyaluronidases and MMPs (150,151). In vitro models have indicated that incorporating HA into gelatin matrices enhances the invasiveness of the follower cells (150). The production of HA by HASs (particularly HAS2) is significantly activated during invasion into the HA-rich ECM (151). The CD44 gene, which is a representative HA receptor, shares a close relationship with HAS, suggesting that there may be crosstalk between these two genes that stimulates signal cascades for glioblastoma follower cell invasion (152). Therefore, the invasion of glioblastoma follower cells is primarily influenced by the HA-rich ECM environments. This is mainly due to the high involvement of CD44 receptors and HASs (151,153,154).

According to extensive data, chemotactic cell migration guided by soluble signalling molecules facilitates invasion and metastasis (155,156). Chemoattractants are soluble proteins secreted into the extracellular space. These molecules are believed to be retained in this space by binding to glycosaminoglycans in the ECM, thereby establishing immobilized concentration gradients. Chemotactic gradients tend to be local and transient in nature. Furthermore, cells can navigate through complex topologies with self-generated chemotaxis (156). In self-generated gradients, follower cells produce an outwards-facing gradient by breaking down an attractive chemical, which serves as a guiding cue for leader cells. Leader cells need chemical gradients generated by follower cells in order to respond to external signals (157). As leader cells interact with their surroundings, their local gradient moves with them, resulting in directed movement that is exceptionally robust and capable of operating over long distances. However, if the attractant level is too high, then the follower cells cannot migrate but instead break down the molecule until the concentration is low enough for a gradient to be observed (157). Additionally, if the cell leading wave does not have enough leader cells, the attractant is left behind, attracting more follower cells. In summary, cells show chemotaxis over long distances by moving in waves, with saturating chemoattractants in front and low levels behind (158). Without physically visiting their environment, follower cells acquire information about their surroundings (156,159) (Fig. 4B). Chemotaxis is a crucial concept for the understanding of the physiology of cells since it helps elucidate matrix composition.

It is believed that chemotaxis promotes the invasion and metastasis of follower cells. When a follower cell migrates, it senses a gradient of external chemoattractants, of which chemokines, chemotactic growth factors and lysophosphatidic acid (LPA) are major families (118,156,160). For example, as zebrafish posterior lateral line primordium collectively migrate, follower cells sense the attractant, CXCL12a, to migrate efficiently (118). The CXCL12/CXCR4 pathway also influences breast cancer cell metastasis to the lungs (161). Follower cells are CXCR4⁺ and invade along CXCL12 gradients into organs expressing CXCL12 (for example, the lymph nodes and lungs). Moreover, when malignant lymphocytes are exposed to CCL19 gradients, their surface is stripped of CCR7, resulting in the retraction of protrusions and loss of polarity, which indicates follower cell behaviour. On the basis of intermediate gradients of CCL19, follower cells migrate towards the invasion front in cell migration (162). In addition, chemical gradients formed by EGF uptake can guide the movement of malignant epithelial cells within confined spaces (156). However, Muinonen-Martin et al (160) noted that rather than acting as chemoattractants that could guide melanoma follower cells, growth factors acted as accessory factors that increased cell speed and chemotaxis efficiency, regulating melanoma cell behaviour. As such, the chemoattractant relay is rearranged to generate positional information using positive feedback. Furthermore, soluble chemicals are either degraded by enzymes or scavenged by decoy receptors (via endocytic internalization). In addition, follower cells are strongly induced to invade outwards by the LPA gradient (160). It has been demonstrated that follower cells can degrade LPA during melanoma metastasis, resulting in a gradient that the cells then respond to by migrating (160). Furthermore, expression of the LPA receptor (LPAR) in follower cells promotes metastasis and cell growth in metastatic breast cancer xenografts (163,164). An instrumental factor in the metastasis of pancreatic ductal adenocarcinoma cells from primary tumours is the neural Wiskott-Aldrich syndrome protein, which controls the recognition of LPA gradients by controlling LPAR1 (165). LPAR signalling mediates actin-myosin contractility and control of cell orientation, as well as matrix remodelling.

According to a mathematical analysis of the tracks of cells, follower cells are chemotactic and are attracted directionally by attractants (166). As follower cells respond to chemotactic gradients, they can alter them, resulting in robust chemotactic gradients. Furthermore, follower cells can reduce attractant concentrations when the attractant concentrations are too high. Depending on their needs follower cells alter themselves accordingly by inducing enzymes or cell division (166). However, the role of follower cells in generating chemokine gradients and their potential regulation by endocytosis have not yet, to the best of our knowledge, been studied.

Among neighbouring cells, Notch signalling can coordinate divergent cell fate, which is termed lateral inhibition (167). Typically, follower cells upregulate Notch1 and Jagged1 (Jag1) expression, while leader cells upregulate Delta-like 4 (Dll4) expression to promote collective invasion (13,92,168). Notch also coordinates the adoption of similar follower cell fates by modulating lateral induction, a process through which it promotes the acquisition of comparable cell fates in adjacent cells (11,169). Through lateral inhibition, Notch-Delta signalling suppresses leader cell behaviour in follower cells. A ligand can bind to the Notch receptor in one of two ways: Delta-like or Jagged-like. In response to ligand-receptor binding and forces originating from endocytosis, the Notch receptor undergoes a specific conformational change, releasing the Notch intracellular domain (NICD) into the cytoplasm, leading to lateral inhibition and induction, which regulate collective invasion (170). NICD translocates to the nucleus and forms an active transcriptional complex with the DNA-binding protein, Rbpj, and the coactivator, Mastermind-like (Maml). Moreover, the NICD-Rbpj-Maml complex regulates the transcription of the Notch receptor and its ligands, thereby promoting the transcription of Hey/Hes1, an inhibitor of Delta but an activator of Jagged and Notch target genes (171,172). Therefore, lateral inhibition is observed between follower and leader cells when Notch-Delta signalling is dominant compared with Notch-Jagged signalling, while lateral induction takes place amongst follower cells when Notch-Jagged signalling is dominant (173). Moreover, Notch1 exhibits a higher affinity to Dll4 than to Jag1 following Fringe-mediated glycosylation of Notch1 (174). Based on the results of a study using mathematical modelling of Notch-Delta-Jagged signalling, Fringe proteins glycosylate the Notch receptor, resulting in a conformational change in the extracellular domain (175). Fringe can stabilize leader and follower cells by inhibiting Notch-Jagged binding, whereas its absence may shift the balance towards Notch-Jagged signalling (176).

VEGF stimulates leader cells by binding VEGFR-2 or VEGFR-3 in the microenvironment (177). Leader cells express Dll4 following VEGFR signalling, which activates intercellular Notch signalling to inhibit adjacent follower cells from turning into leader cells by targeting Notch1 (178-180). In follower cells, Notch signalling inhibits VEGFR function. Conversely, leader cells increase VEGFA secretion, which induces follower cell motility and invasion (89). In Drosophila oogenesis, upregulating Rac expression or PDGF/VEGF receptor expression can make follower cells switch positions from posterior to anterior and maintain their position as precursor cells, controlling migration throughout clusters (89). A landscape perspective on how Notch signalling affects leader and follower cell stability and transition may be a useful future direction of study.

Compared with follower cells, leader cells display a variety of mitotic defects, the most prominent of which is cytokinetic instability (11). Follower cells are proliferative and may be able to rescue defective leader cells during collective movement. A study of NSCLC cells isolated using SaGA technology found that gene expression in follower and leader cells was different (11). In follower cells, lysine demethylase 5B (KDM5B) mutations lead to phenotypic heterogeneity, and expression of ARP3 K240R promotes invasion, even in less invasive follower cells, and confers leader cell behaviour (12).

In a previous study, through a SaGA-based capture and amplification procedure, wild-type KDM5B was selectively enriched in leader cells, while mutant KDM5B L685W was selectively enriched in follower cells (12). KDM5B is a lysine demethylase enzyme responsible for catalysing the elimination of di- and trimethylation from histone H3 molecules that have been methylated at K4 (known as H3K4me2 and H3K4me3, respectively). KDM5B, functioning as a transcriptional repressor, promotes the leader cell phenotype by restricting follower cell behaviour (12). The mutation of KDM5B directly impacts invasive behaviour, serving as a unique epigenetic regulator that affects multiple pathways (181,182). Follower cells contain mutations in L685W near the zinc finger structural domain of KDM5B, which is essential for demethylase activity (183). Overexpression of KDM5B L685W, however, may enhance collective migration behaviour by enhancing heterogeneity and resulting in the emergence of cells with follower characteristics (184). Luminal breast cancer cells with upregulated KDM5B expression possess enhanced phenotypic stability, while cells with KDM5B depletion or repression exhibit enhanced transcriptional plasticity and can overcome therapeutic resistance (12).

The K240R mutation in ARP3, an essential component of the ARP2/3 complex, may interfere with its ubiquitylation at K240, thus resulting in either reduced ARP3 turnover or augmented activity (185). This mutation has been associated with enhanced leader cell behaviour, since it amplifies directional cellular protrusion events and accelerates the speed of cell motility, allowing the cell to detect signals that direct migration more easily. Further experiments are necessary to investigate the conjecture that ARP3 K240R is resistant to ubiquitylation, and if so, ascertain its effect on leader cell behaviour. Genetic heterogeneity can be identified in an invasion model using multiple cell lines and cancer types. Collective invasion may be influenced by a combination of multiple genetic and epigenetic changes rather than by isolated changes alone. Finally, identifying these key changes may support clinical judgement by monitoring relevant predictive biomarkers.

Leader and follower cells have been shown to exhibit metabolic heterogeneity (186,187), and these cells alter their metabolic activity to sustain their growth. As described by Warburg (188), cancer cells can reprogram their glucose metabolism so that glycolysis is their primary energy metabolism, even under aerobic conditions. Collective cellular invasion is accompanied by a metabolic shift towards glycolysis (94). Compared with leader cells, follower cells exhibit higher glucose uptake and glycolysis, as well as less dependence on oxidative phosphorylation (OXPHOS) (189,190). Although migrating cell populations do not share the same metabolic pathways, they do maintain a highly coordinated energetic state. Moreover, migrating cells have appropriate mechanisms to adjust their metabolism accordingly.

Glycolysis is sustained by glucose transporter 1 (GLUT1) expression in follower cells, while mitochondrial respiration is sustained by active pyruvate dehydrogenase (PDH) expression in leader cells (184). Specifically, follower cells exhibit a higher level of GLUT1 expression and glucose uptake than leader cells during collective NSCLC invasion in vitro (190). GLUT1 is ubiquitous in all tumour types that have poor patient prognosis (191) and maintains glucose uptake by cancer cells (192-194). Follower cells may divert glucose uptake to the pentose phosphate pathway (PPP), which supports ribulobiogenesis and proliferation without altering citric acid cycle flux. The PPP begins with glucose-6-phosphate dehydrogenase, which is also more highly expressed in follower cells (190). In response to a decrease in glycolytic intermediates entering glycolysis and the PPP, follower cells switch from proliferation to invasion (190). In leader cells, GLUT1 expression is reduced by regulators, such as tumour suppressor p53 and hypoxia-inducible factor-1 (195,196). By contrast, GLUT1 upregulation in leader cells hinders their collective invasive ability (195-198). In follower cells, mitochondria are primarily found around the nucleus, whereas in leader cells, they are more common at the edge of the cytoplasm (190). The presence of mitochondria at the edge of the cytoplasm suggests a higher energy demand in the leading edge, where cellular protrusions and migratory activity occur. As PDH activity increases, mitochondrial distribution to the periphery of the cell increases (190). Since dichloroacetate (DCA) inhibits PDH kinases, DCA-treated follower cells have lower levels of phosphorylated PDH at position S293 and increase invasion, particularly in chains, similar to leader cells (190).

However, Zhang et al (19) found that leader cells rely more on glucose and have higher cellular energy levels than follower cells in breast cancer invasion models, which are used to drive collective invasion, which in turn gradually depletes energy levels. The metabolic profile of invasive cells can be affected by many factors, such as molecular heterogeneity, the microenvironment and the mode of migration (187). For example, it is possible that the metabolic pathway dictated by the density or sparsity of the microenvironment determines whether tumour cells increase glycolysis and decrease mitochondrial oxidative respiration during invasion. As a result of OXPHOS at the invasion front, ATP could be produced more efficiently to meet the energy demands of collective invasion. This process requires sufficient cellular energy levels in leader cells, as opposed to follower cells that produce less energy through glycolysis (199). As soon as a leader cell exhausts its available ATP, it exchanges its position with a follower cell, and the frequency of this exchange increases in denser collagen matrices (19). Hence, cancer invasion is facilitated by metabolic shifts between leader cells and follower cells. Cancer cells are reprogrammed to maintain proliferation and invasion in a dysregulated environment. Whether collective migration is proliferative or invasive is determined by two distinct metabolic preferences, and therefore, two distinct phenotypes should be taken into account to prevent the metabolic plasticity that can drive invading cells: Glycolysis and OXPHOS.

In summary, CIL, biomechanics, remodelling of the ECM, chemotaxis, lateral inhibition and the genetic and metabolic heterogeneity associated with follower cells were discussed in the present review. These mechanisms determine movement polarity by identifying leader and follower cells and guiding cancer cells to acquire follower-like or leader-like morphology, function and behaviour during collective invasion. The follower cells receive signals from the leader cells and the microenvironment due to intracellular and intercellular signalling cascades as well as mechanotransduction. The signals are then transmitted to the entire mass of cells. When collective movements occur, follower cells maintain their phenotype and consolidate the status of the leader cell. Additionally, cell behaviour and fate can be changed by biomechanics and the microenvironment, resulting in follower cells adopting a leadership phenotype, which ultimately leads to genetic changes. In addition, follower cells may take over the positions of leader cells when their energy level drops below a certain level. For cells to move collectively, follower and leader cells must coordinate their movements to be controlled by physical (mechanical) and chemical (signalling) interactions.

However, the factors that drive follower cell formation and the mechanisms that regulate follower cell migration remain unknown. Currently, the impact of the molecular mechanism of cadherin mechanotransduction on the behaviour of leader-follower cells remains mostly unclear. To improve understanding of the interaction between cadherin junctions and cell mechanics, researchers should focus on the interaction between cadherin junctions and Rho-GTPases. In vivo, mechanocoupling is spatially controlled in a number of ways, such as by cell polarity and ECM remodelling. The more complex and physiologically relevant collective migration within 3D matrices is being replicated in a growing number of in silico models. The use of computational models can provide insights into the migration of follower cells and overcome limitations associated with experimental research (200,201). Furthermore, it is unclear how bioenergetic status affects the emergence of follower cells when taking over from failing leader cells. The metabolic, morphological and migration functions of cells are closely intertwined, so targeting cellular metabolism may provide a novel strategy for treating cancer by inhibiting the production of cellular energy. It is necessary to conduct additional experimental research to investigate the function of follower cells in collective movement and to analyse the mechanism of coordinated invasion.