What makes cells move: Requirements and obstacles for leader cells in collective invasion

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What makes cells move: Requirements and obstacles for leader cells in collective invasion Bing-jun Chena,1, Ya-jie Tangb,2, Ya-ling Tangc,*, Xin-hua Lianga,** a

State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Dept. of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, China b Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan 430068, China c State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Dept. of Oral Pathology, West China Hospital of Stomatology, Sichuan University.China

A R T I C LE I N FO

A B S T R A C T

Keywords: Collective invasion Leader cell K14 Δ;Np63α Dll4 Cysteine protease cathepsin B

Most recently, mounting evidence has shown that cancer cells can invade as a cohesive and multicellular group with coordinated movement, which is called collective invasion. In this cohesive cancer cell group, cancer cells at the front of collective invasion are defined as leader cell that are responsible for many aspects of collective invasion, including sensing the microenvironment, determining the invasion direction, modifying the path of invasion and transmitting information to other cells. To fulfill their dispensable roles, leader cells are required to embark on some specific phenotypes with unusual expression of some proteins and it's very important to investigate into these proteins as they may serve as potential therapeutic targets. Here, in this review we will summarize current knowledge on four emerging proteins highly expressed in leader cells including K14, ΔNp63α, Dll4 and cysteine protease cathepsin B (CTSB), with a focus on their important roles in collective invasion and special mechanisms by which they promote collective invasion.

1. Introduction Cancer cells invasion as a cohesive, multicellular group in a coordinated manner is named collective cell invasion, in which the junctions between cancer cells are still retained and cancer cells are adherent to neighbouring cells during invasion [1–5]. Through collective invasion, cancer cells can invade in the same direction at the same speed through the surrounding tissue [1] with cadherin mediating cellcell junctions and with or without epithelial to mesenchymal transition (EMT) [6,7]. Collective invasion is different from single cell invasion including mesenchymal invasion and amoeboid invasion. In mesenchymal invasion cancer cells undergoing EMT can acquire a mesenchymal-like phenotype and constitutively invade with high levels of cell-

matrix adhesion and proteolysis of surrounding tissues while in amoeboid invasion cancer cells can invade into surrounding tissues with their cortical actomyosin contractililty [8,9]. Recently increasing evidence suggests that collective invasion is one of the invaison modes depending on cell-type-specific mechanisms and induction by the microenvironment [10] and plays an important role in the process of carcinogenesis, progression and distant dissemination of various kinds of human carcinoma including breast cancer, lung cancer, rhabdomyosarcoma and oral squamous cell carcinoma [1,3,11]. Cancer cells at the front of collective invasion are defined as leader cells while cancer cells located at the back of the cluster are defined as follower cells, which means that the definition of leader cells and follower cells is only based on their relative positions within the

* Corresponding author. Institutional address: Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology (Sichuan University), No.14, Sec.3, Renminnan Road, Chengdu, Sichuan, 610041, China. ** Corresponding author. Institutional address: Department of Oral Pathology, West China Hospital of Stomatology (Sichuan University), No.14, Sec.3, Renminnan Road, Chengdu, Sichuan, 610041, China. E-mail addresses: [email protected] (B.-j. Chen), [email protected] (Y.-j. Tang), [email protected] (Y.-l. Tang), [email protected] (X.-h. Liang). 1 Institutional address: West China School of Stomatology (Sichuan University), No.14, Sec.3, Renminnan Road, Chengdu Sichuan 610041, People's Republic of China. 2 Institutional address: Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan 430068, China.

https://doi.org/10.1016/j.yexcr.2019.06.026 Received 24 January 2019; Received in revised form 15 June 2019; Accepted 23 June 2019 0014-4827/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Bing-jun Chen, et al., Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2019.06.026

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including K14 [25,26]. These indicated that K14 + cancer cells have invasive capability and K14 overexpression is strongly associated with poor prognosis. Recently, it was observed that leader cells at tumor-stromal interface strongly expressed multiple basal epithelial markers among which K14 was the most frequently expressed. K14 + cancer cells led the majority of collective invasion in the form of invasive strands and tumor nests in both primary tumors and lung metastases. After knockdown of K14 in tumor organoids, there were large regions with borders displaying a rounded morphology and lack of collective invasive units and there was no association between fibrillar collagen and leader cells [7]. In support of these observations, polyoma virus middle T oncogene (MMTV-PyMT) mouse models of breast cancer [27] reveal that luminal breast cancer cells expressing K14 could become leader cells to initiate collective invasion and K14 + cells were enriched through major stages of metastasis including collective invasion, locally dissemination, circulating tumor cell (CTC) clusters and micrometastases. Consistent with mouse models, K14 + cancer cells in primary human breast tumors were also enriched in collective invasion fronts and generally, higher K14 staining intensity was correlated with higher histologic grade [7]. Within salivary adenoid cystic carcinoma (SACC) patients, the expression of K14 in invasive fronts also turned out to be significantly associated with perineural invasion, local regional recurrence and distant metastasis and the prognosis of K14 + cases was worse than that of K14-cases [28]. This suggested that K14 is necessary for the formation of multicellular invasive strands, cell-cell adhesion and cell-matrix adhesion and thus is important in persistent collective invasion. K14 mediating the mechanistic functions of leader cells is a significantly important mechanism to regulate collective invasion, including regulating protrusion formation, stabilizing desmosomes and hemidesmosomes, promoting cell-matrix and cell-cell adhesion junctions and maintaining cancer cells stiffness and stability (Fig. 1).It has been demonstrated that keratin intermediate filaments can influence the directed protrusive formation of leader cells within the collectively migrating cell clusters by mechanically responsive linkage with cadherins [29], suggesting the functional significance of keratin-associated cadherin adhesions in collective migration. Genetic loss-of-function studies in physiological mouse models of cancers have suggested loss of desmosome proteins and desmosome-mediated adhesion is associated with cancer development and progression [30]. There is evidence showing that K14 can help to stabilize desmosomes by sequestering PKC-α in the cytoplasm and lack of keratins can lead to destabilization of desmosomes through PKC-α mediated desmoplakin phosphorylation and re-expression of the keratin pair K5/14 reconstitutes both desmosome localization at the plasma membrane and epithelial adhesion [31,32]. In addition, by integrating the transcriptome data for K14 knockdown cancer cells with RNA-seq enrichment data for K14 + cancer cells, it has been demonstrated that Dsg3, which encodes the major desmoglein in desmosomes, was significantly enriched in K14 + cells and positively regulated by Krt14 transcript levels. And the absence of keratins also leads to the loss of plectin in the hemidesmosomal plaque, scattering the hemidesmosome transmembrane core along the basement membrane zone, reducing cell-matrix adhesion and further study suggests that keratins can stabilize hemidesmosomes through regulating plectin-β4-integrin interaction and β4-integrin phosphorylation [33,34]. Similarly, as for breast cancer cells collective invasion, DAVID gene ontology (GO) analysis for K14 + cancer cells also indicated increased expression of both cell-cell and cell-matrix adhesion genes in K14 + cells, including MPDZ, TNC, HSPG2, ITGA2, POSTN, CXADR, LAMA3, CD44, COL7A1, PKP1, DSG3, CTGF, FAT2, DSC3, COL12A1, TNN, ANTXR1, DST, ABL2 [35]. Keratin filaments are also the main component of mechanical resilience of keratinocytes and a highly significant softening is found in keratin-deficient keratinocytes [36]. And keratin-free cells show higher cell deformability [37]. These findings suggest a unique role of keratins as a major player of cell

multicellular group. Leader cells can sense the microenvironment by external cues generated by extracelluar matrix (ECM), soluble chemotactic factors and neighbouring cells, determine the direction, modify and enlarge the path of invasion by affecting the shape of the matrix fibres and proteolysis of ECM and transmit information to follower cells by mature focal adhesions and longitudinal acto-myosin cables so that follower cells can move behind leader cells in the same direction [5]. Additionally, leader cells are polarized along the direction of migration and show an elongated morphology with dynamic actin-based protrusive structures including finger-like filopodia and ruffling lamellipodia [12–14]. Apart from moving behind leader cells and forming the majority of the multicellular cluster, follower cells are also responsible for the polarization of leader cells and maintaining their leadership through contact inhibition of locomotion. To fulfill their dispensable roles in collective invasion, the leader cells needs to embark on some specific phenotypes with some proteins highly expressed only in leader cells. And these proteins are involved in many important aspects of collective invasion performed by leader cells such as mediating cell-cell adhesion, promoting partial EMT, restricting leadership to leader cells, and performing proteolysis of surrounding tissues. We hypothesize that investigating these proteins expressed in leader cells will not only help to distinguish leader cells from other cells, but also provide significant insight into elucidating the nature of collective invasion, exploring mechanisms initiating collective invasion and prominent signaling pathway involved in collective invasion and seeking possible therapeutic targets. Recently the research about collective invasion is mainly focused on elucidating the existence of collective invasion and its important role in various kinds of human carcinoma but the research about distinctive molecular mechanisms of leader cells and how these mechanisms promote the distinctive phenotypes of leader cells is still lacking. Here, we briefly review several proteins highly expressed in leader cells including K14, ΔNp63α, Dll4 and cysteine protease cathepsin B (CTSB), especially focused on their abnormal expression and important roles in collective invasion and special mechanisms by which they promote collective invasion. 1.1. K14 mediating cell-cell adhesion When collectively invading into surrounding tissues, the cohesive cancer cells cluster can be formed and maintained by individual cells adhering to their neighbouring cells by cell-cell junctions, which are mechanically both flexible and stable to secure cancer cells’ position and function. During collective invasion, cancer cells can apply a range of cell-cell junction mechanisms including adherens junction orchestrated by both E-cadherin and N-cadherin, labile or transient cell-cell interactions mediated by Ig super family members and ephrins/EpH receptor systems and gap junction through which connexins may enable communication between cancer cells [6,15–19]. However, the molecular mechanism of cell-cell junctions in collective invasion remains still unclear and recent evidence of K14 promoting collective invasion by regulating cell-cell adhesion [7] may provide new cues to elucidate junction nature. Cytokeratin-14 (CK14), also known as keratin 14 (K14), is a member of the type I keratin family of intermediate filament proteins and is usually expressed in basal epithelial cells and usually found as a heterodimer with type II keratin 5. In various kinds of tumors, such as lung squamous cell carcinoma [20] and transitional cell carcinoma [21], the appearance and elevated expression of K14 was strongly associated with higher grade and stage of carcinoma, also indicating different degrees of unfavourable prognosis [21,22]. Just like highly migratory normal mammary embryonic cells expressing K14 [23], the highly invasive breast cancer cell lines such as MDA-MB-468, MDA-MB-436, and BPLER are also K14+ [24]. Experimental models have proposed that mutations in several genetic drivers of breast cancer, such as BRCA1, can alter luminal progenitors into basal subtypes resulting in transformed phenotype with cancer cells expressing basal cytokeratins 2

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Fig.1. K14 mediating the mechanistic function of leader cells：K14 is involved in mediating the mechanistic function of leader cells, which is a significantly important mechanism to regulate collective invasion. 1) K14 can influence the directed protrusion formation of leader cells by mechanically responsive linkage with cadherins. 2) K14 can also help to stabilize desmosomes by sequestering PKC-α in the cytoplasm. 3) K14 can stabilize hemidesmosomes through regulating plectin-β4-integrin interaction and β4-integrin phosphorylation. 4) K14 can promote cell-cell and cell-matrix adhesion junctions by promoting related genens expression.5) K14 also plays a unique role as a major player to maintain cell stiffness to influence cancer invasion.

collagen I fiber density in extracellular matrix relative to noninvasive regions, which means K14 + cells could obtain leader cell behaviors when cultured in collagen-I-rich local microenvironment [7].

stiffness to influence cancer invasion, by which alterations in K14 may compromise leader cells function and collective invasion. Challenging the single-metastasis concept, it has been reported that breast cancer cells can disseminate collectively into the surrounding ECM [3,38,39]and tumor cell clusters exist in circulation [40], suggesting when initiating and completing the metastatic process, collectively invading cancer cells could cooperate as a cohesive multicellular unit. Using ROSAmT/mG; MMTV-PyMT, a mammary tumor model, it was observed that cancer cells expressing K14 induced local dissemination, intravasation and distal seeding as a multicellular cluster to form breast cancer metastases. In locally disseminated clusters, CTC clusters and micrometastases, K14 + cells accounted for 59%, 94%, > 50% respectively [35]. Furthermore, K14 + cancer cells have been shown to be important for distinct steps in metastasis such as the formation of metastatic niche, vascular remodeling, and immunosurveillance. K14 knockdown tumors in host mice had a seven-fold reduction in the mean number of metastases and the expression of 14 genes with two-fold or greater significant enrichment was also strongly associated with Krt14 transcript levels. Among them, there were 9 genes that were previously reported in metastasis regulation including Adam Ts1, Tnc, Jag1, Cav1, Card10, Ereg, Lgr5, Slpi and Ptgs2 [41–45]. Furthermore, K14 knockdown could alter expression of 1584 genes, among which, Tnc, Adam Ts1, Jag1 and Birc5 can elevate metastatic niche remodeling and metastasis survival [46–48]. GO analysis showed that K14 + cells were also enriched for transcripts encoding TNC, POSTN and CTGF, proteins that are required for the metastatic niche [46,49] and were depleted for a number of genes associated with the regulation of the immune system including immune response, MHC class II antigen presentation and Tcell activation [35]. However it needs further investigation to find out the underlying signaling pathway between K14 and these dysregulated genes, which may help to illuminate how K14 regulates collective cell invasion. K14 + phenotype is a differentiation state exhibited by leader cells and is influenced by the characteristics of the microenvironment. Individual cells at the basal surface could directly convert from K14- to K14 + states and induction of this basal epithelial program could occur in diverse microenvironment. However, the conversion of latent invasive potential into actual invasive leader cell behaviors needs specific microenvironment. In vivo, K14 + cancer cells in invasive regions with protrusive morphology were associated with an about 8-fold increase in

1.2. ΔNp63α promoting partial EMT Together with K14, p63 was proved to be expressed by leader cells at the front of collective invasion in breast cancers and plays an essential part for leader cells to lead collective invasion despite being expressed in a small minority of the tumor cells. In 3D culture of breast cancer cells, knocking down p63 could lead to a decrease in the number of protrusive cancer cells and result in rounded cell borders and thus an obvious impairment in collective invasion. By comparison with K14knock down cancer cell organoids, knocking down p63 resulted in fewer protrusive cancer cells and more rounded cell borders, suggesting p63 has K14-independent functions in collective invasion [7]. In vitro, both immunofluorescence and Western blots showed SACR2 cells, a kind of head and neck squamous carcinoma cells, were positive for p63 and actively migrating leader cells were detected to protrude into the surrounding microenvironment and were followed by other cancer cells, eliciting a strong impression of collective invasion [50]. But the precise mechanism of p63 promoting collective invasion still remains unclear and warrants further investigation. Recently, ΔNp63α, one of six isoforms encoded by TP63 gene [51], has been found to promote collective invasion by inducing partial EMT in leader cells, which may shed light on why knocking down p63 can compromise collective invasion. The epithelial–mesenchymal transition (EMT) has been shown to be a process by which epithelial cells lose their cell polarity and cell-cell junctions to gain migratory and invasive properties and facilitate cancer cells detachment, invasion into surrounding tissues and dissemination, usually accompanying the expression of EMT-inducing transcription factors (EMT-TFs) including TWIST, SNAIL, SLUG and ZEB1 [52–54]. Actually, EMT is not an all-or-none response, but a rather multi-state process, resulting in intermediate phenotypes ranging from purely epithelial phenotype to purely mesenchymal phenotype. Colocalization of distinct markers of both epithelial and messenchymal phenotypes defines a hybrid epithelial/messenchymal phenotype of EMT, indicating cells that have passed only partly through EMT [55]. Unlike single cell invasion after a full EMT, collective invasion involves a 3

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Fig.2. ΔNp63α can induce partial EMT in collective invasion:a| EMT is not an all-or-none response, but a rather multi-state process, resulting in intermediate phenotypes ranging from purely epithelial phenotype to purely mesenchymal phenotype. Through partial EMT, leader cells can retain some epithelial characteristics and remain attached to their neighbours and simultaneously undergo a differentiation toward embryonic features with elevated mobility, invasion and metastasis properties. The epithelial and mesenchymal cell markers commonly used by EMT researchers are listed. b| ΔNp63α could increase Slug and Axl expression selectively to promote EMT and change cancer cells into mesenchymal-like phenotype with elongated morphology, protrusive structures and elevated motility. And the parallel activation of miR-205 by ΔNp63α could silence ZEB1 and ZEB2 to prevent further EMT and retain cell-cell adhesions. As a result, a partial EMT programme is induced in cancer cells and confer cancer cells with a hybrid epithelial/messenchymal phenotype.

moving cancer cells have lower membranous localization of E-cadherin and higher vimentin cytoplasmic levels [68,69]. In metastatic breast and prostate cancer and melanoma, circulating tumor cells in patients’ blood moving collectively as clusters have a hybrid mesenchymal/ epithelial phenotype and display both adhesion (an epithelial trait) and migration (a mesenchymal trait) [70]. Furthermore, a study conducted on various tumor types, including pancreatic ductal adenocarcinoma (PDAC), lung adenocarcinoma and invasive breast ductal cancer, suggests single-cell migration is very rare and cancer cell invasion relies mostly on collective cell invasion [6], which in turn suggests that epithelial cell–cell adhesions persist. Hence, these findings imply that not all the cells become purely mesenchymal, which is indicative of partial EMT. The expression of EMT-related markers further support the correlation between collective invasion and partial EMT. In BLBC, ΔNp63α can increase Slug and Axl expression and silence ZEB1 and ZEB2 at the same time, resulting in partial EMT and conferring cancer cells with a hybrid epithelial/mesenchymal state [59]. In breast cancer, elevated interstitial fluid pressure (IFP) can increase the expression of Snail, vimentin and E-cadherin to promote collective invasion [71]. In lung cancer, collectively moving cancer cells display decreased levels of membranous β-catenin [72], while in colorectal cancer and oral squamous cell carcinomas cancer cells display reduced levels of miR-200 family, which is the inhibitor of ZEB1 and ZEB2 [73]. On the other hand, there is also evidence showing that no EMT programme is involved in collective invasion. Cheung and colleagues found an epithelial program was maintained in leader cells and they did not typically express Twist, Slug, or vimentin [7]. Preventing Twist1-induced EMT by inhibiting TGFβ-signaling can switch invasion mode from single cell invasion to collective invasion in immortalized human mammary epithelial cells (HMLE) [74]. Interestingly, as was found in some studies, EMT occurring in carcinoma associated fibroblasts (CAFs) not in leader cells themselves can promote collective invasion. Cancer cells that have not undergone EMT can use the mesenchymal characteristics of CAFs to remodel the matrix and make a path that cancer cells can use to collectively invade [75]. In lung adenocarcinoma, vimentin has been proved to be expressed in CAFs and maintain heterotypic cancer cellCAF interactions to promote collective invasion of cancer cells that are vimentin-negative and E-cadherin-positive, indicating a lack of cancer cell EMT [76]. It needs further investigation to illuminate under what circumstances EMT is a prerequisite for collective invasion and the signaling text in cancer cells and the microenvironment especially CAFs may play an important role.

cohesive, multicellular cancer cell group, in which leader cells still maintain E-cadherin-based junctions and have elongated morphology with protrusive structures at the same time [12–14,56]. This phenotypic state of leader cells is a hybrid mesenchymal/epithelial state, indicating a partial EMT programme induced in leader cells [57]. During this process, moving tumor cell clusters may still maintain cell-cell contacts and simultaneously undergo a differentiation toward embryonic features with elevated mobility and invasion properties [58]. Recently, it has been reported that ΔNp63α can induce collective invasion by partial EMT in basal-like breast cancer(BLBC), in which it can selectively activate several EMT-TFs and in parallel keep epithelial characters [59](Fig. 2). Investigation into MCFDCIS cells (a BLBC population) transfected with 879 miRNA mimics demonstrated that ΔNp63α could increase Slug and Axl expression selectively to change cancer cells into mesenchymal-like phenotype with elevated motility and the parallel activation of miR-205 by ΔNp63α could silence ZEB1 and ZEB2 to prevent further EMT and retain cell-cell adhesions, conferring BLBC cells at the invasive front with a hybrid epithelial/mesenchymal state to initiate collective invasion [59]. When initiating collective invasion, highly motile basal-like breast cancer cells can still maintain epithelial traits such as expressing E-cadherin and K14 [7,60] while expressing a subset of mesenchymal genes whose expression is amplified during induction of EMT [61–63], which indicates BLBC cells are conferred with partial EMT. Gene expression analysis, ChIP-seq and functional analysis of BLBC cells proposed that apart from Slug and Axl, ΔNp63α can also selectively up-regulate the expression of a cohort of genes including FAT2, SNCA, CA12, CPNE8, NEK1 to promote cancer cells motility [64]. In explants derived from a mouse model of breast cancer, ΔNp63α is proved to be necessary for cellular protrusion formation [7]. ΔNp63α dependent induction of FAT2 and Slug can reduce E-cadherin localized to cell-cell adhesions and induce the formation of F-actin containing cellular protrusions extending into the ECM in leader cells and thus to initiate collective invasion [64]. Apart from breast cancer, ΔNp63α induced coexpression of FAT2 and Slug can also promote the mobility of diverse tumor cells including lung squamous cell carcinoma, bladder cancer and prostate adenocarcinoma [65–67]. And elevated expression of FAT2 and Slug induced by ΔNp63α is correlated with poor clinical outcome in HER2-/ER-breast cancer patients and non-small cell lung cancer patients [64]. However, it is still not very clear whether collective invasion requires EMT. On the one hand, studies show that EMT is involved in collective invasion and the specific hybrid mesenchymal/epithelial state of cancer cells indicates the existence of partial EMT. In invasive ductal breast cancer and tongue squamous cell carcinoma, collectively 4

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1.3. Dll4 restricting leadership to leader cells It is obvious that leader cells maintaining and restricting leadership to themselves is significant for controlling the number of leader cells and thus promoting efficient communication between leader cells when discussing invasion direction. Recently, it has been reported that Dll4 is involved in restricting leadership to leader cells through Notch signaling pathway both in collective migration and collective invasion. As one of Notch ligands, Dll4 can interact with Notch receptor and the interaction of Dll4 and its receptors between physically adjacent cells can induce the Notch signaling pathway, which is important for cell-cell communication [77,78] and plays a major role in the regulation of development in many organ systems such as cardiovascular system [79] where the activation of Notch signaling pathway can induce cells to initiate collective migration. In sprouting angiogenesis, vascular endothelial cells (VECs) could move backwards and forwards showing a unique mode of collective cell movement that turned out to be regulated by interaction between adjacent VECs via Dll4-Notch-1 signaling pathway [80,81]. This coordinated specification, proliferation and collective migration of leading and following VECs can drive nascent blood vessel formation [82–84] and investigation into tumor angiogenesis shows that there is a VEGFR–Notch feedback network to influence leader cell selection and collective migration in tumor angiogenesis [85,86]. Gradients of vascular endothelial growth factor (VEGF) in microenvironment can activate vascular endothelial growth factor recptor-2 (VEGFR-2) or VEGFR-3 of leader cells to induce leader cell formation and directed migration [87]. Moreover, VEGFR signaling pathway can up-regulate the expression of Dll4 in leader cells, which then targets Notch-1 on adjacent follower cells [81] to stimulate intracellular Notch signaling pathway in follower cells then to downregulate VEGFR function in follower cells and prevent them from becoming leader cells (Fig. 3) [81,88–90]. This activation of Notch signaling pathway between leader cells and follower cells can restrict leadership to leader cells, which is important for direction decision in collective migration. Inhibition of Notch signaling pathway by blockade of the expression of Dll4 resulted in notably increased tumor vascularity with enhanced angiogenic sprouting and branching, which paradoxically, led to poor perfusion, increased hypoxia and decreased tumor growth in murine tumor models [85]. Similarly, blocking NotchDll4 signaling with a Dll4-selective antibody could confer VECs with hyperproliferative state, cause defective cell fate specification or differentiation, result in increased non-productive vascularity and thus deregulate tumor growth [88]. Just like collective migration, using a double-stranded locked nucleic acid (dsLNA) biosensor capable to investigate gene expression in breast cancer cell spheroids during 3D collective invasion, leader cells are observed to display higher expression of Dll4 while follower cells are observed to display higher expression of Notch-1. Dll4 expression in leader cells was elevated during spheroids sprouting, extending into the surrounding collagen, deforming the collagen fibers and then initiating subsequent collective invasion. Exogenous JAG1 could activate Notch signaling pathway and decrease the number of sprouts per spheroid, suggesting Dll4-Notch-1 signaling pathway could impair leader cell formation to regulate collective invasion [91] just like the suppressive role of Notch signaling in mediating collective migration of VECs and human mammary epithelial cells [81,88–90]. In the investigation of non-small cell lung cancer tumor spheroids, a vascular mimicry displayed by leader cells and follower cells was observed during collective tumor cell invasion, which shows VEGFR was up-regulated in leader cells with VEGF signaling significantly increased in leader cells and VEGF signaling could impact leader cells promoting follower cells motility and collective invasion. Additionally, just like tumor angiogenesis Notch-1 was up-regulated in follower cells whereas Dll4 was upregulated in leader cells. However, inhibition of Notch1 did not promote leader cell formation but blocked initiation of collective invasion instead [92], which is contrary to the suppressive role of Notch

Fig.3. Dll4-Notch-1 signaling pathway can influence leader cells selection and collective migration. 1) VEGF activate VEGFR-2 or VEGFR-3 of leader cells to induce leader cell formation and directed migration. 2) VEGF signaling also increases expression of Dll4 in leader cells. 3) Dll4 then targets Notch-1 on adjacent follower cells to activate Notch signaling pathway. 4) After Notch signaling pathway activation, Notch intracellular domain (NICD) is released from the cell membrane by γ-secretase complex. 5) NICD translocates into the nucleus and then binds to CBF1/Suppressor of Hairless/Lag-1 (CSL) to form Notch/CSL coactivator complexes. 6) The complexes then upregulate Hairy and Enhancer of Split (HES) proteins that can finally suppress the expression of VEGFR2 in follower cells.

signaling pathway of regulating collective migration in angiogenesis [81,88–90] and breast cancer [91] suggesting a non-canonical vascular signaling program in non-small cell lung cancer. We hypothesize that the specific function of Dll4-Notch-1 signaling pathway is dependant on the cancer cell type, signaling text in cancer cells and the microenvironment of different kinds of cancer. There also exists a complex negative feedback loop between Notch1 and ΔNp63α. ΔNp63α expression can be suppressed by Notch1 through down-regulation of selected interferon-responsive genes, including IRF7 and/or IRF3. And in turn, ΔNp63α can function as a modulator of Notch1-dependent transcription and function, with Hes-1 as one of its direct targets [93]. In consideration of the relationship between ΔNp63α and Notch1, investigating the underlying crosstalk between ΔNp63α and Dll4-Notch1 signaling pathway may help to enrich their roles in regulating collective invasion.

1.4. CTSB performing proteolysis of surrounding tissues As leader cells move through the microenvironment, they modify and enlarge the path of invasion, during which traction force exerted on the ECM through focal adhesions can affect the shape of the matrix fibres and then multiple sets of proteolytic enzyme secreted by the leader cells can cut and remodel ECM fibres to facilitate collective invasion [5]. The proteolysis of surrounding tissues performed by leader cells mediates both physical and chemical modification of the ECM, including generating invasion tracks that exert the least resistance to 5

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surrounding matrix with 3D proteolysis imaging confirming that CTSB overexpression in leader cells promoted matrix proteolysis(Fig. 4). When treated with doxycycline, CTSB expression in cancer cells was amplified and an increase of mean sprout length and number of sprouts per spheroid was detected at the same time. However, when treated with doxycycline, CTSB expression in macrophages was also amplified but it had no effect on tumor spheroid sprouting [115]. It is confirmed that CTSB plays an indispensable role in proteolytic network of cancer cells that consists of urokinase-type plasminogen activator (uPA) and several matrix metalloproteinases [104]. And in PyMT mouse model of breast cancer, CTSB overexpression could change transcription of many ECM-modifying enzymes such as lysyl oxidase whose activity was higher upon CTSB overexpression [115]. Among these ECM-modifying enzymes, many proteases play an essential role in collective invasion, such as MMP-14 (MT1-MMP) and MMP2. MMP-14 can mediate collagenolysis and selectively realign sterically impeding fibers into microtracks to initiate collective invasion [116]. In neuroblastoma tumors, MMP-2 is important for substrate type cells to insert into layers of neuronal type cells and initiate collective invasion and using specific MMP2 inhibitor could significantly reduce the invasiveness [117]. Through the proteolytic network, CTSB can indirectly increase the activity of MMP-14 and MMP-2 by inactivating related endogenous inhibitors, which indicates that CTSB may have a more complicated role in collective invasion due to its modulatory role in the proteolytic network of cancer cells. CTSB can also activate or inactivate some important signaling pathways and kinase cascades that turn out to be involved in collective invasion. In collective invasion, activation of focal adhesion kinase (FAK) can promote the extension of membrane protrusion of leader cells at tumor-stromal interface, thus promoting cancer cells to adhere to ECM and participating in proteolysis of ECM [5]. In meningiomas, it was revealed that down regulation of CTSB can inhibit FAK expression both in vitro and in vivo leading to less invasiveness of cancer cells and this effect can be reversed by overexpression of CTSB [118]. Taken together, CTSB may have an influence on initiating collective invasion through modulating cell-matrix adhesions and proteolysis of ECM by regulating the expression of FAK. Furthermore, apart from invasion-promoting role of extracellular CTSB, lysosomal CTSB could exert anti-tumor effects as it is widely accepted that CTSB can lead to cell death by lysosomal membrane permeabilization [119] and emerging evidence suggests that lysosomal membrane permeabilization and release of CTSB into the cytoplasm could be a potential therapeutic approach [120]. This adverse effect of CTSB indicates that distribution of CTSB can have completely different impact on cancer invasion and may confer CTSB with more complicated roles in collective invasion.

invasion and changing the composition of the matrix by cleaving CD44, growth factors, ECM structural proteins and cell-surface integrins to create a chemotactic gradient through the cell group and change the nature of the engaged integrins, which has an impact on the invasive behaviour of the followers cells, increasing the polarized organization of the cell group [5,94–96]. Recently, it has been reported that cysteine protease cathepsin B (CTSB) is involved in the proteolysis of surrounding tissues during collective invasion. Apart from its proteolytic function, CTSB can also regulate the proteolytic network consisting of urokinase-type plasminogen activator (uPA) and matrix metalloproteinases and it can mediate some important signaling pathways and kinase cascades involved in collective invasion, indicating CTSB may play a more complicated role in collective invasion. As a member of lysosomal cysteine proteases family, cysteine protease cathepsin B (CTSB) [97] in human is encoded by the CTSB gene and is composed of a heavy chain of 25–26 kDa and a light chain of 5 kDa, which are linked by a dimer of disulfide [88,89]. In addition to its important role in intracellular proteolysis [98], CTSB also has a pivotal role in the proteolysis of extracellular matrix (ECM), intercellular communication disruption and decreasing protease inhibitor expression to enhance the activity of corresponding proteases, including matrix metalloproteinases (MMP) and urokinase [98,99]. CTSB can be expressed in many kinds of cells at a normal level whereas overexpression of CTSB in both genomic and proteomic leavels is related to invasion and metastasis of several cancers. CTSB overexpression was first observed in human breast cancer [100] and since then abnormal activity and distribution of CTSB and related endogenous inhibitors has been detected in both mesenchymal and epithelial cancers such as melanoma, bladder, brain, breast, colorectal, gastric, lung and prostate cancers [100–105]. CTSB is important for collective invasion as a key role to break down ECM components and regulate proteolytic network and signaling pathways. Tumor-associated macrophages localized at the invasive front that comigrate with cancer cells [106,107] could transform to the M2-like phenotype [108] and promote tumor progression by secreting chemokines and breaking down ECM [109]. CTSB overexpression has been observed in tumor-associated macrophages to support their proneoplastic phenotype [110]. CTSB overexpression is also detected in cancer cells and is distributed to invadosomes, caveolae, and other plasma membrane at specific microdomains and cleave surrounding ECM components including collagen I, collagen IV, fibronectin and laminin，accelerating ECM remodeling and promoting cancer cells to invade into surrounding tissues and the vasculature [111–113]. Interestingly, in contrast to the widely accepted opinion that collective invasion can be mediated by interactions between stromal cells and cancer cells [114], a mouse model of breast cancer with forced overexpression of CTSB shows that it was actually overexpression of CTSB in leader cells not in macrophages that enhanced collective invasion, suggesting CTSB-mediated collective invasion may be a mode of action induced by a direct cancer cell-inherent ability. Cancer cell spheroids in that model can develop multicellular strands permeating into the

2. Conclusions Taken together, during collective invasion leader cells will embark on distinct phenotypes with some special proteins highly expressed that Fig.4. CTSB-mediated collagenolysis promotes collective invasion. CTSB is distributed to invadosomes, caveolae, and other plasma membrane of leader cells. CTSB performs proteolysis of surrounding collagen fibers and paves a path of the least mechanical resistance. The path generated by an individual leader cell are used by follower cells to form a collective invasive strand.

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are involved in many aspects such as mediating cell-cell junctions, initiating partial EMT, regulating leader cells formation and performing proteolysis of surrounding tissues. It seems that Dll4-Notch-1 pathway can play completely opposite roles in regulating collective invasion in different tumors. Is this the inherent feature of Dll4-Notch-1 pathway or the differential influence of microenvironment on tumor cells? With regard to EMT, is EMT necessary for cancer cells to initiate collective invasion? Whether undergoing partial EMT or full EMT is determined by cell-intrinsic mechanisms or the microenvironment? Further investigations are required to elucidate these events. Despite accounting for a small number of cancer cells, leader cells are always indispensable in promoting collective invasion. However, researches linking single cells or clonal phenotypes with genomic, transcriptomic and epigenomic data have been limited due to experimental challenges and phenotypic plasticity of cancer cells. An emerging technique termed spatiotemporal genomic and cellular analysis (SaGA) can isolate and amplify phenotypically distinct subpopulations from a large and heterogeneous population [92]. We expect that this emerging technique will drive some new insights into the special proteins expressed in leader cells or follower cells and help to elucidate their specific functions in collective invasion and perhaps seek therapeutic approaches of interfering the expression of these proteins.

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[8] [9] [10] [11]

[12] [13] [14]

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Declarations Ethics approval and consent to participate: Not applicable.

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Consent for publication [20]

Not applicable. Availability of data and material

[21]

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

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Conflicts of interest [23]

The authors declare that they have no competing interests.

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Funding This work was supported by National Natural Science Foundation of China grants (Nos. 81672672, 81772891, 81572650 and 81621062), and by National Program on Key Research Project of China (2016YFC0902700).

[25]

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Authors' contributions [27]

BJC wrote the paper; YJT revised the manuscript; YLT and XHL revised and determined the final version. All authors read and approved the final manuscript.

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