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Metastasis is the leading cause of cancer-related mortality; however, a complete understanding of the molecular programs driving the metastatic cascade is lacking. Metastasis is dependent on collective invasion—a developmental process exploited by many epithelial cancers to establish secondary tumours and promote widespread disease. The key drivers of collective invasion are "Leader Cells", a functionally distinct subpopulation of cells that direct migration, cellular contractility, and lead trailing or follower cells. While a significant body of research has focused on leader cell biology in the traditional context of collective invasion, the influence of metastasis-promoting leader cells is an emerging area of study. This review provides insights into the expanded role of leader cells, detailing emerging evidence on the hybrid epithelial–mesenchymal transition (EMT) state and the phenotypical plasticity exhibited by leader cells. Additionally, we explore the role of leader cells in chemotherapeutic resistance and immune evasion, highlighting their potential as effective and diverse targets for novel cancer therapies. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) remains a challenge in Neuro‐oncology, with a poor prognosis showing only a 5% survival rate beyond two years. This is primarily due to its aggressiveness and intra‐tumoral heterogeneity, which limits complete surgical resection and reduces the efficacy of existing treatments. The existence of oncostreams—neuropathological structures comprising aligned spindle‐like cells from both tumor and non‐tumor origins‐ is discovered earlier. Oncostreams are closely linked to glioma aggressiveness and facilitate the spread into adjacent healthy brain tissue. A unique molecular signature intrinsic to oncostreams, with overexpression of key genes (i.e., COL1A1, ACTA2) that drive the tumor's mesenchymal transition and malignancy is also identified. Pre‐clinical studies on genetically engineered mouse models demonstrated that COL1A1 inhibition disrupts oncostreams, modifies TME, reduces mesenchymal gene expression, and extends survival. An in vitro model using GFP+ NPA cells to investigate how various treatments affect oncostream dynamics is developed. Analysis showed that factors such as cell density, morphology, neurotransmitter agonists, calcium chelators, and cytoskeleton‐targeting drugs influence oncostream formation. This data illuminate the patterns of glioma migration and suggest anti‐invasion strategies that can improve GBM patient outcomes when combined with traditional therapies. This work highlights the potential of targeting oncostreams to control glioma invasion and enhance treatment efficacy. [ABSTRACT FROM AUTHOR]

Abstract Glioblastoma (GBM) remains a challenge in Neuro‐oncology, with a poor prognosis showing only a 5% survival rate beyond two years. This is primarily due to its aggressiveness and intra‐tumoral heterogeneity, which limits complete surgical resection and reduces the efficacy of existing treatments. The existence of oncostreams—neuropathological structures comprising aligned spindle‐like cells from both tumor and non‐tumor origins‐ is discovered earlier. Oncostreams are closely linked to glioma aggressiveness and facilitate the spread into adjacent healthy brain tissue. A unique molecular signature intrinsic to oncostreams, with overexpression of key genes (i.e., COL1A1, ACTA2) that drive the tumor's mesenchymal transition and malignancy is also identified. Pre‐clinical studies on genetically engineered mouse models demonstrated that COL1A1 inhibition disrupts oncostreams, modifies TME, reduces mesenchymal gene expression, and extends survival. An in vitro model using GFP+ NPA cells to investigate how various treatments affect oncostream dynamics is developed. Analysis showed that factors such as cell density, morphology, neurotransmitter agonists, calcium chelators, and cytoskeleton‐targeting drugs influence oncostream formation. This data illuminate the patterns of glioma migration and suggest anti‐invasion strategies that can improve GBM patient outcomes when combined with traditional therapies. This work highlights the potential of targeting oncostreams to control glioma invasion and enhance treatment efficacy.

Metastasis is the leading cause of cancer-related mortality; however, a complete understanding of the molecular programs driving the metastatic cascade is lacking. Metastasis is dependent on collective invasion—a developmental process exploited by many epithelial cancers to establish secondary tumours and promote widespread disease. The key drivers of collective invasion are “Leader Cells”, a functionally distinct subpopulation of cells that direct migration, cellular contractility, and lead trailing or follower cells. While a significant body of research has focused on leader cell biology in the traditional context of collective invasion, the influence of metastasis-promoting leader cells is an emerging area of study. This review provides insights into the expanded role of leader cells, detailing emerging evidence on the hybrid epithelial–mesenchymal transition (EMT) state and the phenotypical plasticity exhibited by leader cells. Additionally, we explore the role of leader cells in chemotherapeutic resistance and immune evasion, highlighting their potential as effective and diverse targets for novel cancer therapies.

• Periostin expression in CAFs is regulated by YAP activation in breast tumor. • Periostin is required for age-related mammary gland stiffening and tumour growth. • Periostin deficiency impairs matrix stiffening-triggered breast tumor cell collective invasion. • Periostin-mediated outside-in mechanotransduction is required for breast tumor cell collective invasion. Matrix rigidity is a critical contributor to tumor progression; however, whether and how matrix stiffness modulates the collective invasion of tumor cells remain unknown. Here we demonstrate that increased matrix stiffness activates YAP to promote the secretion of periostin (POSTN) in cancer-associated fibroblasts, which in turn augments the matrix rigidity of mammary glands and breast tumor tissues by facilitating collagen crosslinking. Moreover, decreased tissue stiffening resulted from the POSTN deficiency impairs peritoneal metastatic potential of orthotopic breast tumors. Increased matrix stiffness also promotes three-dimensional (3D) collective breast tumor cell invasion via multicellular cytoskeleton remodeling. POSTN triggers the integrin/FAK/ERK/Cdc42/Rac1 mechanotransduction pathway during 3D collective invasion of breast tumor. Clinically, high POSTN expression correlates with high collagen levels in breast tumors and cooperatively determines the metastatic recurrence potential in breast cancer patients. Collectively, these findings indicate that matrix rigidity promotes 3D collective invasion of breast tumor cells via the YAP-POSTN-integrin mechanotransduction signaling. [ABSTRACT FROM AUTHOR]

The invasion of cancer cells into interstitial tissues in a cohesive unit is termed collective invasion, and it is important for the invasion and metastasis of salivary adenoid cystic carcinoma (SACC). However, the underlying mechanisms regulating SACC collective invasion are still poorly understood. Here, we found that SACC tissues exhibited remarkable FAT1 downregulation and YAP1 upregulation at the invasive front, which was closely associated with collective invasion and distant metastasis. Decreasing FAT1 expression significantly activated the YAP1 signaling pathway and promoted collective invasion. Moreover, miR-183-5p was identified as the candidate regulator of FAT1 by bioinformatic analysis and an online database algorithm. A dual luciferase reporter experiment further confirmed that miR-183-5p directly targeted the FAT1 3′-UTR to reduce FAT1 expression. Increasing or decreasing miR-183-5p expression promoted or attenuated collective invasion, which was reversed by YAP1 siRNA or FAT1 siRNA, respectively. In addition, knocking down miR-183-5p reduced tumor burden and attenuated collective invasion in vivo. Together, these results suggest that the miR-183-5p/FAT1/YAP1 signaling pathway is a critical driver of SACC collective invasion, revealing a novel therapeutic target. [Display omitted] • FAT1 downregulation and YAP1 upregulation are related to metastasis. • Multicellular and single cell methods were used to evaluate collective invasion. • miR-183-5p can directly target the FAT1 3′-UTR to activate the YAP1 signaling pathway. • Knocking down miR-183-5p prevented tumor growth and collective invasion in vivo. [ABSTRACT FROM AUTHOR]

Abstract Background Mounting evidence suggests that solid tumors display the features of collective invasion, however, the molecular mechanisms are far from clear. This study aims to verify the role and the underlying mechanisms of CD147 in collective invasion in hepatocellular carcinoma. Methods Immunostaining was used to analyze human hepatocellular carcinoma specimens and three-dimensional cultures. Three-dimensional invasion model was established to mimic in vivo invasion. RNA-sequencing was used to identify downstream effectors. Results Human hepatocellular carcinoma underwent collective invasion and CD147 was observed to be upregulated at the invasive front of tumor cell groups. CD147 was demonstrated to promote collective invasion using the modified three-dimensional invasion model, which recapitulated the main features of collective invasion. Through transcriptome analysis and enzyme activity assay, we found that CD147 enhanced cathepsin B expression and activity. Upregulated cathepsin B in hepatocellular carcinoma cells facilitated migration and invasion, which mediated CD147-induced invasive phenotype in hepatocellular carcinoma. In terms of mechanism, we found that CD147 promoted cathepsin B transcription by activating β-catenin signaling as a result of reduced GSK-3β expression. Furthermore, we found that elevated expression of CD147 as well as cathepsin B were correlated with poor prognosis in patients with hepatocellular carcinoma. Conclusions CD147 promotes hepatocellular carcinoma cells collective invasion via upregulating cathepsin B expression and targeting CD147 would be valuable for the development of novel therapeutic modalities against invasion and metastasis of cancer.

The metastatic process is arduous. Cancer cells must escape the confines of the primary tumor, make their way into and travel through the circulation, then survive and proliferate in unfavorable microenvironments. A key question is how cancer cells overcome these multiple barriers to orchestrate distant organ colonization. Accumulating evidence in human patients and animal models supports the hypothesis that clusters of tumor cells can complete the entire metastatic journey in a process referred to as collective metastasis. Here we highlight recent studies unraveling how multicellular coordination, via both physical and biochemical coupling of cells, induces cooperative properties advantageous for the completion of metastasis. We discuss conceptual challenges and unique mechanisms arising from collective dissemination that are distinct from single cell-based metastasis. Finally, we consider how the dissection of molecular transitions regulating collective metastasis could offer potential insight into cancer therapy. [ABSTRACT FROM AUTHOR]

In this study, we explore the dynamic process of colorectal cancer progression, emphasizing the evolution toward a more metastatic phenotype. The term "evolution" as used in this study specifically denotes the phenotypic transition toward a higher metastatic potency from well-formed glandular structures to collective invasion, ultimately resulting in the development of cancer cell buddings at the invasive front. Our findings highlight the spatial correlation of this evolution with tumor cell senescence, revealing distinct types of senescent tumor cells (types I and II) that play different roles in the overall cancer progression. Type I senescent tumor cells (p16INK4A+/CXCL12+/LAMC2−/MMP7−) are identified in the collective invasion region, whereas type II senescent tumor cells (p16INK4A+/CXCL12+/LAMC2+/MMP7+), representing the final evolved form, are prominently located in the partial-EMT region. Importantly, type II senescent tumor cells associate with local invasion and lymph node metastasis in colorectal cancer, potentially affecting patient prognosis. [Display omitted] • Cellular senescence is associated with gradual phenotypic transition of cancer cells • Evolutionary path of cancer cells includes two types (I and II) of senescent tumor cells • Type II senescent tumor cells express MMP7 and are associated with higher metastatic potency Park et al. unravel the spatial heterogeneity of colorectal cancer and gradual phenotypic transition from the center to the invasive front. They demonstrated that cellular senescence is associated with this evolutional transition. Type II senescent tumor cells (p16INK4A+/LAMC2+/MMP7+) were found in the invasive front and are associated with higher metastatic potency. [ABSTRACT FROM AUTHOR]

Bladder cancer is a common type of cancer, and one with high recurrence rate and overall monitoring and treatment costs. It was developed and tested a three-dimensional agent-based computational model of the urothelium, a purpose-built Cellular Potts Model, to describe both a healthy urothelium and the initiation, development and invasion stages of a bladder cancer. This work is focused on the identification of the conditions in which tumor cells can cross, by mechanical means, the basement membrane and invade the bladder lamina propria. It is demonstrated how specific mechanical properties of the cancer cells, as their stiffness and adhesion to neighboring cells, heavily determine the probability of transition from a non-invasive to an invasive tumor. In particular, it is shown how an increased cellular adhesion of tumor cells to neighbor cells and/or extracellular fibers drives a collective migration, increasing the tumor growth rate and its invasion capacity. It is proposed that these physical characteristics should be considered as potential therapeutic targets to control the tumor growth and its progress to a more dangerous condition.

Tumor dissemination into the surrounding stroma is the initial step in a metastatic cascade. Invasion into stroma is a non-autonomous process for cancer, and its progression depends upon the stage of cancer, as well as the cells residing in the stroma. However, a systems framework to understand how stromal fibroblasts resist, collude, or aid cancer invasion has been lacking, limiting our understanding of the role of stromal biology in cancer metastasis. We and others have shown that gene perturbation in stromal fibroblasts can modulate cancer invasion into the stroma, highlighting the active role stroma plays in regulating its own invasion. However, cancer invasion into stroma is a complex higher-order process and consists of various sub-phenotypes that together can result in an invasion. Stromal invasion exhibits a diversity of modalities in vivo. It is not well understood if these diverse modalities are correlated, or they emanate from distinct mechanisms and if stromal biology could regulate these characteristics. These characteristics include the extent of invasion, formation, and persistence of invasive forks by cancer as opposed to a collective frontal invasion, the persistence of invading velocity by leader cells at the tip of invasive forks, etc. We posit that quantifying distinct aspects of collective invasion can provide useful suggestions about the plausible mechanisms regulating these processes, including whether the process is regulated by mechanics or by intercellular communication between stromal cells and cancer. Here, we have identified the sub-characteristics of invasion, which might be indicative of broader mechanisms regulating these processes, developed methods to quantify these metrics, and demonstrated that perturbation of stromal genes can modulate distinct aspects of collective invasion. Our results highlight that the genetic state of stromal fibroblasts can regulate complex phenomena involved in cancer dissemination and suggest that collective cancer invasion into stroma is an outcome of the complex interplay between cancer and stromal fibroblasts. [ABSTRACT FROM AUTHOR]

Cancer cells collectively invading as a cohesive and polarized group is termed collective invasion, which is a fundamental property of many types of cancers. In this multicellular unit, cancer cells are heterogeneous, consisting of two morphologically and functionally distinct subpopulations, leader cells and follower cells. Leader cells at the invasive front are responsible for exploring the microenvironment, paving the way, and transmitting information to follower cells. Here, in this review, we will describe the important role of leader cells in collective invasion and the emerging underlying mechanisms of leader cell formation including intrinsic properties and the support from neighboring cells. It will help us to elucidate the essence of collective invasion and provide new anticancer therapeutic clues. [ABSTRACT FROM AUTHOR]

Interaction of cancer cells with cancer‐associated fibroblasts (CAFs) plays critical roles in tumor progression. Recently we proposed a new tumor invasion mechanism in which invasive cancer cells individually migrate on elongate protrusions of CAFs (CAF fibers) in 3‐D collagen matrix. In this mechanism, cancer cells interact with fibronectin fibrils assembled on CAFs mainly through integrin‐α5β1. Here we tested whether this mechanism is applicable to the collective invasion of cancer cells, using two E‐cadherin‐expressing adenocarcinoma cell lines, DLD‐1 (colon) and MCF‐7 (breast). When hybrid spheroids of DLD‐1 cells with CAFs were embedded into collagen gel, DLD‐1 cells collectively but very slowly migrated through the collagen matrix in contact with CAFs. Epidermal growth factor and tumor necrosis factor‐α promoted the collective invasion, possibly by reducing the E‐cadherin junction, as did the transforming growth factor‐β inhibitor SB431542 by stimulating the outgrowth of CAFs. Transforming growth factor‐β itself inhibited the cancer cell invasion. Efficient collective invasion of DLD‐1 cells required large CAF fibers or their assembly as stable adhesion substrates. Experiments with function‐blocking Abs and siRNAs confirmed that DLD‐1 cells adhered to fibronectin fibrils on CAFs mainly through integrin‐α5β1. Anti‐E‐cadherin Ab promoted the single cell invasion of DLD‐1 cells by dissociating the E‐cadherin junction. Although the binding affinity of MCF‐7 cells to CAFs was lower than DLD‐1, they also collectively invaded the collagen matrix in a similar fashion to DLD‐1 cells. Our results suggest that the direct interaction with CAFs, as well as environmental cytokines, contributes to the collective invasion of cancers. [ABSTRACT FROM AUTHOR]

Localization of RNAs at protrusive regions of cells is important for single-cell migration on two-dimensional surfaces. Protrusionenriched RNAs encode factors linked to cancer progression, such as the RAB13 GTPase and the NET1 guanine nucleotide exchange factor, and are regulated by the tumor-suppressor protein APC. However, tumor cells in vivo often do not move as single cells but rather utilize collective modes of invasion and dissemination. Here, we developed an inducible system of three-dimensional (3D) collective invasion to study the behavior and importance of protrusion-enriched RNAs. We find that, strikingly, both the RAB13 and NET1 RNAs are enriched specifically at the invasive front of leader cells in invasive cell strands. This localization requires microtubules and coincides with sites of high laminin concentration. Indeed, laminin association and integrin engagement are required for RNA accumulation at the invasive front. Importantly, perturbing RNA accumulation reduces collective 3D invasion. Examination of in vivo tumors reveals a similar localization of the RAB13 and NET1 RNAs at potential invasive sites, suggesting that this mechanism could provide a targeting opportunity for interfering with collective cancer cell invasion. [ABSTRACT FROM AUTHOR]

Background: Mounting evidence suggests that solid tumors display the features of collective invasion, however, the molecular mechanisms are far from clear. This study aims to verify the role and the underlying mechanisms of CD147 in collective invasion in hepatocellular carcinoma. Methods: Immunostaining was used to analyze human hepatocellular carcinoma specimens and three-dimensional cultures. Three-dimensional invasion model was established to mimic in vivo invasion. RNA-sequencing was used to identify downstream effectors. Results: Human hepatocellular carcinoma underwent collective invasion and CD147 was observed to be upregulated at the invasive front of tumor cell groups. CD147 was demonstrated to promote collective invasion using the modified three-dimensional invasion model, which recapitulated the main features of collective invasion. Through transcriptome analysis and enzyme activity assay, we found that CD147 enhanced cathepsin B expression and activity. Upregulated cathepsin B in hepatocellular carcinoma cells facilitated migration and invasion, which mediated CD147-induced invasive phenotype in hepatocellular carcinoma. In terms of mechanism, we found that CD147 promoted cathepsin B transcription by activating β-catenin signaling as a result of reduced GSK-3β expression. Furthermore, we found that elevated expression of CD147 as well as cathepsin B were correlated with poor prognosis in patients with hepatocellular carcinoma. Conclusions: CD147 promotes hepatocellular carcinoma cells collective invasion via upregulating cathepsin B expression and targeting CD147 would be valuable for the development of novel therapeutic modalities against invasion and metastasis of cancer. [ABSTRACT FROM AUTHOR]

Collective invasion of cancer cells is the key process of circulating tumor cell (CTC) cluster formation, and greatly contributes to metastasis. Cancer stem‐like cells (CSC) have a distinct advantage of motility for metastatic dissemination. To verify the role of CSC in the collective invasion, we performed 3D assays to investigate the collective invasion from cancer cell spheroids. The results demonstrated that CSC can significantly promote both collective and single‐cell invasion. Further study showed that CSC prefer to move outside and lead the collective invasion. More interestingly, approximately 60% of the leader CSC in collective invasion co–expressed both epithelial and mesenchymal genes, while only 4% co–expressed in single invasive CSC, indicating that CSC with hybrid epithelial/mesenchymal phenotype play a key role in cancer cell collective invasion. [ABSTRACT FROM AUTHOR]

Both chemical and mechanical determinants adapt and react throughout the process of tumor invasion. In this study, a cell-based model is used to uncover the growth and invasion of a three-dimensional solid tumor confined within normal cells. Each cell is treated as a spheroid that can deform, migrate, and proliferate. Some fundamental aspects of tumor development are considered, including normal tissue constraints, active cellular motility, homotypic and heterotypic intercellular interactions, and pressure-regulated cell division as well. It is found that differential motility between cancerous and normal cells tends to break the spheroidal symmetry, leading to a finger instability at the tumor rim, while stiff normal cells inhibit tumor branching and favor uniform tumor expansion. The heterotypic cell-cell adhesion is revealed to affect the branching geometry. Our results explain many experimental observations, such as fingering invasion during tumor growth, stiffness inhibition of tumor invasion, and facilitation of tumor invasion through cancerous-normal cell adhesion. This study helps understand how cellular events are coordinated in tumor morphogenesis at the tissue level. [ABSTRACT FROM AUTHOR]

Cancer cells can invade as a population in various cancer tissues. This phenomenon is called collective invasion, which is associated with the metastatic potential and prognosis of cancer patients. The collectiveness of cancer cells is necessary for collective invasion. However, the mechanism underlying the generation of collectiveness by cancer cells is not well known. In this study, the phenomenon of contact following, where neighboring cells move in the same direction via intercellular adhesion, was investigated. An experimental system was created to observe the two-dimensional invasion using a collagen gel overlay to study contact following in collective invasion. The role of integrin-β1, one of the major extracellular matrix (ECM) receptors, in contact following was examined through the experimental system. Integrin-β1 was localized to the intercellular site in squamous carcinoma cells. Moreover, the intercellular adhesion and contact following were suppressed by treatment of an integrin-β1 inhibitory antibody. ECM proteins such as laminin-332 and type-XVII collagen were also localized to the intercellular site and critical for contact following. Collectively, it was demonstrated that the activity of integrin-β1 and expression of ECM proteins in the intercellular site promote contact following in the collective invasion of a cancer cell population. • Integrin-β1 is localized to the intercellular site in a cancer cell population. • Integrin-β1 activity promotes contact following in collective invasion. • ECM proteins are localized to the intercellular site in a cancer cell population. • Expressions of ECM proteins are required for contact following in collective invasion. [ABSTRACT FROM AUTHOR]

Collective invasion is an important strategy of cancers of epithelial origin, including colorectal cancer (CRC), to infiltrate efficiently into local tissues as collective cell groups. Within the groups, cells at the invasive front, called leader cells, are highly polarized and motile, thereby providing the migratory traction that guides the follower cells. However, its underlying mechanisms remain unclear. We have previously shown that signaling emanating from the receptor tyrosine kinase Ror2 can promote invasion of human osteosarcoma cells and that intraflagellar transport 20 (IFT20) mediates its signaling to regulate Golgi structure and transport. Herein, we investigated the role of Ror2 and IFT20 in collective invasion of CRC cells, where Ror2 expression is either silenced or nonsilenced. We show by cell biological analyses that IFT20 promotes collective invasion of CRC cells, irrespective of expression and function of Ror2. Intraflagellar transport 20 is required for organization of Golgi‐associated, stabilized microtubules, oriented toward the direction of invasion in leader cells. Our results also indicate that IFT20 promotes reorientation of the Golgi apparatus toward the front side of leader cells. Live cell imaging of the microtubule plus‐end binding protein EB1 revealed that IFT20 is required for continuous polarized microtubule growth in leader cells. These results indicate that IFT20 plays an important role in collective invasion of CRC cells by regulating organization of Golgi‐associated, stabilized microtubules and Golgi polarity in leader cells. Our results indicate that intraflagellar transport 20 promotes collective invasion of colorectal cancer cells, irrespective of expression status and function of Ror2, through organization of Golgi‐associated, stabilized microtubules, oriented toward the direction of invasion in cells at the invasive front. [ABSTRACT FROM AUTHOR]

Abstract Collective invasion drives the spread of multicellular cancer groups, into the normal tissue surrounding several epithelial tumors. Collective invasion recapitulates various aspects of the multicellular organization and collective migration that take place during normal development and repair. Collective migration starts with the specification of leader cells in which a polarized, migratory phenotype is established. Leader cells initiate and organize the migration of follower cells, to allow the group of cells to move as a cohesive and polarized unit. Leader-follower specification is essential for coordinated and directional collective movement. Forces exerted by cohesive cells represent key signals that dictate multicellular coordination and directionality. Physical forces originate from the contraction of the actomyosin cytoskeleton, which is linked between cells via cadherin-based cell-cell junctions. The cadherin complex senses and transduces fluctuations in forces into biochemical signals that regulate processes like cell proliferation, motility and polarity. With cadherin junctions being maintained in most collective movements the cadherin complex is ideally positioned to integrate mechanical information into the organization of collective cell migration. Here we discuss the potential roles of cadherin mechanotransduction in the diverse aspects of leader versus follower cell specification during collective migration and neoplastic invasion. [ABSTRACT FROM AUTHOR]

Pathological observations show that cancer cells frequently invade the surrounding stroma in collective groups rather than through single cell migration. Here, we studied the role of the actin‐binding protein Girdin, a specific regulator of collective migration of neuroblasts in the brain, in collective cancer cell migration. We found that Girdin was essential for the collective migration of the skin cancer cell line A431 on collagen gels as well as their fibroblast‐led collective invasion in an organotypic culture model. We provide evidence that Girdin binds to β‐catenin that plays important roles in the Wnt signaling pathway and in E‐cadherin‐mediated cell‐cell adhesion. Girdin‐depleted cells displayed scattering and impaired E‐cadherin‐specific cell‐cell adhesion. Importantly, Girdin depletion led to impaired cytoskeletal association of the β‐catenin complex, which was accompanied by changes in the supracellular actin cytoskeletal organization of cancer cell cohorts on collagen gels. Although the underlying mechanism is unclear, this observation is consistent with the established role of the actin cytoskeletal system and cell‐cell adhesion in the collective behavior of cells. Finally, we showed the correlation of the expression of Girdin with that of the components of the E‐cadherin complex and the differentiation of human skin cancer. Collectively, our results suggest that Girdin is an important modulator of the collective behavior of cancer cells. This study showed the role of the actin‐binding protein Girdin in collective cancer cell migration. The data showed the involvement of Girdin in E‐cadherin‐specific cell‐cell adhesion and supracellular actin cytoskeletal organization of cancer cell cohorts. The expression of Girdin was also involved in the differentiation of human skin cancer, suggesting that Girdin is a modulator of the collective behavior of cancer cells [ABSTRACT FROM AUTHOR]

Collective cellular invasion in malignant tumours is typically characterized by the cooperative migration of multiple cells in close proximity to each other. Follower cells are led away from the tumour by specialized leader cells, and both cell populations play a crucial role in collective invasion. Follower cells form the main body of the migration system and depend on intercellular contact for migration, whereas leader cells indicate the direction for the entire cell population. Although collective invasion can occur in epithelial and non‑epithelial malignant neoplasms, such as medulloblastoma and rhabdomyosarcoma, the present review mainly provided an extensive analysis of epithelial tumours. In the present review, the cooperative mechanisms of contact inhibition locomotion between follower and leader cells, where follower cells coordinate and direct collective movement through physical (mechanical) and chemical (signalling) interactions, is summarised. In addition, the molecular mechanisms of follower cell invasion and metastasis during remodelling and degradation of the extracellular matrix and how chemotaxis and lateral inhibition mediate follower cell behaviour were analysed. It was also demonstrated that follower cells exhibit genetic and metabolic heterogeneity during invasion, unlike leader cells.

Heterogeneity within tumour cell populations is commonly observed in most cancers. However, its impact on metastatic dissemination, one of the primary determinants of the disease prognosis, remains poorly understood. Working with a simplified numerical model of tumour spheroids, we investigated the impact of mechanical heterogeneity on the onset of tumour invasion into surrounding tissues. Our work establishes a positive link between tumour heterogeneity and metastatic dissemination, and recapitulates a number of invasion patterns identified in vivo, such as multicellular finger-like protrusions. Two complementary mechanisms are at play in heterogeneous tumours. A small proportion of stronger cells are able to initiate and lead the escape of cells, while collective effects in the bulk of the tumour provide the coordination required to sustain the invasive process through multicellular streaming. This suggests that the multicellular dynamics observed during metastasis is a generic feature of mechanically heterogeneous cell populations and might rely on a limited and generic set of attributes.

Mouse colorectal cancer (CRC) models generated by orthotopic microinjection of human CRC cell lines reproduce the pattern of lymphatic, haematological and transcoelomic spread but generate low metastatic efficiency. Our aim was to develop a new strategy that could increase the metastatic efficiency of these models. We used subcutaneous implantation of the human CRC cell lines HCT116 or SW48 prior to their orthotopic microinjection in the cecum of nude mice (SC+ORT). This subcutaneous preconditioning significantly enhanced metastatic dissemination. In the HCT116 model it increased the number and size of metastatic foci in lymph nodes, lung, liver and peritoneum, whereas, in the SW48 model, it induced a shift from non-metastatic to metastatic. In both models the number of apoptotic bodies in the primary tumour in the SC+ORT group was significantly reduced compared with that in the direct orthotopic injection (ORT) group. Moreover, in HCT116 tumours the number of keratin-positive tumour buddings and single epithelial cells increased at the invasion front in SC+ORT mice. In the SW48 tumour model, we observed a trend towards a higher number of tumour buds and single cells in the SC+ORT group but this did not reach statistical significance. At a molecular level, the enhanced metastatic efficiency observed in the HCT116 SC+ORT model was associated with an increase in AKT activation, VEGF-A overexpression and downregulation of β1 integrin in primary tumour tissue, whereas, in SW48 SC+ORT mice, the level of expression of these proteins remained unchanged. In summary, subcutaneous preconditioning increased the metastatic dissemination of both orthotopic CRC models by increasing tumour cell survival and invasion at the tumour invasion front. This approach could be useful to simultaneously study the mechanisms of metastases and to evaluate anti-metastatic drugs against CRC.

Breast cancer undergoes collective tissue invasion and, in experimental models, can collectively metastasize. The prevalence of collective invasion and its contribution to distant metastasis in clinical disease, however, remains poorly defined. We here scored the adipose tissue invasion of primary invasive ductal carcinoma (IDC), expressing E-cadherin, and E-cadherin negative invasive lobular carcinoma (ILC) and identified predominantly collective invasion patterns (86/86 samples) in both carcinoma types. Whereas collective invasion in IDC lesions retained adherens junctions, multicellular clusters and 'Indian files' in ILC, despite the absence of adherens junctions (AJ) proteins E-cadherin and β-catenin, retained CD44 at cell-cell contacts. By histomorphological scoring and semi-automated image analysis, we show that the extent of collective invasion into the adipose tissue correlated with decreased distant metastasis-free survival (5-year follow-up; hazard ratio: 2.32 and 2.29, respectively). Thus, collective invasion represents the predominant invasion mode in breast cancer, develops distinct junctional subtypes in IDC and ILC, and associates with distant metastasis, suggesting a critical role in systemic dissemination. [ABSTRACT FROM AUTHOR]

Background The mechanisms of breast cancer collective invasion are poorly understood limiting the metastasis therapy. The ATPase RUVBL1 is frequently overexpressed in various cancers and plays a crucial role in oncogenic process. We further investigated the role of RUVBL1 in promoting collective invasion and uncovered that targeting RUVBL1 could inhibit metastatic progression. Methods The expression levels of RUVBL1 and ITFG1 were examined by Western blot and qRT-PCR. Co-localization and interaction of RUVBL1 and ITFG1 were determined by immunofluorescence and co-immunoprecipitation. The invasive ability was examined by transwell assay and microfluidic assay. The metastatic and tumorigenic abilities of breast cancer cells were revealed in BALB/c nude mice by xenograft and tail vein injection. Results ATPase RUVBL1 is highly expressed in breast cancer and predicts the poor prognosis. Elevated expression of RUVBL1 is found in high metastatic breast cancer cells. Silencing RUVBL1 suppresses cancer cell expansion and invasion in vitro and in vivo . RUVBL1 interacts with a conserved transmembrane protein ITFG1 in cytoplasm and plasma membrane to promote the collective invasion. Using a microfluidic model, we demonstrated that silencing RUVBL1 or ITFG1 individually compromises collective invasion of breast cancer cells. Conclusion RUVBL1 is a vital regulator for collective invasion. The interaction between RUVBL1 and ITFG1 is required for breast cancer cell collective invasion and progression. General significance Targeting collective invasion promoted by RUVBL1-ITFG1 complex provides a novel therapeutic strategy to improve the prognosis of invasive breast cancer. [ABSTRACT FROM AUTHOR]

Background: Around 5% of all cutaneous squamous cell carcinoma (cSCC) metastasise. Metastases usually locate in regional skin and lymph nodes, suggesting collective cancer invasion. The cellular level of tumour invasion and prognostic parameters remain to be characterised.Methods: We performed immunohistochemical analyses of E-cadherin (marker for collective cancer invasion) and podoplanin (marker for epithelial-mesenchymal transition [EMT], single-cell invasion) expression in 102 samples of metastatic and non-metastatic cSCC and 18 corresponding skin and lymph node metastases to characterise the invasion of cSCC. Immunohistochemical results were retrospectively correlated with clinical data.Results: E-cadherin was highly expressed in metastatic and non-metastatic cSCC and skin metastases. This suggests collective cancer invasion. However, E-cadherin was downregulated in poorly differentiated cSCC and lymph node metastases, suggesting partial EMT. Podoplanin was significantly upregulated in metastatic (p = 0.002) and poorly differentiated (p = 0.003) cSCC. Overexpression of podoplanin represented a statistically independent prognostic factor for disease-free survival (p = 0.014).Conclusion: Collective cancer invasion is likely in cSCC. In lymph node metastases and poorly differentiated cSCC, partial EMT is possible. Podoplanin is an independent prognostic parameter for metastasis. [ABSTRACT FROM AUTHOR]

Clinical pathologists have long been aware that in many types of human malignant tumors, the cells are often connected and form groups of various sizes or 'nests'. In this way, they achieve 'collective invasion' into the surrounding stroma, rather than spreading out individually. Such collective behavior is also a common feature of migration during embryonic and postnatal developmental stages, suggesting there are advantages gained by collective cell migration in the organisms. Recent studies have revealed the mechanisms underlying the collective invasion of cancer cells. These mechanisms differ from those observed in the migration of single cells in culture, including reliance on the epithelial-mesenchymal transition program. Whereas intercellular adhesion appears to be coordinated, cancer cell groups can be heterogenous, including cells that are leaders and those that are followers. There is also interaction with the tumor microenvironment that is a prerequisite for collective invasion of cancer. In this review, we describe recently emerging mechanisms underlying the collective migration of cells, with a particular focus in our studies on the actin-binding protein Girdin/GIV and the transcriptional regulator tripartite motif containing 27. These studies provide new perspectives on the mechanistic analogy between cancer and development. [ABSTRACT FROM AUTHOR]

Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14+ cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14+ epithelial tumor cell clusters disseminate collectively to colonize distant organs. [ABSTRACT FROM AUTHOR]

A fundamental step of tumor metastasis is tumor cell migration away from the primary tumor site. One mode of migration that is essential but still understudied is collective invasion, the process by which clusters of cells move in a coordinated fashion. In recent years, there has been growing interest to understand factors regulating collective invasion, with increasing number of studies investigating the biomechanical regulation of collective invasion. In this review we discuss the dynamic relationship between tumor microenvironment cues and cell response by first covering mechanical factors in the microenvironment and second, discussing the mechanosensing pathways utilized by cells in collective clusters to dynamically respond to mechanical matrix cues. Finally, we discuss model systems that have been developed which have increased our understanding of the mechanical factors contributing to tumor progression., (Copyright © 2023. Published by Elsevier Inc.)

Cancer invasion and metastasis require remodeling of the adjacent extracellular matrix (ECM). In this mini review, we will cover the mechanisms of proteolytic degradation and the mechanical remodeling of the ECM by cancer cells, with a focus on invadopodia. Invadopodia are membrane protrusions unique to cancer cells, characterized by an actin core and by the focal degradation of ECM via matrix metalloproteases (MMPs). While ECM can also be remodeled, at lower levels, by focal adhesions, or internal collagen digestion, invadopodia are now recognized as the major mechanism for MMP-dependent pericellular ECM degradation by cancer cells. Recent evidence suggests that the completion of epithelial-mesenchymal transition may be dispensable for invadopodia and metastasis, and that invadopodia are required not only for mesenchymal, single cell invasion, but also for collective invasion. During collective invasion, invadopodia was then shown to be located in leader cells, allowing follower cells to move via cooperation. Collectively, this suggests that invadopodia function may be a requirement not only for later steps of metastasis, but also for early invasion of epithelial cells into the stromal tissue. Over the last decade, invadopodia studies have transitioned into in 3D and in vivo settings, leading to the confirmation of their essential role in metastasis in preclinical animal models. In summary, invadopodia may hold a great potential for individual risk assessment as a prognostic marker for metastasis, as well as a therapeutic target., (© 2022 IOP Publishing Ltd.)

Multicellularity evolved in fungi and animals, or the opisthokonts, from their common amoeboflagellate ancestor but resulted in strikingly distinct cellular organizations. The origins of this multicellularity divergence are not known. The stark mechanistic differences that underlie the two groups and the lack of information about ancestral cellular organizations limits progress in this field. We discovered a new type of invasive multicellular behavior in Fonticula alba , a unique species in the opisthokont tree, which has a simple, bacteria-feeding sorocarpic amoeba lifestyle. This invasive multicellularity follows germination dependent on the bacterial culture state, after which amoebae coalesce to form dynamic collectives that invade virgin bacterial resources. This bacteria-dependent social behavior emerges from amoeba density and allows for rapid and directed invasion. The motile collectives have animal-like properties but also hyphal-like search and invasive behavior. These surprising findings enrich the diverse multicellularities present within the opisthokont lineage and offer a new perspective on fungal origins. • Unexpected bacterial-state-dependent culture conditions for Fonticula alba • A multicellular invasion of bacterial food resources that is distinct from fruiting • A leader-led invasive collectivity that is an emergent property • Insights into the origins of invasive hyphal and fruiting multicellularity in dikarya Toret et al. delve into the biology of the Fonticula alba , a unique cellular slime mold that is sister to fungi. They discover a cryptic, emergent, and invasive multicellular organization with parallels to invasive hyphae. This study sheds light on the multicellular organizations that occur within the fungi-animal divergence. [ABSTRACT FROM AUTHOR]

Purpose: Cancer-associated fibroblasts (CAFs) contribute to tumor progression through multiple pathways. However, the effect of CAFs on gene expression in lung cancer has been largely unknown. Here we systematically compared the gene expression changes in lung cancer cells induced by normal fibroblasts and CAFs. Methods: Wound healing and cell proliferation assays were used to identify the property of CAFs used in this study. We used cDNA microarray analysis to compare gene expression in lung cancer cells cultured with either conditioned medium (CM) from lung CAFs or normal lung fibroblasts, the result of which was confirmed by RT-PCR and Western blot analysis. Immunohistochemistry on tissue sections from lung cancers was conducted to further confirm the results of cDNA microarray analysis. Results: The expression of many genes was upregulated in cancer cells by CAF CM, particularly cell adhesion molecules, integrins, and anti-apoptotic protein Bcl-2. Expression of integrins appeared to be upstream from Bcl-2. We identified transforming growth factor-β as a candidate factor that induced the expression of those genes in cancer cells. Immunohistochemical studies of clinical lung cancer tissues revealed that integrins and Bcl-2 were more highly expressed in the leading cells (LCs) than in the following cells, at the invasive front of cancer nests, which are adjacent to or in proximity to the stroma. Furthermore, the expression of integrins and Bcl-2 in LCs had a tendency to correlate with the clinical stage of cancer progression, including lymph node metastasis. Conclusions: Our results suggest that CAFs promote lung cancer progression partly through the direct regulation of gene expression in the LCs of invasive cancer nests. [ABSTRACT FROM AUTHOR]

The metastatic invasion of cancer cells from the primary lesion into the adjacent stroma is a key step in cancer progression, and is associated with poor outcome. The principles of cancer invasion have been experimentally addressed in various in vitro models; however, key steps and mechanisms in vivo remain unclear. Here, we establish a modified skin-fold chamber model for orthotopic implantation, growth and invasion of human HT-1080 fibrosarcoma cells, dynamically reconstructed by epifluorescence and multiphoton microscopy. This strategy allows repeated imaging of tumor growth, tumor-induced angiogenesis and invasion, as either individual cells, or collective strands and cell masses that move along collagen-rich extracellular matrix and coopt host tissue including striated muscle strands and lymph vessels. This modified window model will be suited to address mechanisms of cancer invasion and metastasis, and related experimental therapy. [ABSTRACT FROM AUTHOR]

Understanding the modes and mechanisms of tumor cell invasion is key to developing targeted therapies against metastatic disease. In vitro assays modeling tumor progression have primarily been optimized for studying classical single-cell migration through an epithelial-mesenchymal transition (EMT). Although experimental and clinical histopathological evidence has revealed that tumor invasion is plastic and that epithelial carcinomas can invade by a range of modes that vary from single, mesenchyme-like cells, all the way to cohesive, collective units, few in vitro assays have been designed to assess these modes specifically. Thus, we have developed a Matrigel-Collagen I overlay assay that is suitable for identifying and quantifying both collective and mesenchymal invasion. This three-dimensional (3D) culture assay utilizes the features of Matrigel and Collagen I to mimic the laminin-rich basement membrane and the stiff, fibrillar Collagen I tumor microenvironment allowing for spheroid invasion to be assessed at the interface between these two matrix components., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Metastasis of cancer cells leads to 90% of lethality among cancer patients. A crucial step in the hematogenous spread of metastatic cancer is the detachment of cells from the primary tumor followed by invasion through nearby blood vessels (Wong and Hynes. Cell Cycle 5(8):812-817, 2006). This is common to several solid tumors, including medulloblastoma (Van Ommeren et al. Brain Pathol 30:691-702, 2020). Because invasion is a crucial step in metastasis, the development of assays studying invasion are important for identifying antimetastatic drugs. There is always a need to develop better 3D in vitro models that not only mimic the complexity of in vivo architecture of solid tumors and their microenvironment, but are also simple to execute in medium to high throughput. We developed an in vitro coculture invasion assay that relies on the binary interaction between cancer cells and endothelial cells for research on tumor invasion and antimetastatic drug discovery. The goal of the current protocol is to use the simplicity of a two-dimensional endothelial cell culture to create a gel-free physiological substratum that can facilitate cancer cell invasion from a 3D cancer spheroid. This provides a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. Using this assay, we can compare the effect of invasion inhibitors/activators on cancer spheroids. The results are analyzed by manual scoring of images for the presence or absence of sprouting from cancer spheroids. This enables simple and fast analysis of metastasis, which facilitates multiparameter examination., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)