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Significance Specific RNAs are enriched at protrusive regions of migrating cells. This localization is important for cell migration on 2D surfaces. However, in vivo, tumor cells navigate complex 3D environments often in collective groups. Here, we investigated protrusion-enriched RNAs during collective 3D invasion. We show that specific RNAs exhibit a striking accumulation at the front of invasive leader cells. We provide insights into the mechanism underlying RNA accumulation at the invasive front, and we further demonstrate that it is required for efficient 3D invasion of tumor cells. We additionally observe RNA enrichment at invasive sites of in vivo tumors, supporting the physiological relevance of this mechanism and suggesting a targeting opportunity for perturbing cancer cell invasion., Localization of RNAs at protrusive regions of cells is important for single-cell migration on two-dimensional surfaces. Protrusion-enriched 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.

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., This study established a simple collective cancer invasion model in which E‐cadherin‐expressing cancer cells collectively invaded the collagen matrix in contact with bundles of cancer‐associated fibroblast fibers through integrin‐α5β1/fibronectin interaction. The results suggest that the fibrosis and environmental cytokines contribute to the collective invasion of cancers in vivo.

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., To verify the role of CSC in the collective invasion, we performed 3D assays for investigating the collective invasion from cancer cell spheroids. The results demonstrated that CSC act as leaders to promote collective invasion with hybrid epithelial/mesenchymal phenotype. Our present observations may predict a new strategy for cancer metastasis, including interventions targeting CSC.

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.

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.

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. Electronic supplementary material The online version of this article (doi:10.1007/s10585-017-9858-6) contains supplementary material, which is available to authorized users.

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.

Networks of nanotubes and microtubules are highly valued in cellular communication, and collective cancer movement has been revealed to be associated with cell information exchange. In the present study, cellular communication was demonstrated to participate in mammosphere growth, differentiation and collective invasion. By promoting differentiation, networks of cells and microtubule-like structures were verified. Analyses of cell cycle progression, stemness markers and gene expression indicated that mammospheres had collective characteristics of stemness and differentiation. Invasion assays revealed that networks of microtubule-like structures promoted collective invasion. Conversely, using anti-angiogenic intervention, the growth of stem-like mammospheres and cellular communication links were effectively inhibited. In vivo experiments revealed that cellular communication promoted tumor growth and metastasis through the formation of nodular fusion, cluttered microtubule-like structures and cancer stem cells, as well as vascular niches. In conclusion, the present results demonstrated that a network of cells and structures were largely present in mammosphere cellular communication in vitro and in vivo. Therefore, blocking cellular communication may prove beneficial in halting the progression of mammary tumors.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer. Virtually all women with DCIS are treated, despite evidence suggesting up to half would remain with stable, non-threatening, disease. Overtreatment thus presents a pressing issue in DCIS management. To understand the role of the normally tumour suppressive myoepithelial cell in disease progression we present a 3D in vitro model incorporating both luminal and myoepithelial cells in physiomimetic conditions. We demonstrate that DCIS-associated myoepithelial cells promote striking myoepithelial-led invasion of luminal cells, mediated by the collagenase MMP13 through a non-canonical TGF beta - EP300 pathway. In vivo, MMP13 expression is associated with stromal invasion in a murine model of DCIS progression and is elevated in myoepithelial cells of clinical high-grade DCIS cases. Our data identify a key role for myoepithelial-derived MMP13 in facilitating DCIS progression and point the way towards a robust marker for risk stratification in DCIS patients.