Fibroblast-derived MT1-MMP promotes tumor progression in vitro and in vivo

BACKGROUND: Identification of fibroblast derived factors in tumor progression has the potential to provide novel molecular targets for modulating tumor cell growth and metastasis. Multiple matrix metalloproteases (MMPs) are expressed by both mesenchymal and epithelial cells within head and neck squamous cell carcinomas (HNSCCs), but the relative importance of these enzymes and the cell source is the subject of controversy. METHODS: The invasive potential of HNSCC tumor cells were assessed in vitro atop type I collagen gels in coculture with wild-type (WT), MMP-2 null, MMP-9 null or MT1-MMP null fibroblasts. A floor of mouth mouse model of HNSCC was used to assess in vivo growth after co-injection of FaDu tumor cells with MMP null fibroblasts. RESULTS: Here we report changes in tumor phenotype when FaDu HNSCCs cells are cocultured with WT, MMP-2 null, MMP-9 null or MT1-MMP null fibroblasts in vitro and in vivo. WT, MMP-2 null and MMP-9 null fibroblasts, but not MT1-MMP null fibroblasts, spontaneously invaded into type I collagen gels. WT fibroblasts stimulated FaDu tumor cell invasion in coculture. This invasive phenotype was unaffected by combination with MMP-9 null fibroblasts, reduced with MMP-2 null fibroblasts (50%) and abrogated in MT1-MMP null fibroblasts. Co-injection of FaDu tumor cells with fibroblasts in an orthotopic oral cavity SCID mouse model demonstrated a reduction of tumor volume using MMP-9 and MMP-2 null fibroblasts (48% and 49%, respectively) compared to WT fibroblasts. Consistent with in vitro studies, MT1-MMP null fibroblasts when co-injected with FaDu cells resulted in a 90% reduction in tumor volume compared to FaDu cells injected with WT fibroblasts. CONCLUSION: These data suggest a role for fibroblast-derived MMP-2 and MT1-MMP in HNSCC tumor invasion in vitro and tumor growth in vivo

Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes.

A considerable body of research indicates that mammary gland branching morphogenesis is dependent, in part, on the extracellular matrix (ECM), ECM-receptors, such as integrins and other ECM receptors, and ECM-degrading enzymes, including matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). There is some evidence that these ECM cues affect one or more of the following processes: cell survival, polarity, proliferation, differentiation, adhesion, and migration. Both three-dimensional culture models and genetic manipulations of the mouse mammary gland have been used to study the signaling pathways that affect these processes. However, the precise mechanisms of ECM-directed mammary morphogenesis are not well understood. Mammary morphogenesis involves epithelial 'invasion' of adipose tissue, a process akin to invasion by breast cancer cells, although the former is a highly regulated developmental process. How these morphogenic pathways are integrated in the normal gland and how they become dysregulated and subverted in the progression of breast cancer also remain largely unanswered questions

Adult Subependymal Neural Precursors, but Not Differentiated Cells, Undergo Rapid Cathodal Migration in the Presence of Direct Current Electric Fields

BACKGROUND: The existence of neural stem and progenitor cells (together termed neural precursor cells) in the adult mammalian brain has sparked great interest in utilizing these cells for regenerative medicine strategies. Endogenous neural precursors within the adult forebrain subependyma can be activated following injury, resulting in their proliferation and migration toward lesion sites where they differentiate into neural cells. The administration of growth factors and immunomodulatory agents following injury augments this activation and has been shown to result in behavioural functional recovery following stroke. METHODS AND FINDINGS: With the goal of enhancing neural precursor migration to facilitate the repair process we report that externally applied direct current electric fields induce rapid and directed cathodal migration of pure populations of undifferentiated adult subependyma-derived neural precursors. Using time-lapse imaging microscopy in vitro we performed an extensive single-cell kinematic analysis demonstrating that this galvanotactic phenomenon is a feature of undifferentiated precursors, and not differentiated phenotypes. Moreover, we have shown that the migratory response of the neural precursors is a direct effect of the electric field and not due to chemotactic gradients. We also identified that epidermal growth factor receptor (EGFR) signaling plays a role in the galvanotactic response as blocking EGFR significantly attenuates the migratory behaviour. CONCLUSIONS: These findings suggest direct current electric fields may be implemented in endogenous repair paradigms to promote migration and tissue repair following neurotrauma

A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis

Extracellular matrix interactions have essential roles in normal physiology and many pathological processes. Although the importance of extracellular matrix interactions in metastasis is well documented, systematic approaches to identify their roles in distinct stages of tumorigenesis have not been described. Here we report a novel-screening platform capable of measuring phenotypic responses to combinations of extracellular matrix molecules. Using a genetic mouse model of lung adenocarcinoma, we measure the extracellular matrix-dependent adhesion of tumour-derived cells. Hierarchical clustering of the adhesion profiles differentiates metastatic cell lines from primary tumour lines. Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8 or laminin. We show that these molecules correlate with human disease and that their interactions are mediated in part by α3β1 integrin. Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified extracellular matrix and integrin interactions that could serve as therapeutic targets.National Institutes of Health (U.S.) (Grant K99-CA151968)National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service AwardStand Up To Cancer (SU2C/AACR)David H. Koch Institute for Integrative Cancer Research at MIT (CTC Project)Harvard Stem Cell Institute (SG-0046-08-00)National Cancer Center (Postdoctoral Fellowship)National Cancer Institute (U.S.) (U54CA126515)National Cancer Institute (U.S.) (U54CA112967)Howard Hughes Medical InstituteMassachusetts Institute of Technology. Ludwig Center for Molecular Oncolog

Identification of membrane-type 1 matrix metalloproteinase tyrosine phosphorylation in association with neuroblastoma progression

<p>Abstract</p> <p>Background</p> <p>Neuroblastoma is a pediatric tumor of neural crest cells that is clinically characterized by its variable evolution, from spontaneous regression to malignancy. Despite many advances in neuroblastoma research, 60% of neuroblastoma, which are essentially metastatic cases, are associated with poor clinical outcome due to the lack of effectiveness of current therapeutic strategies. Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), an enzyme involved in several steps in tumor progression, has previously been shown to be associated with poor clinical outcome for neuroblastoma. Based on our recent demonstration that MT1-MMP phosphorylation is involved in the growth of fibrosarcoma tumors, we examined the potential role of phosphorylated MT1-MMP in neuroblastoma progression.</p> <p>Methods</p> <p>Tyrosine phosphorylated MT1-MMP was immunostained on tissue microarray samples from 55 patients with neuroblastoma detected by mass screening (known to be predominantly associated with favourable outcome), and from 234 patients with standard diagnosed neuroblastoma. In addition, the effects of a non phosphorylable version of MT1-MMP on neuroblastoma cell migration and proliferation were investigated within three-dimensional collagen matrices.</p> <p>Results</p> <p>Although there is no correlation between the extent of tyrosine phosphorylation of MT1-MMP (pMT1-MMP) and MYCN amplification or clinical stage, we observed greater phosphorylation of pMT1-MMP in standard neuroblastoma, while it is less evident in neuroblastoma from mass screening samples (P = 0.0006) or in neuroblastoma samples from patients younger than one year (P = 0.0002). <it>In vitro </it>experiments showed that overexpression of a non-phosphorylable version of MT1-MMP reduced MT1-MMP-mediated neuroblastoma cell migration and proliferation within a three-dimensional type I collagen matrix, suggesting a role for the phosphorylated enzyme in the invasive properties of neuroblastoma cells.</p> <p>Conclusion</p> <p>Overall, these results suggest that tyrosine phosphorylated MT1-MMP plays an important role in neuroblastoma progression and that its expression is preferentially observed in tumor specimens from neuroblastoma patients showing poor clinical outcome.</p

Collagen I but not Matrigel matrices provide an MMP-dependent barrier to ovarian cancer cell penetration

Abstract Background The invasive potential of cancer cells is usually assessed in vitro using Matrigel as a surrogate basement membrane. Yet cancer cell interaction with collagen I matrices is critical, particularly for the peritoneal metastatic route undertaken by several cancer types including ovarian. Matrix metalloprotease (MMP) activity is important to enable cells to overcome the barrier constraints imposed by basement membranes and stromal matrices in vivo. Our objective was to compare matrices reconstituted from collagen I and Matrigel as representative barriers for ovarian cancer cell invasion. Methods The requirement of MMP activity for ovarian cancer cell penetration of Matrigel and collagen matrices was assessed in 2D transwell and 3D spheroid culture systems. Results The broad range MMP inhibitor GM6001 completely prevented cell perforation of polymerised collagen I-coated transwell membranes. In contrast, GM6001 decreased ES-2 cell penetration of Matrigel by only ~30% and had no effect on HEY cell Matrigel penetration. In 3D culture, ovarian cancer cells grown as spheroids also migrated into surrounding Matrigel matrices despite MMP blockade. In contrast, MMP activity was required for invasion into 3D matrices of collagen I reconstituted from acid-soluble rat-tail collagen I, but not from pepsin-extracted collagen I (Vitrogen/Purecol), which lacks telopeptide regions. Conclusion Matrigel does not form representative barriers to ovarian cancer cells in either 2D or 3D culture systems. Our findings support the use of collagen I rather than Matrigel as a matrix barrier for invasion studies to better approximate critical interactions and events associated with peritoneal metastasis

The distinct roles of the nucleus and nucleus-cytoskeleton connections in three-dimensional cell migration

Cells often migrate in vivo in an extracellular matrix that is intrinsically three-dimensional (3D) and the role of actin filament architecture in 3D cell migration is less well understood. Here we show that, while recently identified linkers of nucleoskeleton to cytoskeleton (LINC) complexes play a minimal role in conventional 2D migration, they play a critical role in regulating the organization of a subset of actin filament bundles – the perinuclear actin cap - connected to the nucleus through Nesprin2giant and Nesprin3 in cells in 3D collagen I matrix. Actin cap fibers prolong the nucleus and mediate the formation of pseudopodial protrusions, which drive matrix traction and 3D cell migration. Disruption of LINC complexes disorganizes the actin cap, which impairs 3D cell migration. A simple mechanical model explains why LINC complexes and the perinuclear actin cap are essential in 3D migration by providing mechanical support to the formation of pseudopodial protrusions

Cells Assemble Invadopodia-Like Structures and Invade into Matrigel in a Matrix Metalloprotease Dependent Manner in the Circular Invasion Assay

The ability of tumor cells to invade is one of the hallmarks of the metastatic phenotype. To elucidate the mechanisms by which tumor cells acquire an invasive phenotype, in vitro assays have been developed that mimic the process of cancer cell invasion through basement membrane or in the stroma. We have extended the characterization of the circular invasion assay and found that it provides a simple and amenable system to study cell invasion in matrix in an environment that closely mimics 3D invasion. Furthermore, it allows detailed microscopic analysis of both live and fixed cells during the invasion process. We find that cells invade in a protease dependent manner in this assay and that they assemble focal adhesions and invadopodia that resemble structures visualized in 3D embedded cells. We propose that this is a useful assay for routine and medium throughput analysis of invasion of cancer cells in vitro and the study of cells migrating in a 3D environment

Dynamic and influential interaction of cancer cells with normal epithelial cells in 3D culture

BACKGROUND: The cancer microenvironment has a strong impact on the growth and dynamics of cancer cells. Conventional 2D culture systems, however, do not reflect in vivo conditions, impeding detailed studies of cancer cell dynamics. This work aims to establish a method to reveal the interaction of cancer and normal epithelial cells using 3D time-lapse. METHODS: GFP-labelled breast cancer cells, MDA-MB-231, were co-cultured with mCherry-labelled non-cancerous epithelial cells, MDCK, in a gel matrix. In the 3D culture, the epithelial cells establish a spherical morphology (epithelial sphere) thus providing cancer cells with accessibility to the basal surface of epithelia, similar to the in vivo condition. Cell movement was monitored using time-lapse analyses. Ultrastructural, immunocytochemical and protein expression analyses were also performed following the time-lapse study. RESULTS: In contrast to the 2D culture system, whereby most MDA-MB-231 cells exhibit spindle-shaped morphology as single cells, in the 3D culture the MDA-MB-231 cells were found to be single cells or else formed aggregates, both of which were motile. The single MDA-MB-231 cells exhibited both round and spindle shapes, with dynamic changes from one shape to the other, visible within a matter of hours. When co-cultured with epithelial cells, the MDA-MB-231 cells displayed a strong attraction to the epithelial spheres, and proceeded to surround and engulf the epithelial cell mass. The surrounded epithelial cells were eventually destroyed, becoming debris, and were taken into the MDA-MB-231 cells. However, when there was a relatively large population of normal epithelial cells, the MDA-MB-231 cells did not engulf the epithelial spheres effectively, despite repeated contacts. MDA-MB-231 cells co-cultured with a large number of normal epithelial cells showed reduced expression of monocarboxylate transporter-1, suggesting a change in the cell metabolism. A decreased level of gelatin-digesting ability as well as reduced production of matrix metaroproteinase-2 was also observed. CONCLUSIONS: This culture method is a powerful technique to investigate cancer cell dynamics and cellular changes in response to the microenvironment. The method can be useful for various aspects such as; different combinations of cancer and non-cancer cell types, addressing the organ-specific affinity of cancer cells to host cells, and monitoring the cellular response to anti-cancer drugs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12935-014-0108-6) contains supplementary material, which is available to authorized users