The chemoinvasion assay: a method to assess tumor and endothelial cell invasion and its modulation

Invasive and metastatic cells, as well as endothelial cells, must cross basement membranes (BMs) in order to disseminate or to form new blood vessels. The chemoinvasion assay using the reconstituted BM Matrigel in Boyden blind-well chambers is a very rapid, easy, inexpensive and flexible test that can be used to quantify the invasive potential of most cell types; it can be applied to detect the migratory activity associated with matrix degradation and can also be adapted to study the selective degrading activity on different matrix substrates. Transwell inserts can also be used. Once the optimal experimental conditions are empirically determined for specific cellular models, the chemoinvasion assay can be used for the screening of inhibitors of invasiveness and angiogenesis, or to select for invasive cellular populations. This protocol can be completed in 9 h.     

Zebrafish embryo as a tool to study tumor/endothelial cell cross-talk

Tumor/endothelial cell cross-talk plays a pivotal role in the growth, neovascularization and metastatic dissemination of human cancer. Recent observations have shown that the teleost zebrafish (Danio rerio) may represent a powerful experimental platform in cancer research. Various tumor models have been established in zebrafish adults, juveniles, and embryos and novel genetic tools and high resolution in vivo imaging techniques have been exploited. In particular, grafting of mammalian tumor cells in zebrafish embryo body may simulate early stages of tumor development, neovascularization, and local invasion whereas the injection of cancer cells in the bloodstream of zebrafish embryo may allow the study of metastatic homing and colonization. This review focuses on the recent advances in tumor xenotransplantation in zebrafish embryo for the in vivo study of the cancer neovascularization, invasion and metastatic processes. This article is part of a Special Issue entitled: Animal Models of Disease.     

Cell invasion through basement membranes: an anchor of understanding

To metastasize, cancer cells must acquire the ability to breach several basement membrane barriers. Cell invasions through basement membranes also occur during normal development and immune system function, enabling organ formation and cell dispersal. The mechanisms that cells use to cross basement membranes in vivo remain elusive. In cancer and development, these invasions occur in complex and inaccessible environments, which are difficult to study in vivo. Anchor-cell invasion in Caenorhabditis elegans is a simple, visually and experimentally accessible model of basement membrane invasion that is beginning to reveal a network of cellular and molecular control mechanisms that regulate the fundamental cellular process of invasion through basement membranes.     

Zebrafish embryo, a tool to study tumor angiogenesis

Zebrafish (Danio rerio) represents a powerful model system in cancer research. Recent observations have shown the possibility to exploit zebrafish to investigate tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Experimental models have been established in zebrafish adults, juveniles, and embryos, each one with its own advantages and disadvantages. Novel genetic tools and high resolution in vivo imaging techniques are also becoming available in zebrafish. It is anticipated that zebrafish will represent an important tool for chemical discovery and gene targeting in tumor angiogenesis. This review focuses on the recently developed tumor angiogenesis models in zebrafish, with particular emphasis to tumor engrafting in zebrafish embryos.     

Degradation of intact basement membranes by human and murine tumor enzymes

Homogenates from malignant tumors, obtained from surgery specimens or from transplants of Walker 256 carcinosarcoma in rats, contained an enzyme activity capable of degrading intact 3H-acetylated basement membranes from bovine lens. The enzyme activity from murine tumor was purified about 7500-fold by (NH4)2SO4 fractionation, ion exchange and gel chromatography. The apparent molecular weight of the purified enzyme was approximately 50,000. The rate of degradation of 3H-labelled basement membrane by the murine tumor enzyme was reduced by addition of excess type IV collagen, but not of excess type I, type III or type V collagen. These results suggested specificity of this enzyme for type IV collagen. Inhibitors of serine proteinases, thiol proteinases and soybean trypsin inhibitor were without effect on the enzyme activity. Chelators such as 1,10-phenanthroline or EDTA reduced the activity to control levels, indicating that the enzyme activity was due to a metalloproteinase. Chromatographic and electrophoretic separation of the enzymatic products from 3H-labelled basement membrane and type IV collagen indicated that the enzyme activity was due to a type IV collagenase. The use of basement membrane in the native physiological state as a substrate for the study of basement membrane-degrading activity by homogenates of solid malignant tumors offers an in vitro model for the investigation of the metastatic potential of these tumors.     

Initial steps of metastasis: cell invasion and endothelial transmigration

Metastasis is the leading cause of cancer mortality. The metastatic cascade represents a multi-step process which includes local tumor cell invasion, entry into the vasculature followed by the exit of carcinoma cells from the circulation and colonization at the distal sites. At the earliest stage of successful cancer cell dissemination, the primary cancer adapts the secondary site of tumor colonization involving the tumor-stroma crosstalk. The migration and plasticity of cancer cells as well as the surrounding environment such as stromal and endothelial cells are mandatory. Consequently, the mechanisms of cell movement are of utmost relevance for targeted intervention of which three different types have been reported. Tumor cells can migrate either collectively, in a mesenchymal or in an amoeboid type of movement and intravasate the blood or lymph vasculature. Intravasation by the interaction of tumor cells with the vascular endothelium is mechanistically poorly understood. Changes in the epithelial plasticity enable carcinoma cells to switch between these types of motility. The types of migration may change depending on the intervention thereby increasing the velocity and aggressiveness of invading cancer cells. Interference with collective or mesenchymal cell invasion by targeting integrin expression or metalloproteinase activity, respectively, resulted in an amoeboid cell phenotype as the ultimate exit strategy of cancer cells. There are little mechanistic details reported in vivo showing that the amoeboid behavior can be either reversed or efficiently inhibited. Future concepts of metastasis intervention must simultaneously address the collective, mesenchymal and amoeboid mechanisms of cell invasion in order to advance in anti-metastatic strategies as these different types of movement can coexist and cooperate. Beyond the targeting of cell movements, the adhesion of cancer cells to the stroma in heterotypic circulating tumor cell emboli is of paramount relevance for anti-metastatic therapy.     

Double-layered collagen gel hemisphere for cell invasion assay: successful visualization and quantification of cell invasion activity

Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution (containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources: Movies 1-3; 4a and b].     

The significance of focal myoepithelial cell layer disruptions in human breast tumor invasion: a paradigm shift from the "protease-centered" hypothesis

Human breast epithelium and the stroma are separated by a layer of myoepithelial (ME) cells and basement membrane, whose disruption is a prerequisite for tumor invasion. The dissolution of the basement membrane is traditionally attributed primarily to an over-production of proteolytic enzymes by the tumor or the surrounding stromal cells. The results from matrix metalloproteinase inhibitor clinical trials, however, suggest that this "protease-centered" hypothesis is inadequate to completely reflect the molecular mechanisms of tumor invasion. The causes and signs of ME cell layer disruption are currently under-explored. Our studies revealed that a subset of pre- and micro-invasive tumors contained focal disruptions in the ME cell layers. These disruptions were associated with immunohistochemical and genetic alterations in the overlying tumor cells, including the loss of estrogen receptor expression, a higher frequency of loss of heterozygosity, and a higher expression of cell cycle, angiogenesis, and invasion-related genes. Focal ME layer disruptions were also associated with a higher rate of epithelial proliferation and leukocyte infiltration. We propose the novel hypothesis that a localized death of ME cells and immunoreactions that accompany an external environmental insult or internal genetic alterations are triggering factors for ME layer disruptions, basement membrane degradation, and subsequent tumor progression and invasion.     

肿瘤新生血管相关内皮细胞受体研究进展

血管生成对肿瘤的生长,存留和转移是至关重要的,增殖的肿瘤血管内皮细胞不同于静止期内皮细胞,增生血管内皮细胞上特异或高表达的许多受体目前已被鉴定,它们是肿瘤抗血管生成疗法的理想靶点,简要综述了其中主要的几种受体,如血管内皮细胞生长因子（VEGF）受体家族,Tie受体族,整合素受体αvβ3,ATP合成酶等。     

The human tumor stem cell assay revisited

The human tumor stem cell assay (HTSCA) is a bilayer soft agar system for growing fresh human tumor specimens in vitro to determine drug sensitivity and improve our understanding of tumor biology. Recent clinical correlations of 60% accuracy for predicting a positive clinical response and a 90% accuracy for predicting a lack of response to therapeutic agents suggest promising clinical usefulness. However, the clinician should be aware of the assay's inherent pitfalls, such as heterogeneity of the tumor specimen, inability to obtain pure single-cell suspensions, low cloning efficiency, unusual drug dose-dependent survival curves, uncertain validity of in vitro pharmacology, non-standardized criteria for in vitro sensitivity, and the variability of in vitro results. A brief summary of the concepts, potential, and limitations of this assay are discussed.     

Incomplete fusion of the epithelial and endothelial basement membranes in interspecies hybrid glomeruli

Avascular, undifferentiated mouse kidneys transplanted onto quail chorioallantoic membrane differentiate and become vascularized by quail vessels. The glomeruli which form under these conditions consist of mouse podocytes and quail endothelial cells. Immunohistochemistry has shown that the glomerular basement membrane (GBM) has a dual origin, as integral basement membrane components are produced by both podocytes and endothelial cells. In electron microscopy this GBM is composed of two partially separated layers, an epithelial and an endothelial basal lamina which both have a lamina densa and a lamina rara. These two basal laminas are partially fused, but there are large areas where this fusion does not occur. In some places of incomplete fusion, fibrillar extracellular material is seen between and beneath the GBM. It is concluded that basement membrane components derived from the different species can interact partially, but the fusion is incomplete. The abnormal assembly of the epithelial and the endothelial basal laminas might be due to molecular differences between the components produced by the two cell lineages. In spite of the incomplete fusion, the system used serves as a good model-system to study basement membrane formation, since the cells organize in a histiotypic fashion and form true vascularized glomeruli.     

Intrinsic tyrosine fluorescence as a tool to study the interaction of the shaker B "ball" peptide with anionic membranes

Steady-state and time-resolved fluorescence from the single tyrosine in the inactivating peptide of the Shaker B potassium channel (ShB peptide) and in a noninactivating peptide mutant, ShB-L7E, has been used to characterize their interaction with anionic phospholipid membranes, a model target mimicking features of the inactivation site on the channel protein. Partition coefficients derived from steady-state anisotropy indicate that both peptides show a high affinity for anionic vesicles, being higher in ShB than in ShB-L7E. Moreover, differential quenching by lipophilic spin-labeled probes and fluorescence energy transfer using trans-parinaric acid as the acceptor confirm that the ShB peptide inserts deep into the membrane, while the ShB-L7E peptide remains near the membrane surface. The rotational mobility of tyrosine in membrane-embedded ShB, examined from the decay of fluorescence anisotropy, can be described by two different rotational correlation times and a residual constant value. The short correlation time corresponds to fast rotation reporting on local tyrosine mobility. The long rotational correlation time and the high residual anisotropy suggest that the ShB peptide diffuses in a viscous and anisotropic medium compatible with the aliphatic region of a lipid bilayer and support the hypothesis that the peptide inserts into it as a monomer, to configure an intramolecular beta-hairpin structure. Assuming that this hairpin structure behaves like a rigid body, we have estimated its dimensions and rotational dynamics, and a model for the peptide inserted into the bilayer has been proposed.     

Quantitative study of the binding of cysteine proteinases to basement membranes.

Binding of cysteine proteinases of the papain superfamily (papain and cathepsins B, B-like and L) to basement membranes was studied by using the enzymatic activity of these proteinases against their specific fluorogenic substrates. Papain inactivated by E64 was used for Kd determination by competition experiments. The binding was characterized using the following parameters, the equilibrium constant, Kd, and the number of substrate sites, n, values of which were in the range of 10(-7) M and 10(12), respectively. Such results would be of significant interest for the understanding of the biological role of cysteine proteinases in tumour invasion and other types of tissue remodeling.     

The zebrafish/tumor xenograft angiogenesis assay as a tool for screening anti-angiogenic miRNAs

The zebrafish/tumor xenograft angiogenesis assay is used to approach tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Here, we evaluated whether the assay could allow the identification of microRNAs having an anti-angiogenic potential. For that, we transfected DU-145 prostate cancer cells with four microRNAs (miR-125a, miR-320, miR-487b, miR-492) responsive to both anti- and pro-angiogenic stimuli applied to human umbilical vein endothelial cells. After transfection, DU-145 cells were injected close to the developing subintestinal vessels of transgenic Tg(Kdrl:eGFP)s843 zebrafish embryos that express green fluorescent protein under the control of Kdrl promoter. At 72 h post-fertilization, we observed that green fluorescent protein–positive neo-vessels infiltrated the graft of DU-145 transfected with miR-125a, miR-320, and miR-487b. Vice versa, neo-vessel formation and tumor cell infiltration were inhibited when DU-145 cells transfected with miR-492 were used. These results indicated that the zebrafish/tumor xenograft assay was adequate to identify microRNAs able to suppress the release of angiogenic growth factors by angiogenic tumor cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-014-9735-y) contains supplementary material, which is available to authorized users.     

The quartz crystal microbalance as a continuous monitoring tool for the study of endothelial cell surface attachment and growth

The quartz crystal microbalance (QCM) was used to monitor endothelial cell (EC) adhesion on the gold surface of an oscillating quartz crystal contained in a QCM device. A number of parameters were investigated. First, we observed differential QCM O-ring toxicities for ECs. Second, appropriate conditions for cell culture and QCM cell environment were identified that can eliminate large-scale frequency oscillations in the measurements. These artifacts are not due to added cells but originate in the time-dependent evaporation of water. Having eliminated these artifacts, we then demonstrated that the measured steady-state crystal frequency shift, Delta f, and motional resistance shift, DeltaR, were determined by the number of firmly attached ECs requiring trypsinization from the crystal surface. Last, following steady-state attachment of ECs, the EC growth stimulation by fibroblast growth factor was monitored in a continuous fashion by measuring f and R values over a 72 h. period. We observed the Delta f values to increase in a way that reflected the increase in EC number bound to the QCM surface. Following addition of ECs to the QCM, the time-dependent increase in DeltaR can be interpreted in terms of increase by the ECs of the energy dissipation properties of the solution at the solution-gold surface interface. This effect is due to their rapid surface attachment and the elaboration of their cytoskeletal properties. These results indicate that the QCM technique can be used for the study of EC attachment and growth and suggest its potential for the real time study of per unit surface area cell mass distribution dynamics and viscoelastic properties and the cells' responses to stresses or perturbations brought about using biologically active molecules.