A Unique 3D In Vitro Cellular Invasion Assay (original) (raw)
Related papers
Human Tumor Tissue-Based 3D In Vitro Invasion Assays
Methods in molecular biology (Clifton, N.J.), 2018
Here we describe a protocol to utilize human benign leiomyoma tissue in in vitro 3D model that enables an assessment of cell invasion. The chapter also describes detailed instructions for image analysis to quantify the results. Leiomyoma is a benign tumor of the uterus which mimics authentic components of the tumor microenvironment including fibroblasts, vessels, collagen fibers, and extracellular protein composition. The leiomyoma invasion model represents a superior 3D model for cell invasion studies compared to the other non-human organotypic models.
In Vivo Assay for Tumor Cell Invasion
Methods in Molecular Biology, 2009
We describe an in vivo invasion assay that enables the collection of invasive cells from the primary tumor. In addition to determination of the endogenous, unstimulated invasive properties of cells in vivo, the assay can take advantage of the chemotactic properties of cancer cells. Microneedles are filled with a mixture of extracellular matrix components such as Matrigel with or without a chemoattractant such as EGF, and then introduced into the primary tumor of a rat or mouse that is generated either by orthotopic injection of carcinoma cell lines or by a transgene such as polyoma Middle T. Over the course of 4 h the invasive cell population enters the needles while the animal is kept under anesthesia. At the end of the collection time, the invasive cells are extruded from the microneedles and can be analyzed in terms of the number and type of cells that invade in response to defined stimuli. By including pharmacological inhibitors in the needle, signaling pathways contributing to in vivo invasion can also be identified. This assay leads to a better understanding of the cell types and signaling involved in the tumor microenvironment, and has the potential to be applied to a variety of in vivo models.
Two-dimensional vs. three-dimensional in vitro tumor migration and invasion assays
2013
Motility and invasion are key hallmarks that distinguish benign from malignant tumors, enabling cells to cross tissue boundaries, disseminate in blood and lymph and establish metastases at distant sites. Similar properties are also utilized by activated endothelial cells during tumor-induced angiogenesis. It is now appreciated that these processes might provide a rich source of novel molecular targets with the potential for inhibitors to restrain both metastasis and neoangiogenesis. Such therapeutic strategies require assays that can rapidly and quantitatively measure cell movement and the ability to traverse physiological barriers. The need for high-throughput, however, must be balanced by assay designs that accommodate, as far as possible, the complexity of the in vivo tumor microenvironment. This chapter aims to give an overview of some commonly used migration and invasion assays to aid in the selection of a balanced portfolio of techniques for the rapid and accurate evaluation of novel therapeutic agents.
BMC Cancer, 2010
Introduction Tumour phenotype is regulated in a complex fashion as a result of interactions between malignant cells and the tumour stroma. Fibroblasts are the most abundant and perhaps most active part of the tumour stroma. A better understanding of the changes that occur in fibroblasts in response to the presence of malignant cells may lead to the development of new strategies for cancer treatment. We explored the effects of fibroblasts on the growth and invasion of mammary carcinoma tumour cells in vitro and in vivo. Methods In order to analyse secreted factors that affect invasive abilities of breast cancer cells we co-cultured human mammary fibroblasts (HMF3s) and cancer cells (MCF7S1) in three-dimensional (3D) growth conditions devoid of heterogeneous cell-cell contact. To study the possible influence of fibroblasts on MCF7S1 cancer cell growth in vivo we co-injected HMF3s and MCF7S1 cells in Balb/c nu/nu mice. Results In 3D co-culture both HMF3s and MCF7S1 cells demonstrated e...
Breast Cancer Cell Invasion into a Three Dimensional Tumor-Stroma Microenvironment
Scientific Reports, 2016
In this study, to model 3D chemotactic tumor-stroma invasion in vitro, we developed an innovative microfluidic chip allowing side-by-side positioning of 3D hydrogel-based matrices. We were able to (1) create a dual matrix architecture that extended in a continuous manner, thus allowing invasion from one 3D matrix to another, and (2) establish distinct regions of tumor and stroma cell/ECM compositions, with a clearly demarcated tumor invasion front, thus allowing us to quantitatively analyze progression of cancer cells into the stroma at a tissue or single-cell level. We showed significantly enhanced cancer cell invasion in response to a transient gradient of epidermal growth factor (EGF). 3D tracking at the single-cell level displayed increased migration speed and persistence. Subsequently, we analyzed changes in expression of EGF receptors, cell aspect ratio, and protrusive activity. These findings show the unique ability of our model to quantitatively analyze 3D chemotactic invasion, both globally by tracking the progression of the invasion front, and at the single-cell level by examining changes in cellular behavior and morphology using high-resolution imaging. Taken together, we have shown a novel model recapitulating 3D tumor-stroma interactions for studies of real-time cell invasion and morphological changes within a single platform.
Development of a High-Throughput Three-Dimensional Invasion Assay for Anti-Cancer Drug Discovery
PLoS ONE, 2013
The lack of three-dimensional (3-D) high-throughput (HT) screening assays designed to identify anti-cancer invasion drugs is a major hurdle in reducing cancer-related mortality, with the key challenge being assay standardization. Presented is the development of a novel 3-D invasion assay with HT potential that involves surrounding cell-collagen spheres within collagen to create a 3-D environment through which cells can invade. Standardization was achieved by designing a tooled 96-well plate to create a precisely designated location for the cell-collagen spheres and by using dialdehyde dextran to inhibit collagen contraction, maintaining uniform size and shape. This permits automated readout for determination of the effect of inhibitory compounds on cancer cell invasion. Sensitivity was demonstrated by the ability to distinguish varying levels of invasiveness of cancer cell lines, and robustness was determined by calculating the Z-factor. A Z-factor of 0.65 was obtained by comparing the effects of DMSO and anti-β1-integrin antibody, an inhibitory reagent, on the invasion of Du145 cancer cells, suggesting this novel assay is suitable for large scale drug discovery. As proof of principle, the NCI Diversity Compound Library was screened against human invasive cancer cells. Nine compounds exhibiting high potency and low toxicity were identified, including DX-52-1, a compound previously reported to inhibit cell migration, a critical determinant of cancer invasion. The results indicate that this innovative HT platform is a simple, precise, and easy to replicate 3-D invasion assay for anti-cancer drug discovery.
Clinical & experimental metastasis, 2002
Growth of human tumor cells as three-dimensional (3D) multicellular spheroids modifies their invasive properties. Here we study the differences in the biological features of MCF-7, a human breast cancer cell line, and its multidrug resistant variant (MDR-MCF-7) cultured as spheroids or as monolayers. Three-dimensional culture decreased the proliferative rate of both cell lines, reduced the drug sensitivity of MCF-7 cells and did not affect the resistance of MDR-MCF-7 cells. Transmission electron microscopic studies and intercellular junctions labeling showed that MCF-7 spheroids had a junctional system involving E-cadherin, tight-junctions and desmosomes. In MDR-MCF-7 cell spheroids, cell cohesion was mostly due to membrane interdigitations. MDR-MCF-7 cells, but not their parental counterpart, displayed a higher invasive potential when cultured as spheroids, as shown in the Boyden chamber assay. 3D-induced invasiveness was correlated with serine protease and plasminogen activator (P...
An in vitro quantitative assay for tumor cell invasion
Cancer Research
An in vitro quantitative assay of tumor cell invasion is described. The assay measures the rate at which [12sl]iododeoxyuridine-labeledtumor cells migrate through the chorioallantoic membrane of developing chicken em bryos. Invasion chambers were prepared from amber latex cylinders, chorioallantoic membrane, and thread liga tures. The chambers were placed in glass vials to rest on discs of photographic sponge immersed in tissue culture medium. Labeled tumor cells were added to the chamber, and the vial was incubated at 37°. At various time points individual chambers were monitored to determine the number of viable, labeled cells that had passed through the membrane and were present in the medium, on the glass vial, or in the sponge. The application of the assay may be of use in delineat ing the mechanisms of tumor cell invasion. 2 The abbreviations used are: [125)ldUrd, [125l]iododeoxyuridine; CMEM, complete Eagle's minimum essential medium.