Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Optimized in vitro three-dimensional invasion assay for quantifying a wide range of cancer cell invasive behavior.

ABSTRACT: We describe a three-dimensional (3D) in vitro assay for quantifying cancer cell invasion into a 3D microenvironment with defined biochemical and biophysical properties. Researchers can quantify invasion dynamics (e.g., cell motility and directionality) and examine morphological changes during invasion, using live-cell and confocal imaging techniques. Together, these advantages over existing in vitro invasion assays, such as transwell-based assays, provide researchers with a valuable tool to gain insight into the mechanisms regulating cancer cell invasion. For complete details on the use and execution of this protocol, please refer to Padilla-Rodriguez et al. (2018) and Watson et al. (2021).

SUBMITTER: Hill SM

PROVIDER: S-EPMC9256944 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Optimized in vitro three-dimensional invasion assay for quantifying a wide range of cancer cell invasive behavior.

Hill Samantha M SM Padilla-Rodriguez Marco M Clements Amber A Sweetland Jillian A JA Parker Sara S SS Warfel Noel A NA Mouneimne Ghassan G

STAR protocols 20220701 3

We describe a three-dimensional (3D) in vitro assay for quantifying cancer cell invasion into a 3D microenvironment with defined biochemical and biophysical properties. Researchers can quantify invasion dynamics (e.g., cell motility and directionality) and examine morphological changes during invasion, using live-cell and confocal imaging techniques. Together, these advantages over existing in vitro invasion assays, such as transwell-based assays, provide researchers with a valuable tool to gain ...[more]

PMID: 35780429

Similar Datasets

Fabrication of three dimensional patterns of wide dimensional range using microbes and their applications.

Project description:Inspired by the wound healing property of certain trees, we report a novel microbes based additive process for producing three dimensional patterns, which has a potential of engineering applications in a variety of fields. Imposing a two dimensional pattern of microbes on a gel media and allowing them to grow in the third dimension is known from its use in biological studies. Instead, we have introduced an intermediate porous substrate between the gel media and the microbial growth, which enables three dimensional patterns in specific forms that can be lifted off and used in engineering applications. In order to demonstrate the applicability of this idea in a diverse set of areas, two applications are selected. In one, using this method of microbial growth, we have fabricated microlenses for enhanced light extraction in organic light emitting diodes, where densely packed microlenses of the diameters of hundreds of microns lead to luminance increase by a factor of 1.24X. In another entirely different type of application, braille text patterns are prepared on a normal office paper where the grown microbial colonies serve as braille tactile dots. Braille dot patterns thus prepared meet the standard specifications (size and spacing) for braille books.

| S-EPMC4613880 | biostudies-literature

Validated prediction of pro-invasive growth factors using a transcriptome-wide invasion signature derived from a complex 3D invasion assay.

Project description:The invasion of activated fibroblasts represents a key pathomechanism in fibrotic diseases, carcinogenesis and metastasis. Invading fibroblasts contribute to fibrotic extracellular matrix (ECM) formation and the initiation, progression, or resistance of cancer. To construct transcriptome-wide signatures of fibroblast invasion, we used a multiplex phenotypic 3D invasion assay using lung fibroblasts. Microarray-based gene expression profiles of invading and non-invading fibroblasts demonstrated that 1,049 genes were differentially regulated (>1.5-fold). Unbiased pathway analysis (Ingenuity) identified significant enrichment for the functional clusters 'invasion of cells', 'idiopathic pulmonary fibrosis', and 'metastasis'. Matrix metalloprotease 13 (MMP13), transforming growth factor (TGF)-β1, Caveolin (Cav) 1, Phosphatase and Tensin Homolog (Pten), and secreted frizzled-related protein (Sfrp) 1 were among the highest regulated genes, confirmed by qRT-PCR and Western Blotting. We next performed in silico analysis (Ingenuity Pathway Analysis) to predict mediators that induced fibroblast invasion. Of these, TGFβ1, epidermal growth factor (EGF), fibroblast growth factor (FGF) 2, and platelet-derived growth factor (PDGF)-BB were tested in our 3D invasion assay and found to significantly induce invasion, thus validating the transcriptome profile. Accordingly, our transcriptomic invasion signature describes the invading fibroblast phenotype in unprecedented detail and provides a tool for future functional studies of cell invasion and therapeutic modulation thereof using complex phenotypic assays.

| S-EPMC4525140 | biostudies-literature

Quantifying two-dimensional and three-dimensional stereoscopic learning in anatomy using electroencephalography.

Project description:Advances in computer visualization enabling both 2D and 3D representation have generated tools to aid perception of spatial relationships and provide a new forum for instructional design. A key knowledge gap is the lack of understanding of how the brain neurobiologically processes and learns from spatially presented content, and new quantitative variables are required to address this gap. The objective of this study was to apply quantitative neural measures derived from electroencephalography (EEG) to examine stereopsis in anatomy learning by comparing mean amplitude changes in N250 (related to object recognition) and reward positivity (related to responding to feedback) event related to potential components using a reinforcement-based learning paradigm. Health sciences students (n = 61) learned to identify and localize neuroanatomical structures using 2D, 3D, or a combination of models while EEG and behavioral (accuracy) data were recorded. Participants learning using 3D models had a greater object recognition (N250 amplitude) compared to those who learned from 2D models. Based on neurological results, interleaved learning incorporating both 2D and 3D models provided an advantage in learning, retention, and transfer activities represented by decreased reward positivity amplitude. Behavioral data did not have the same sensitivity as neural data for distinguishing differences in learning with and without stereopsis in these learning activities. Measuring neural activity reveals new insights in applied settings for educators to consider when incorporating stereoscopic models in the design of learning interventions.

| S-EPMC6637108 | biostudies-literature

Probing three-dimensional collective cancer invasion with DIGME.

Project description:BackgroundMulticellular pattern formation plays an important role in developmental biology, cancer metastasis and wound healing. While many physical factors have been shown to regulate these multicellular processes, the role of ECM micro-to-meso scale geometry has been poorly understood in 3D collective cancer invasion.ResultsWe have developed a mechanical-based strategy, Diskoid In Geometrically Micropatterned ECM (DIGME). DIGME allows easy engineering of the shape of 3D tissue organoid, the mesoscale ECM heterogeneity, and the fiber alignment of collagen-based ECM all at the same time. We have employed DIGME to study the 3D invasion of MDA-MB-231 diskoids in engineered collagen matrix. We find that the collective cancer invasion is closely regulated by the micro-to-meso scale geometry of the ECM.ConclusionsWe conclude that DIGME provides a simple yet powerful tool to probe 3D dynamics of tissue organoids in physically patterned microenvironments.

| S-EPMC5876692 | biostudies-literature

Quantifying Intracellular Nanoparticle Distributions with Three-Dimensional Super-Resolution Microscopy.

Project description:Super-resolution microscopy can transform our understanding of nanoparticle-cell interactions. Here, we established a super-resolution imaging technology to visualize nanoparticle distributions inside mammalian cells. The cells were exposed to metallic nanoparticles and then embedded within different swellable hydrogels to enable quantitative three-dimensional (3D) imaging approaching electron-microscopy-like resolution using a standard light microscope. By exploiting the nanoparticles' light scattering properties, we demonstrated quantitative label-free imaging of intracellular nanoparticles with ultrastructural context. We confirmed the compatibility of two expansion microscopy protocols, protein retention and pan-expansion microscopy, with nanoparticle uptake studies. We validated relative differences between nanoparticle cellular accumulation for various surface modifications using mass spectrometry and determined the intracellular nanoparticle spatial distribution in 3D for entire single cells. This super-resolution imaging platform technology may be broadly used to understand the nanoparticle intracellular fate in fundamental and applied studies to potentially inform the engineering of safer and more effective nanomedicines.

| S-EPMC10643044 | biostudies-literature

Three-dimensional neurophenotyping of adult zebrafish behavior.

Project description:The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior.

| S-EPMC3049776 | biostudies-literature

Validated prediction of pro-invasive growth factors using a transcriptome-wide invasion signature derived from a complex 3-D invasion assay

Project description:The invasion of activated fibroblasts represents a key pathomechanism in fibrotic diseases, carcinogenesis and metastasis. Here, invading fibroblasts contribute to fibrotic extracellular matrix (ECM) formation and the initiation, progression, or resistance of cancer, respectively. To construct a transcriptome-wide signature of fibroblast invasion, we used a multiplex phenotypic 3D invasion assay using murine lung fibroblasts. Microarray-based gene expression profiles of invading and non-invading fibroblasts were highly distinct: 1049 genes were differentially regulated (>1.5-fold). An unbiased pathway analysis (Ingenuity) identified a significant enrichment for the functional clusters ‘invasion of cells’, ‘idiopathic pulmonary fibrosis (IPF)’ and ‘metastasis’. Particularly, matrix metalloprotease13 (MMP13), transforming growth factor (TGF)β1, Caveolin1 (Cav1), Phosphatase and Tensin Homolog (Pten), and secreted frizzled-related protein1 (Sfrp1) were among the highest regulated genes. In silico analysis by Ingenuity predicted TGFβ1, epidermal growth factor (EGF), fibroblast growth factor2 (FGF2), and platelet-derived growth factor (PDGF)-BB to induce invasion. As such, these growth factors were tested in the 3D invasion assay and displayed a significant induction of invasion, thus validating the transcriptome profile. Accordingly, our transcriptomic invasion signature describes the invading fibroblast phenotype in unprecedented detail and provides a tool for future functional studies of cell invasion and therapeutic modulation thereof.

2015-10-05 | GSE55322 | GEO

An Optimized Transient Dual Luciferase Assay for Quantifying MicroRNA Directed Repression of Targeted Sequences.

Project description:Studies investigating the action of small RNAs on computationally predicted target genes require some form of experimental validation. Classical molecular methods of validating microRNA action on target genes are laborious, while approaches that tag predicted target sequences to qualitative reporter genes encounter technical limitations. The aim of this study was to address the challenge of experimentally validating large numbers of computationally predicted microRNA-target transcript interactions using an optimized, quantitative, cost-effective, and scalable approach. The presented method combines transient expression via agroinfiltration of Nicotiana benthamiana leaves with a quantitative dual luciferase reporter system, where firefly luciferase is used to report the microRNA-target sequence interaction and Renilla luciferase is used as an internal standard to normalize expression between replicates. We report the appropriate concentration of N. benthamiana leaf extracts and dilution factor to apply in order to avoid inhibition of firefly LUC activity. Furthermore, the optimal ratio of microRNA precursor expression construct to reporter construct and duration of the incubation period post-agroinfiltration were determined. The optimized dual luciferase assay provides an efficient, repeatable and scalable method to validate and quantify microRNA action on predicted target sequences. The optimized assay was used to validate five predicted targets of rice microRNA miR529b, with as few as six technical replicates. The assay can be extended to assess other small RNA-target sequence interactions, including assessing the functionality of an artificial miRNA or an RNAi construct on a targeted sequence.

| S-EPMC5611435 | biostudies-literature

A facile and specific assay for quantifying microRNA by an optimized RT-qPCR approach.

Project description:BackgroundThe spatiotemporal expression patterns of microRNAs (miRNAs) are important to the verification of their predicted function. RT-qPCR is the accepted technique for the quantification of miRNA expression; however, stem-loop RT-PCR and poly(T)-adapter assay, the two most frequently used methods, are not very convenient in practice and have poor specificity, respectively.ResultsWe have developed an optimal approach that integrates these two methods and allows specific and rapid detection of tiny amounts of sample RNA and reduces costs relative to other techniques. miRNAs of the same sample are polyuridylated and reverse transcribed into cDNAs using a universal poly(A)-stem-loop RT primer and then used as templates for SYBR® Green real-time PCR. The technique has a dynamic range of eight orders of magnitude with a sensitivity of up to 0.2 fM miRNA or as little as 10 pg of total RNA. Virtually no cross-reaction is observed among the closely-related miRNA family members and with miRNAs that have only a single nucleotide difference in this highly specific assay. The spatial constraint of the stem-loop structure of the modified RT primer allowed detection of miRNAs directly from cell lysates without laborious total RNA isolation, and the poly(U) tail made it possible to use multiplex RT reactions of mRNA and miRNAs in the same run.ConclusionsThe cost-effective RT-qPCR of miRNAs with poly(A)-stem-loop RT primer is simple to perform and highly specific, which is especially important for samples that are precious and/or difficult to obtain.

| S-EPMC3465266 | biostudies-literature

Quantifying two-dimensional filamentous and invasive growth spatial patterns in yeast colonies.

Project description:The top-view, two-dimensional spatial patterning of non-uniform growth in a Saccharomyces cerevisiae yeast colony is considered. Experimental images are processed to obtain data sets that provide spatial information on the cell-area that is occupied by the colony. A method is developed that allows for the analysis of the spatial distribution with three metrics. The growth of the colony is quantified in both the radial direction from the centre of the colony and in the angular direction in a prescribed outer region of the colony. It is shown that during the period of 100-200 hours from the start of the growth of the colony there is an increasing amount of non-uniform growth. The statistical framework outlined in this work provides a platform for comparative quantitative assays of strain-specific mechanisms, with potential implementation in inferencing algorithms used for parameter-rate estimation.

| S-EPMC4342342 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data