Establishment and Molecular Cytogenetic Characterization of a Cell Culture Model of Head and Neck Squamous Cell Carcinoma (HNSCC)

Bauer, Verena; Hieber, Ludwig; Schaeffner, Quirin; Weber, Johannes; Braselmann, Herbert; Huber, R.; Walch, Axel; Zitzelsberger, Horst

doi:10.3390/genes1030388

Cited by 11 publications

(9 citation statements)

References 75 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Black, clear bottom, 384 well HCS Assay Plates (Cat# 781091) were purchased from Greiner Bio-One (Monroe, NC). Cal33 human head and neck squamous cell carcinoma (HNSCC) cells [37, 38] were kindly provided by Dr. Gerard Milano (University of Nice, Nice, France). The cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA), 100 U/ml penicillin and 100 mg/ml streptomycin (HyClone, Logan, UT).…”

Section: Methodsmentioning

confidence: 99%

A metric and workflow for quality control in the analysis of heterogeneity in phenotypic profiles and screens

Gough

Shun

Taylor

et al. 2016

Methods

View full text Add to dashboard Cite

Heterogeneity is well recognized as a common property of cellular systems that impacts biomedical research and the development of therapeutics and diagnostics. Several studies have shown that analysis of heterogeneity: gives insight into mechanisms of action of perturbagens; can be used to predict optimal combination therapies; and to quantify heterogeneity in tumors where heterogeneity is believed to be associated with adaptation and resistance. Cytometry methods including high content screening (HCS), high throughput microscopy, flow cytometry, mass spec imaging and digital pathology capture cell level data for populations of cells. However it is often assumed that the population response is normally distributed and therefore that the average adequately describes the results. A deeper understanding of the results of the measurements and more effective comparison of perturbagen effects requires analysis that takes into account the distribution of the measurements, i.e. the heterogeneity. However, the reproducibility of heterogeneous data collected on different days, and in different plates/slides has not previously been evaluated. Here we show that conventional assay quality metrics alone are not adequate for quality control of the heterogeneity in the data. To address this need, we demonstrate the use of the Kolmogorov-Smirnov statistic as a metric for monitoring the reproducibility of heterogeneity in an SAR screen, describe a workflow for quality control in heterogeneity analysis. One major challenge in high throughput biology is the evaluation and interpretation of heterogeneity in thousands of samples, such as compounds in a cell-based screen. In this study we also demonstrate that three heterogeneity indices previously reported, capture the shapes of the distributions and provide a means to filter and browse big data sets of cellular distributions in order to compare and identify distributions of interest. These metrics and methods are presented as a workflow for analysis of heterogeneity in large scale biology projects.

show abstract

Section: Methodsmentioning

confidence: 99%

A metric and workflow for quality control in the analysis of heterogeneity in phenotypic profiles and screens

Gough

Shun

Taylor

et al. 2016

Methods

View full text Add to dashboard Cite

show abstract

“…We demonstrate the ML method for two examples. For cell survival curves it is demonstrated on colony counts of irradiation experiments with a pair of two human head and neck squamous cell carcinoma (HNSCC) cell lines, CAL33 [ 8 ] and OKF6/TERT1 [ 9 ] which were irradiated with five different doses up to 6 Gy. The second example was taken from [ 4 ].…”

Section: Methodsmentioning

confidence: 99%

CFAssay: statistical analysis of the colony formation assay

et al. 2015

Self Cite

View full text Add to dashboard Cite

BackgroundColony formation assay is the gold standard to determine cell reproductive death after treatment with ionizing radiation, applied for different cell lines or in combination with other treatment modalities. Associated linear-quadratic cell survival curves can be calculated with different methods. For easy code exchange and methodological standardisation among collaborating laboratories a software package CFAssay for R (R Core Team, R: A Language and Environment for Statistical Computing, 2014) was established to perform thorough statistical analysis of linear-quadratic cell survival curves after treatment with ionizing radiation and of two-way designs of experiments with chemical treatments only.MethodsCFAssay offers maximum likelihood and related methods by default and the least squares or weighted least squares method can be optionally chosen. A test for comparision of cell survival curves and an ANOVA test for experimental two-way designs are provided.ResultsFor the two presented examples estimated parameters do not differ much between maximum-likelihood and least squares. However the dispersion parameter of the quasi-likelihood method is much more sensitive for statistical variation in the data than the multiple R2 coefficient of determination from the least squares method.ConclusionThe dispersion parameter for goodness of fit and different plot functions in CFAssay help to evaluate experimental data quality. As open source software interlaboratory code sharing between users is facilitated.AvailabilityThe package is available at http://www.bioconductor.org/packages/release/bioc/html/CFAssay.html.

show abstract

“…37 Cal33 cells are of epithelial origin that display chromosomal rearrangements and copy number changes which mirror the cytogenetic status of primary HNSCC. 36 To configure the pSTAT3 and pSTAT1 assays for the IXU-automated HCS platform, we developed sample preparation protocols, image acquisition procedures, and image analysis settings such that only minor adjustments to the laser power and/or PMT gain were required to ensure that the Hoechst stained nuclei and fluorescent pSTAT3/ pSTAT1 staining did not saturate the PMT detectors. Similarly, after we had established and optimized the parameter settings of the TE image analysis module (Fig.…”

Section: Discussionmentioning

confidence: 99%

High-Content pSTAT3/1 Imaging Assays to Screen for Selective Inhibitors of STAT3 Pathway Activation in Head and Neck Cancer Cell Lines

Johnston

SenMalabika

Hua-yun

et al. 2014

ASSAY and Drug Development Technologies

View full text Add to dashboard Cite

The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is hyperactivated in most cancers and represents a plausible therapeutic target. In the absence of STAT3-selective smallmolecule inhibitors, we sought to develop pSTAT3/1 high-content imaging (HCS) assays to screen for selective inhibitors of STAT3 pathway activation in head and neck squamous cell carcinomas (HNSCC) tumor cell lines. Based on the expression of the interleukin-6 (IL-6)Ra and gp130 subunits of the IL-6 receptor complex and STAT3, we selected the Cal33 HNSCC cell line as our model. After developing image acquisition and analysis procedures, we rigorously investigated the cytokine activation responses to optimize the dynamic ranges of both assays and demonstrated that the pan-Janus kinase inhibitor pyridone 6 nonselectively inhibited pSTAT3 and pSTAT1 activation with 50% inhibition concentrations of 7.19 -4.08 and 16.38 -8.45 nM, respectively. The optimized pSTAT3 HCS assay performed very well in a pilot screen of 1,726 compounds from the Library of Pharmacologically Active Compounds and the National Institutes of Health clinical collection sets, and we identified 51 inhibitors of IL-6-induced pSTAT3 activation. However, only three of the primary HCS actives selectively inhibited STAT3 compared with STAT1. Our follow-up studies indicated that the nonselective inhibition of cytokine induced pSTAT3 and pSTAT1 activation by G-alpha stimulatory subunit-coupled G-protein-coupled receptor agonists, and forskolin was likely due to cyclic adenosine monophosphatemediated up-regulation of suppressors of cytokine signaling 3. Azelastine, an H 1 receptor antagonist approved for the treatment of seasonal allergic rhinitis, nonallergic vasomotor rhinitis, and ocular conjunctivitis, was subsequently confirmed as a selective inhibitor of IL-6-induced pSTAT3 activation that also reduced the growth of HNSCC cell lines. These data illustrate the power of a chemical biology approach to lead generation that utilizes fully developed and optimized HCS assays as phenotypic screens to interrogate specific signaling pathways.

show abstract

Establishment and Molecular Cytogenetic Characterization of a Cell Culture Model of Head and Neck Squamous Cell Carcinoma (HNSCC)

Cited by 11 publications

References 75 publications

A metric and workflow for quality control in the analysis of heterogeneity in phenotypic profiles and screens

A metric and workflow for quality control in the analysis of heterogeneity in phenotypic profiles and screens

CFAssay: statistical analysis of the colony formation assay

High-Content pSTAT3/1 Imaging Assays to Screen for Selective Inhibitors of STAT3 Pathway Activation in Head and Neck Cancer Cell Lines

Contact Info

Product

Resources

About