Quantitation of Macropinocytosis in Glioblastoma Based on High-Content Analysis

2021 ◽  
Author(s):  
Bo Wang ◽  
Xuan Yao ◽  
Qiang Dong ◽  
Xiao-fang Wang ◽  
Hang Yin ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Content Analysis ◽  
Tumor Microenvironment ◽  
High Throughput ◽  
Extracellular Fluid ◽  
Visual Evaluation ◽  
Time Saving ◽  
The Past ◽  
High Content Analysis ◽  
High Throughput Assay

Abstract Background: Macropinocytosis serves as an internalization pathway for extracellular fluid, albumin and dissolved molecules. Assessing macropinocytosis has been challenging in the past because manual acquisition in combination with visual evaluation of images is laborious, making it difficult for high-throughput applications. So, there is a need to develop sensitive and specific methods. Methods: This paper proposed a quantitative and time-saving method for macropinocytosis detection based on high-content analysis (HCA). Meanwhile, cell proliferation was tested by means of CCK8. Results: The term “macropinosome index” was defined to estimate macropinocytosis and allow comparison between different cell lines and treatments. Furthermore, we demonstrated that macropinocytosis can promote Glioblastoma (GBM) cells survival in glutamine deficient conditions which resemble tumor microenvironment. Conclusions: HCA represents a novel, non-subjective and high-throughput assay for macropinocytosis assessment. Besides, Gln deprivation increased the macropinosome index in GBM cells, which points to the possible exploitation of this process in the design of GBM therapies.

scholarly journals A Novel High Throughput High Content Analysis Assay for Intermediate Filament Perturbing Drugs

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Bernard Michael Corfe ◽  
Joanna Chowdry ◽  
Gareth J. Griffiths ◽  
Rod P. Benson
Keyword(s):  
Content Analysis ◽  
High Throughput ◽  
Intermediate Filament ◽  
High Content Analysis

High Content Analysis of an In Vitro Model for Metabolic Toxicity

2014 ◽  
Vol 19 (10) ◽  
pp. 1402-1408 ◽  
Author(s):  
Stephanie D. Cole ◽  
Janna S. Madren-Whalley ◽  
Albert P. Li ◽  
Russell Dorsey ◽  
Harry Salem
Keyword(s):  
Content Analysis ◽  
High Throughput ◽  
Metabolic Activation ◽  
Hepatic Metabolism ◽  
Cell Types ◽  
Multiple Organ ◽  
Compound Libraries ◽  
High Content Analysis ◽  
Calcein Am

In vitro models that accurately and rapidly assess hepatotoxicity and the effects of hepatic metabolism on nonliver cell types are needed by the U.S. Department of Defense and the pharmaceutical industry to screen compound libraries. Here, we report the first use of high content analysis on the Integrated Discrete Multiple Organ Co-Culture (IdMOC) system, a high-throughput method for such studies. We cultured 3T3-L1 cells in the presence and absence of primary human hepatocytes, and exposed the cultures to 4-aminophenol and cyclophosphamide, model toxicants that are respectively detoxified and activated by the liver. Following staining with calcein-AM, ethidium homodimer-1, and Hoechst 33342, high content analysis of the cultures revealed four cytotoxic endpoints: fluorescence intensities of calcein-AM and ethidium homodimer-1, nuclear area, and cell density. Using these endpoints, we observed that the cytotoxicity of 4-aminophenol in 3T3-L1 cells in co-culture was less than that observed for 3T3-L1 monocultures, consistent with the known detoxification of 4-aminophenol by hepatocytes. Conversely, cyclophosphamide cytotoxicity for 3T3-L1 cells was enhanced by co-culturing with hepatocytes, consistent with the known metabolic activation of this toxicant. The use of IdMOC plates combined with high content analysis is therefore a multi-endpoint, high-throughput capability for measuring the effects of metabolism on toxicity.

scholarly journals Multidimensional Profiling of CSF1R Screening Hits and Inhibitors

2011 ◽  
Vol 16 (9) ◽  
pp. 1007-1017 ◽  
Author(s):  
Joost C. M. Uitdehaag ◽  
Cecile M. Sünnen ◽  
Antoon M. van Doornmalen ◽  
Nikki de Rouw ◽  
Arthur Oubrie ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Active Form ◽  
Dissociation Rate ◽  
The Past ◽  
Compound Libraries ◽  
Competitive Kinetics ◽  
Homogeneous Assay ◽  
High Throughput Assay ◽  
Stimulating Factor

Over the past years, improvements in high-throughput screening (HTS) technology and compound libraries have resulted in a dramatic increase in the amounts of good-quality screening hits, and there is a growing need for follow-on hit profiling assays with medium throughput to further triage hits. Here the authors present such assays for the colony-stimulating factor 1 receptor (CSF1R, Fms), including tests for cellular activity and a homogeneous assay to measure affinity for inactive CSF1R. They also present a high-throughput assay to measure target residence time, which is based on competitive binding kinetics. To better fit koff rates, they present a modified mathematical model for competitive kinetics. In all assays, they profiled eight reference inhibitors (imatinib, sorafenib, sunitinib, tandutinib, dasatinib, GW2580, Ki20227, and J&J’s pyrido[2,3-d]pyrimidin-5-one). Using the known biochemical selectivities of these inhibitors, which can be quantified using metrics such as the selectivity entropy, the authors have determined which assay readout best predicts hit selectivity. Their profiling shows surprisingly that imatinib has a preference for the active form of CSF1R and that Ki20227 has an unusually slow target dissociation rate. This confirms that follow-on hit profiling is essential to ensure that the best hits are selected for lead optimization.

KCNN4 promotes invasion and metastasis through the MAPK/ERK pathway in hepatocellular carcinoma

2019 ◽  
Vol 68 (1) ◽  
pp. 68-74
Author(s):  
Qiu-Ting Li ◽  
Yi-Ming Feng ◽  
Zun-Hui Ke ◽  
Meng-Jun Qiu ◽  
Xiao-Xiao He ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Wound Healing ◽  
Content Analysis ◽  
Mapk Pathway ◽  
Migration And Invasion ◽  
Erk Pathway ◽  
Invasion And Metastasis ◽  
High Content Analysis ◽  
Hcc Cells

Hepatocellular Carcinoma (HCC) is one of the most common malignancies in the world, and is well-known for its bad prognosis. Potassium calcium-activated channel subfamily N member 4 (KCNN4) is a type of intermediate conductance calcium-activated potassium channel, and increasing evidence suggests that KCNN4 contributes to the regulation of invasion and metastasis in a number of cancers. However, its clinical significance and biological function remain unclear in the HCC disease process. In this study, the expression levels of KCNN4 in 86 HCC samples were compared with corresponding paracancerous tissues. sh-RNA was used to reduce the expression of KCNN4 in Hep3B HCC cells in vitro; this was confirmed by Real time-PCR and western blotting. Wound healing, transwell assays and high content analysis were performed to investigate the tumor-promoting characteristics of KCNN4 in Hep3B HCC cells. As results, KCNN4 expression was significantly associated with preoperative serum alpha-fetoprotein level (p=0.038) and TNM stage (p=0.039). Additionally, patients with high KCNN4 amplification in HCC tissue exhibited shorter disease-free survival, whereas there was no statistical significance between KCNN4 amplification and overall survival. Wound healing and transwell assays showed that knockdown of KCNN4 expression could reduce migration and invasion abilities of HCC cells. High content analysis result showed that down-regulated KCNN4 could inhibit the ability of HCC cell proliferation. The mitogen-activated protein kinase (MAPK) pathway is active in cell proliferation, differentiation, migration, senescence, and apoptosis. Matrix metallopeptidase 9 and extracellular signal regulated kinase 1/2 (ERK1/2) were important biomarkers of MAPK/ERK pathway, knockdown of KCNN4 reduced the expression of MMP9 and ERK1/2. These findings showed that KCNN4 promotes HCC invasion and metastasis through the MAPK/ERK pathway.

scholarly journals High content analysis methods enable high throughput nematode discovery screening for viability and movement behavior in a multiplex sample in response to natural product treatment

2018 ◽  
Author(s):  
Jennifer M. Petitte ◽  
Mary H. Lewis ◽  
Tucker K. Witsil ◽  
Xiang Huang ◽  
John W. Rice
Keyword(s):  
Content Analysis ◽  
Natural Product ◽  
High Throughput ◽  
Statistical Significance ◽  
Microscopic Analysis ◽  
Movement Behavior ◽  
Large Sample ◽  
Large Numbers ◽  
Treatment Data ◽  
High Content Analysis

AbstractMonitoring nematode parasite movement and mortality in response to various treatment samples usually involves tedious manual microscopic analysis. High Content Analysis instrumentation enables rapid and high throughput collecting of large numbers of treatment data on huge numbers of individual worms. These large sample sizes and increased sample diversity result in robust, reliable results with increased statistical significance. These methods would be applicable to relevant human, crop, or animal worm parasites.

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