scholarly journals Complex Tumor Spheroids, a Tissue-Mimicking Tumor Model, for Drug Discovery and Precision Medicine

2021 ◽  
pp. 247255522110383
Author(s):  
Gurmeet Kaur ◽  
David M. Evans ◽  
Beverly A. Teicher ◽  
Nathan P. Coussens
Keyword(s):  
Drug Discovery ◽  
Precision Medicine ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cell Types ◽  
Response To Therapy ◽  
Cancer Drug ◽  
Malignant Cells ◽  
Pancreatic Cell

Malignant tumors are complex tissues composed of malignant cells, vascular cells, structural mesenchymal cells including pericytes and carcinoma-associated fibroblasts, infiltrating immune cells, and others, collectively called the tumor stroma. The number of stromal cells in a tumor is often much greater than the number of malignant cells. The physical associations among all these cell types are critical to tumor growth, survival, and response to therapy. Most cell-based screens for cancer drug discovery and precision medicine validation use malignant cells in isolation as monolayers, embedded in a matrix, or as spheroids in suspension. Medium- and high-throughput screening with multiple cell lines requires a scalable, reproducible, robust cell-based assay. Complex spheroids include malignant cells and two normal cell types, human umbilical vein endothelial cells and highly plastic mesenchymal stem cells, which rapidly adapt to the malignant cell microenvironment. The patient-derived pancreatic adenocarcinoma cell line, K24384-001-R, was used to explore complex spheroid structure and response to anticancer agents in a 96-well format. We describe the development of the complex spheroid assay as well as the growth and structure of complex spheroids over time. Subsequently, we demonstrate successful assay miniaturization to a 384-well format and robust performance in a high-throughput screen. Implementation of the complex spheroid assay was further demonstrated with 10 well-established pancreatic cell lines. By incorporating both human stromal and tumor components, complex spheroids might provide an improved model for tumor response in vivo.

scholarly journals Electroporation Knows No Boundaries

2015 ◽  
Vol 20 (8) ◽  
pp. 932-942 ◽  
Author(s):  
Christin Luft ◽  
Robin Ketteler
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Efficiency ◽  
Target Identification ◽  
Cell Types ◽  
Rnai Technology ◽  
Drug Target Identification ◽  
Interfering Rna ◽  
Calcium Phosphate Precipitation

The discovery of RNA interference (RNAi) has enabled several breakthrough discoveries in the area of functional genomics. The RNAi technology has emerged as one of the major tools for drug target identification and has been steadily improved to allow gene manipulation in cell lines, tissues, and whole organisms. One of the major hurdles for the use of RNAi in high-throughput screening has been delivery to cells and tissues. Some cell types are refractory to high-efficiency transfection with standard methods such as lipofection or calcium phosphate precipitation and require different means. Electroporation is a powerful and versatile method for delivery of RNA, DNA, peptides, and small molecules into cell lines and primary cells, as well as whole tissues and organisms. Of particular interest is the use of electroporation for delivery of small interfering RNA oligonucleotides and clustered regularly interspaced short palindromic repeats/Cas9 plasmid vectors in high-throughput screening and for therapeutic applications. Here, we will review the use of electroporation in high-throughput screening in cell lines and tissues.

Abstract 4206: Utilizing a Drosophila high throughput screening technology for cancer drug discovery

2010 ◽  
Author(s):  
Elena M. Gracheva ◽  
Eduardo J. Martinez ◽  
Jill Thompson ◽  
Dominick Thompson ◽  
Ross L. Cagan
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cancer Drug ◽  
Cancer Drug Discovery ◽  
Screening Technology

scholarly journals Identification by High-Throughput Screening of Viridin Analogs as Biochemical and Cell-Based Inhibitors of the Cell Cycle–Regulated Nek2 Kinase

2010 ◽  
Vol 15 (8) ◽  
pp. 918-927 ◽  
Author(s):  
Daniel G. Hayward ◽  
Yvette Newbatt ◽  
Lisa Pickard ◽  
Eilis Byrne ◽  
Guojie Mao ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Human Tumor ◽  
Tumor Cell Line ◽  
Cancer Drug ◽  
Human Tumor Cell ◽  
Cancer Drug Discovery ◽  
Centrosome Separation

Nek2 is a serine/threonine protein kinase that localizes to the centrosome and is implicated in mitotic regulation. Overexpression of Nek2 induces premature centrosome separation and nuclear defects indicative of mitotic errors, whereas depletion of Nek2 interferes with cell growth. As Nek2 expression is upregulated in a range of cancer cell lines and primary human tumors, inhibitors of Nek2 may have therapeutic value in cancer treatment. The authors used a radiometric proximity assay in a high-throughput screen to identify small-molecule inhibitors of Nek2 kinase activity. The assay was based on the measurement of the radiolabeled phosphorylated product of the kinase reaction brought into contact with the surface of wells of solid scintillant-coated microplates. Seventy nonaggregating hits were identified from approximately 73,000 compounds screened and included a number of toxoflavins and a series of viridin/wortmannin-like compounds. The viridin-like compounds were >70-fold selective for Nek2 over Nek6 and Nek7 and inhibited the growth of human tumor cell lines at concentrations consistent with their biochemical potencies. An automated mechanism-based microscopy assay in which centrosomes were visualized using pericentrin antibodies confirmed that 2 of the viridin inhibitors reduced centrosome separation in a human tumor cell line. The data presented show that pharmacological inhibition of Nek2 kinase results in the expected phenotype of disruption to centrosome function associated with growth inhibition and further supports Nek2 as a target for cancer drug discovery.

scholarly journals Establishing a High-Throughput and Automated Cancer Cell Proliferation Panel for Oncology Lead Optimization

2013 ◽  
Vol 18 (9) ◽  
pp. 1043-1053 ◽  
Author(s):  
Ming Lei ◽  
Humberto Ribeiro ◽  
Garrett Kolodin ◽  
James Gill ◽  
Yu-Sun Wang ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Proliferation ◽  
Drug Discovery ◽  
Tumor Cell ◽  
Cell Lines ◽  
High Throughput ◽  
Lead Optimization ◽  
Cancer Drug ◽  
Compound Identification ◽  
Proliferation Assays

Tumor cell proliferation assays are widely used for oncology drug discovery, including target validation, lead compound identification, and optimization, as well as determination of compound off-target activities. Taking advantage of robotic systems to maintain cell culture and perform cell proliferation assays would greatly increase productivity and efficiency. Here we describe the establishment of automated systems for high-throughput cell proliferation assays in a panel of 13 human tumor cell lines. These cell lines were selected from various types of human tumors containing a broad range of well-characterized mutations in multiple cellular signaling pathways. Standard procedures for cell culture and assay performance were developed and optimized in each cell line. Moreover, in-house developed software (i.e., Toolset, Curvemaster, and Biobars) was applied to analyze the data and generate data reports. Using tool compounds, we have shown that results obtained through this panel exhibit high reproducibility over a long period. Furthermore, we have demonstrated that this panel can be used to identify sensitive and insensitive cell lines for specific cancer targets, to drive cellular structure-activity relationships, and to profile compound off-target activities. All those efforts are important for cancer drug discovery lead optimization.

scholarly journals Development of a High-Throughput Fluorescence Polarization Assay to Detect Inhibitors of the FAK–Paxillin Interaction

2019 ◽  
Vol 25 (1) ◽  
pp. 21-32 ◽  
Author(s):  
Timothy Marlowe ◽  
Carlos Alvarado ◽  
Andrew Rivera ◽  
Felicia Lenzo ◽  
Rohini Nott ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Focal Adhesion ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Drug Target ◽  
Scaffolding Protein ◽  
Cancer Drug ◽  
Screening Assay ◽  
High Throughput Screening Assay

Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and its critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening have exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein–protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion–associated protein. We designed a tetramethylrhodamine (TAMRA)-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z’-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified three compounds that show dose-dependent competition of FAT–paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.

Upscaling and Automation of Electrophysiology: Toward High Throughput Screening in Ion Channel Drug Discovery

2003 ◽  
Vol 9 (1) ◽  
pp. 49-58
Author(s):  
Margit Asmild ◽  
Nicholas Oswald ◽  
Karen M. Krzywkowski ◽  
Søren Friis ◽  
Rasmus B. Jacobsen ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Ion Channel ◽  
High Throughput ◽  
High Throughput Screening
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