Cell Based Assays Using Ipscs For Drug Development And Testing

Author : You Li
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (14 download)

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Download or read book In Vitro 3D Fluorescent Cell-based Assay Reporting Gene Regulation for High-throughput Drug Screening written by You Li and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Traditional cell-based assays have a high failure rate of identifying effective compounds in drug discovery. To address this problem, recent drug research has focused on engineering cancer cells that can indicate specific drug molecular mechanisms of action and developing cancer models that can more accurately represent tumor physiology. Survivin and telomerase reverse transcriptase (TERT), two important cancer biomarkers overexpressed in 85% of tumor tissues, play crucial roles in anti-apoptosis and unlimited cell proliferation in carcinogenesis, as well as drug resistance in cancer therapy. In this research, we have developed survivin and TERT reporter gene assays for high-throughput screening (HTS) of drugs downregulating these genes as anticancer therapeutics. Cancer cells were engineered to express fluorescent proteins controlled by TERT and survivin promoters, respectively, and were cultured in 3-dimensional (3D) scaffolds in microbioreactors to investigate drug effects by monitoring changes in culture fluorescence in real time. The novel 3-D assays offered molecular and physiological predictions of gene-specific responses to drugs, thus facilitating cancer drug discovery and development. Using survivin as the target, a dual-color fluorescent reporter gene assay performed in 3-D culture was developed. To demonstrate the drug's specific effect on the survivin promoter, MCF-7 breast cancer cells were engineered to express enhanced green fluorescent protein (EGFP) driven by a survivin promoter. The cell also expressed red fluorescent protein (RFP) under the control of a cytomegalovirus promoter as an internal control and to indicate cell number. The specific survivin expression was assessed by the green fluorescence normalized to the red fluorescence, which was used as an indicator to assess drug’s regulation on survivin. The internal control compensated for the variations in cell number, cell culture environment, fluorescent protein property, detection setting, and even for the reading interference from tested compounds, resulting in a more accurate evaluation of drug effect. The assay correctly detected the downregulation and upregulation of several anticancer compounds and demonstrated its reliability for high-throughput screening of survivin-targeted drugs. In this work, a 3-D cancer cell-based hTERT promoter assay was also developed for anticancer drug screening. Different lengths of the hTERT promoter were cloned with EGFP as reporting cassettes and were introduced in multiple cancer cell lines. The selected MCF7 reporter cell was able to correctly respond to the known hTERT inhibitors, pristimerin, epigallocatechin gallate and n-butylidenephthalide. The reporter cells were cultured on 3-D scaffolds to resemble in vivo tumor physiology and present in vivo like responses. The 3-D hTERT promoter assay was verified by pristimerin, and subsequently, other five anticancer compounds, including doxorubicin, cisplatin, paclitaxel, blasticidin, and tamoxifen, were tested for their effects on hTERT. The developed assay achieved an overall accuracy rate of over 80%, which demonstrated the feasibility of using 3D culture and EGFP as a reporter to screen hTERT inhibitors. In addition to the development of cancer cell-based assays, hTERT as a pluripotency marker to predict embryotoxic compounds during tissue development was also evaluated. The hTERT-EGFP reporting cassettes were introduced into mouse embryonic stem cells D3. The reportability was assessed by the corelated expression changes of EGFP and endogenous TERT during neural differentiation and after receiving toxicant stimuli. Our results showed that EGFP was able to indicate drug effect on human TERT expression level during ESC differentiation, which, however, was not always consistent with internal mouse TERT (mTERT) due to species difference. The results confirmed that drug effects on TERT were species-specific, especially at the beginning of differentiation. In conclusion, hTERT in mESCs reacted to embryotoxic compounds and the engineered mESC-hTERT-EGFP cells could be used for human teratogen prediction.