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Development of 3D Cell-Based Assay for High Throughput Screening of Cancer Drugs

Xin, Xin
http://rave.ohiolink.edu/etdc/view?acc_num=osu1492700405342723

Abstract Details

2017, Doctor of Philosophy, Ohio State University, Chemical Engineering.
Survivin, a member of inhibitor of apoptosis family (IAP), is overexpressed in tumor tissues and plays an important role in tumor resistance to chemo-and radiation therapies. In this work, a three-dimensional (3D) cell-based survivin promoter assay was developed for cancer drug screening. Breast cancer MCF-7 cells were engineered to express enhanced green fluorescent protein (EGFP) controlled by a human survivin promoter and CMV promoter, respectively. The fluorescent cells were cultured in a novel 3D polymer-based scaffold in a 40-microbioreactor platform (40-MBR) with capacities of high throughput performance and real-time monitoring of EGFP signals. The EGFP signal driven by the survivin promoter was closely correlated with survivin gene expression in MCF-7 cells treated with YM155, a small-molecule survivin promoter suppressant. In addition, the drug effectiveness of other antitumor agents on survivin expression and their drug cytotoxicity were successfully evaluated by this assay. These findings confirmed that the novel survivin promoter assay could be used as a reliable and high throughput tool to monitor the drug efficiacy on survivin promoter activity, which could play as a novel biomarker for early-stage cancer drug discovery. A novel dual fluorescence system using EGFP and red fluorescent protein (Ds-red) signals was then developed in the 3D cell culture platform, 40-MBR. The dual fluorescence system was constructed by the cell coculture between the EGFP-exressing and Ds-red-expressing MCF-7 cells. The fluorescence kinetics of Ds-red signal was firstly investigated in the 40-MBR. The results indicated that similar with previous studies on EGFP, Ds-red could be utilized to detect cell number and evaluate drug cytotoxicity in the 3D platform. Furthermore, cytotoxicity study and survivin promoter assay demonstrated that both EGFP and Ds-red in a dual fluorescence system were sensitive enough to monitor corresponding cell proliferation. These results suggested that the novel dual fluorescence system provided a simple, real-time and high throughput method for individual cell detection in 3D coculture assay, which might be used for a variety of research purposes. Generally, cancer cells are surrounded by various types of normal cells in tumor microenvironment. Close interactions between tumor and surrounding stromal cells greatly affect cancer’s aggressiveness and responses to antitumor drugs. In this study, a novel 3D multicellular tumor model with cell coculture between breast cancer cell (MCF-7) and mouse fibroblasts (NIH-3T3) was developed to better stimulate tumor microenvironment for cancer drug screening. The cell coculture was performed in novel 3D static and microfluidic bioreactors, using the dual fluorescence system for individual cell monitoring. The results showed that the drug resistance of MCF-7 was significantly enhanced by the cocultured NIH-3T3 at the exposure of antitumor agents. The cancer drugs were also shown as cancer-specific reagents due to their relatively lower cytotoxicity to NIH-3T3 versus MCF-7. These results demonstrated our novel 3D coculture assay could better mimic stromal effects in tumor microenvironment and serve as a high throughput platform with improved predictability and accuracy of in vitro drug screening.
Shang-tian Yang (Advisor)
David Wood (Committee Member)
Jeffrey Chalmers (Committee Member)
198 p.
Biomedical Engineering; Chemical Engineering
3D cell based assay; high throughput screening of cancer drugs

Recommended Citations

Citations

  • Xin, X. (2017). Development of 3D Cell-Based Assay for High Throughput Screening of Cancer Drugs [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492700405342723

    APA Style (7th edition)

  • Xin, Xin. Development of 3D Cell-Based Assay for High Throughput Screening of Cancer Drugs. 2017. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1492700405342723.

    MLA Style (8th edition)

  • Xin, Xin. "Development of 3D Cell-Based Assay for High Throughput Screening of Cancer Drugs." Doctoral dissertation, Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492700405342723

    Chicago Manual of Style (17th edition)

osu1492700405342723
901
© 2017, some rights reserved.Creative Commons License Development of 3D Cell-Based Assay for High Throughput Screening of Cancer Drugs by Xin Xin is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.