Publication Date

2018-11-12

Availability

Embargoed

Embargo Period

2020-11-11

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2018-10-22

First Committee Member

Deo Sapna

Second Committee Member

Sylvia Daunert

Third Committee Member

Richard Cote

Fourth Committee Member

Xin-Hai Pei

Fifth Committee Member

Eleonore Beurel

Abstract

The intrinsic sensitivity and low detection limits of bioluminescence have labeled this technique as a powerful tool for the detection of biomolecules at the cellular level, and real-time visualization of biological processes within living organisms. In our studies, we seek to develop novel bioluminescence-based detection and imaging techniques, and further investigate the feasibility and efficacy of these techniques in two major fields of oncology research: “Detection of circulating tumor cells” and “Image-guided therapy” Circulating tumor cells (CTCs) have shown potential in monitoring cancer prognosis and treatment response as well as providing novel insight into personalized medicine for guiding treatment decisions. However, widespread utilization of CTC assays in a clinical setting is still challenged due to the cumbersome procedure of current techniques. Herein, we report a high-sensitive bioluminescence-based assay by genetically fusing Gaussia luciferases with antibody mimetics for rapid detection of viable CTCs and subsequent downstream analysis of those captured CTCs (ex vivo culture expansion and single-cell analysis). Our assay showed the high specificity and sensitivity in detection of CTCs from tumor-bearing mice with no cell toxicity observed which subsequently allows expansion of those cells in culture for CTC biobanks. Moreover, incorporating a fluorescent modality with our bioluminescence-based assay to generate a dual bioluminescence/fluorescence sensing platform enabled precise identification and isolation of individual CTCs from breast cancer patients for single-cell analysis using current fluorescence-based single-cell isolation devices after initiating the bioluminescent CTC detection. Tumor recurrence is a common cause of treatment failure due to incomplete resection of tumor cells that remain undetected outside of the treatment area. Therefore, developing a technique to identify the presence of residual tumor cells within the tumor bed followed by eliminating those residual cells is highly desired. Here, we developed the self-luminous theranostic nanoparticles by leveraging bioluminescence imaging and photothermal therapy to achieve highly sensitive detection and accurate ablation of tumor cells. To accomplish this, biotinylated Gaussia luciferases (GLuc) were generated and attached on the streptavidin coated gold nanorods (AuNR). In order to improve spatial resolution with depth and signal-to-noise ratio of bioluminescence imaging in vivo, we created a detection system based on a BRET-mediated red-shifted GLuc with a cancer specific protease-activable property. This approach showed better penetration of emitted light and the increase of bioluminescence intensity in the presence of matrix metalloproteinases (MMPs), which are highly expressed in tumors. We demonstrated that our GLuc-AuNR has (1) high signal-to-noise ratio of a bioluminescent signal as compared to other fluorescence counterparts used in tumor imaging, (2) highly selective targeting with incorporation of antibody mimetics (DARPins) against tumor-specific antigens, as well as (3) efficient photothermal therapy upon the near-infrared irradiation.

Keywords

Bioluminescence; Antibody-mimetic; Circulating Tumor Cell; Theranostics

Available for download on Wednesday, November 11, 2020

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