Fluorescent Probe Interrogation of a Cytoprotective Cancer-linked Oxidoreductase in Two-Dimensional Human Cell Cultures and Solid Tumor Mimics

Fluorescence molecular imaging is an emerging field with potential to aid in optically guided surgery for cancer treatment. To overcome the drawbacks of always-on fluorescent probes, such as indocyanine green, activatable fluorescent probes are being developed that allow high signal-to-background imaging for better discrimination of cancerous cells from normal cells. For activation, turn-on fluorescent probes rely on the presence of biomarkers such as antibodies, cell-surface receptors or enzymes that are highly specific to tumor cells. hNQO1 (human NAD(P)H: quinone oxidoreductase isoenzyme 1) is considered as one of such cancer-associated biomarkers that are present intracellularly in cancers of multiple origins. It helps detoxify quinones by bypassing highly reactive and toxic semiquinone radical intermediates and prevents the oxidative stress generated from redox cycling. Overexpression of this enzyme in solid tumors and its catalytic ability to provide two-electron reduction processes are being exploited in the development of therapeutic prodrugs that generate anticancer intermediates upon hNQO1 action. Activation of fluorescently silent hNQO1-activatable molecular probes synthesized in McCarley research lab is imparted by a similar two-electron reduction, which we use for sensing the enzyme in cancer cells. The goal of this research is the use of a molecular probe as an hNQO1 sensor to evaluate the distribution and functionality of hNQO1 enzyme in microregions of multicellular tumor spheroids (MCTSs) that mimic solid tumors. The ability to examine for possible spatially dependent enzyme activity in tumors is of great value in the development of imaging and chemotherapeutic agents that are activated by hNQO1. This dissertation will address research to date, including: (1) spectroscopic and enzyme kinetics evaluation of a molecular probe and its subsequent application in hNQO1-expressing cell monolayers for the determination of the viability of its activation; (2) investigation of the mechanism of probe uptake; (3) overcoming the disadvantages of two-dimensional cell culture systems, namely the lack of features associated with real tissues, including their complex microenvironment and cellular heterogeneity, by study of probe activation and hNQO1 activity/presence within three-dimensional cell cultures; and (4) evaluation of the relationship between NQO1 expression and invasive phenotype in an in vitro tumor model for ovarian cancer (OVCAR-5)