Abstract 3265: Multispectral fluorescence imaging in a mouse model of tongue carcinogenesis
Authors: Hellebust Anne , Rosbach Kelsey , Wu Jessica , Nguyen Jennifer , Schwarz Richard , Gillenwater Ann , Vigneswaran Nadarajah , Richards-Kortum Rebecca 
Published: June 2012

Abstract
The purpose of this study was to evaluate multispectral imaging of autofluorescence and targeted fluorescent contrast agents for non-invasive diagnosis of epithelial dysplasia and cancer using a mouse model of oral tongue carcinogenesis. In humans, dysplasia and cancer in the oral cavity lead to a loss of stromal autofluorescence, increased epithelial cell metabolism, and a higher nuclear-to-cytoplasmic ratio. Mice fed the DNA adduct forming 4-nitroquinoline 1-oxide (4NQO) in their drinking water exhibit the histologic changes and patterns of biomarker expression observed in patient specimens. Thus, this model provides an opportunity to explore and optimize non-invasive optical imaging techniques.
Two inbred strains (CBA and C57BL/6; n = 30) of mice were given 4NQO (100μg/mL) in their drinking water for 16 weeks. Mice were euthanized between weeks 8-22 and tongues were harvested for ex-vivo widefield and high-resolution imaging. Widefield autofluorescence images were obtained. A fluorescent glucose analog, 2-NBDG, and fluorescent nuclear dye, proflavine, were then applied topically, and images were again acquired. Fresh tissue was sliced into 200 micron thick transverse sections for high resolution confocal imaging. After imaging, tongues were fixed with 10% formalin, embedded in paraffin and processed for histopathologic studies.
For each specimen, a pathology map was created to delineate tissue as normal, dysplasia, or invasive carcinoma as determined by pathology. Widefield and high resolution images were analyzed by pathology grade. Confocal imaging of fresh tissue sections revealed an increase in average nuclear size and nuclei per unit area in neoplastic regions of specimens stained with proflavine. Similarly, an increase in 2-NBDG cytoplasmic staining was observed in confocal images from neoplastic regions of specimens compared to control tissue. In widefield imaging, both 2-NBDG and proflavine stained tongues revealed increased fluorescence intensity in neoplastic regions. The average intensity of 2-NBDG stained tongues exhibiting dysplasia was twofold that of control tongues. Autofluorescence intensity increased for exposed mice relative to controls. The majority of fluorescence in this model system is linked to epithelial cells and little stromal fluorescence is observed. We hypothesize that this increase in autofluorescence is associated with increased mitochondrial cofactors NADH and FAD. This is in contrast to results with human oral mucosa, where widefield images show loss of fluorescence due to the loss of stromal collagen fluorescence associated with neoplasia.
Autofluorescence and fluorescence from topically applied dyes for optical imaging reveal quantitative differences between normal and neoplastic tongue lesions. These findings may be used to optimize non-invasive optical molecular imaging tests to improve early detection of oral cancer and its precursors in the clinic.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3265. doi:10.1158/1538-7445.AM2011-3265