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Background: The development of noninvasive molecular imaging approaches has the potential to improve management of cancer. Methods: In this study, we demonstrate the potential of noninvasive topical delivery of an epidermal growth factor-Alexa 647 (EGF-Alexa 647) conjugate to image changes in epidermal growth factor receptor expression associated with oral neoplasia. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of EGF-Alexa 647 in a variety of model systems, including cells, three-dimensional tissue cultures, and intact human tissue specimens using wide-field and high-resolution fluorescence imaging. Data were collected from 17 different oral cancer patients: eight pairs of normal and abnormal biopsies and nine resected tumors were examined. Results: The EGF-dye conjugate can be uniformly delivered throughout the oral epithelium with a penetration depth exceeding 500 µ m and incubation time of less than 30 minutes. After EGF-Alexa 647 incubation, the presence of oral neoplasia is associated with a 1.5- to 6.9-fold increase in fluorescence contrast compared with grossly normal mucosa from the same patient with both wide-field and high-resolution fluorescence imaging. Conclusions: Results illustrate the potential of EGF-targeted fluorescent agents for in vivo molecular imaging, a technique that may aid in the diagnosis and characterization of oral neoplasia and allow real-time detection of tumor margins.

In this longitudinal study, a mouse model of 4-nitroquinoline 1-oxide chemically induced tongue carcinogenesis was used to assess the ability of optical imaging with exogenous and endogenous contrast to detect neoplastic lesions in a heterogeneous mucosal surface. Widefield autofluorescence and fluorescence images of intact 2-NBDG-stained and proflavine-stained tissues were acquired at multiple time points in the carcinogenesis process. Confocal fluorescence images of transverse fresh tissue slices from the same specimens were acquired to investigate how changes in tissue microarchitecture affect widefield fluorescence images of intact tissue. Widefield images were analyzed to develop and evaluate an algorithm to delineate areas of dysplasia and cancer. A classification algorithm for the presence of neoplasia based on the mean fluorescence intensity of 2-NBDG staining and the standard deviation of the fluorescence intensity of proflavine staining was found to separate moderate dysplasia, severe dysplasia, and cancer from non-neoplastic regions of interest with 91% sensitivity and specificity. Results suggest this combination of noninvasive optical imaging modalities can be used in vivo to discriminate non-neoplastic from neoplastic tissue in this model with the potential to translate this technology to the clinic.

This prospective pilot study evaluates the potential of high-resolution fiber optic microscopy (HRFM) to identify lymph node metastases in breast cancer patients. 43 lymph nodes were collected from 14 consenting breast cancer patients. Proflavine dye was topically applied to lymph nodes ex vivo to allow visualization of nuclei. 242 images were collected at 105 sites with confirmed histopathologic diagnosis. Quantitative statistical features were calculated from images, assessed with one-way ANOVA, and were used to develop a classification algorithm with the goal of objectively discriminating between normal and metastatic tissue. A classification algorithm using mean image intensity and skewness achieved sensitivity of 79% (27/34) and specificity of 77% (55/71). This study demonstrates the technical feasibility and diagnostic potential of HRFM with fluorescent contrast in the ex vivo evaluation of lymph nodes from breast cancer patients.

We have developed a high-resolution fluorescence microendoscope capable of imaging sub-cellular morphology in vivo, in real-time. We report our latest results in clinical studies for early cancer detection in the cervix, oral cavity, and esophagus.

Three probes targeting molecular or morphologic characteristics of cancer were topically applied to freshly resected oral tissue. Optical contrast was used to predict the region of neoplasia; predicted regions agree well with histopathology maps.

Cytotoxicity assays play a central role in studying the function of immune effector cells such as cytolytic T lymphocytes (CTL) and natural killer (NK) cells. Traditionally, cytotoxicity assays have been performed using 51Chromium (51Cr) and Calcein release assays. The assays involve labeling tumor cells (target) with radioisotope or fluorescent dyes, when the target cells are subjected to cytolysis by CTLs or NK cells (effector), they releases the entrapped labels into the media upon lysis. The amount of labels in the media is measured to determine the level of cytotoxicity the effectors have induced. These traditional methods may generate inconsistent results due to low sensitivity caused by poor loading efficiency and high spontaneous release of the reagents. In this work, we demonstrate a novel cytotoxicity assay using the Celigo imaging cytometry method. Utilizing imaging cytometry, direct cell counting of live fluorescent target cells can be performed, which is a direct method for assessment of cytotoxicity. Human NK cells from one healthy donor were used as effectors, and K562 (suspension) and IMR32 (adherent) were used as the target cells. Both target cells were first stained with Calcein AM, and seeded at 10,000 cells/well in a standard 96-well microplate. The donor NK cells were then added to each well at Effector-to-Target (E:T) ratios 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125:1. The 96 well plate was then scanned and analyzed using Celigo imaging cytometer at t = 1, 2, 3, and 4 h to measure the% lysis of target cells. The results showed increasing% lysis as incubation time and E:T ratio increased. The propose Celigo imaging cytometry is an accurate and simple method for direct quantification of cytotoxicity. Citation Format: Leo Li-Ying Chan, Kelsey Rosbach, Srinivas S. Somanchi, Dean Lee. Quantification of natural killer cell-mediated cytotoxicity using Celigo imaging cytometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1303. doi:10.1158/1538-7445.AM2015-1303

Cytotoxicity assays play a central role in studying the function of immune effector cells such as cytolytic T lymphocytes (CTL) and natural killer (NK) cells. Traditionally, cytotoxicity assays have been performed using 51Chromium (51Cr) and Calcein release assays. The assays involve labeling tumor cells (target) with radioisotope or fluorescent dyes, when the target cells are subjected to cytolysis by CTLs or NK cells, they releases the entrapped labels into the media upon lysis. These traditional methods may generate inconsistent results due to low sensitivity caused by poor loading efficiency and high spontaneous release of the reagents. In this work, we demonstrate a novel high-throughput direct cell counting method for cytotoxicity assay using the Celigo imaging cytometry method. Human NK cells from one healthy donor were used as effectors, and K562 and IMR32 were used as the target cells. Both target cells were first stained with Calcein AM, and seeded at in a 96-well microplate. NK cells were then added to each well at various Effector-to-Target (E:T) ratios. The 96 well plate was then scanned and analyzed using the Celigo to measure the % lysis of target cells at different time points. The results showed increasing % lysis as incubation time and E:T ratio increased. The propose Celigo imaging cytometry is an accurate and simple method for direct quantification of cytotoxicity, which can be an attractive method for both academic and clinical research.

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

Background: Early detection of dysplasia and cancer in the oral cavity dramatically improves patient survival, however, early stage disease can be difficult to identify with a conventional oral examination. Non-invasive, molecular-specific optical imaging has the potential to rapidly and simultaneously assess expression of multiple biomarkers that indicate neoplastic changes. Imaging biomarker expression may aid not only in early detection, but also in determination of an appropriate surgical margin, ensuring that all neoplastic tissue is removed without removing excess normal tissue. Methods: Three contrast agents were topically applied to freshly resected oral lesions (n=20) for 20 minutes. One contrast agent targets overexpression of epidermal growth factor receptor and is composed of epidermal growth factor peptide labeled with a fluorescent dye. Another is a fluorescently labeled glucose that targets increased metabolism due to increased expression and activity of GLUT transporters. The third intercalates with nucleic acids to allow quantification of metrics such as nuclear size and inter-nuclear distance. After incubation, both widefield and high-resolution fluorescence images were collected for analysis. Widefield imaging increases sensitivity of detection by surveying a large region, while high-resolution imaging increases specificity by interrogating a smaller field of view in more detail. Using an auto-threshold function in Matlab software, the widefield fluorescence images were converted to binary. This binary image was smoothed to identify a contiguous region, which was outlined as a potential “margin.” This potential margin was overlaid onto a pathology map created through intensive review of H&E slides with an expert head and neck pathologist to assess agreement of the predicted margin to the gold standard. Quantitative features from high-resolution images were analyzed in Matlab and used to create an objective classification algorithm to classify images as either normal or neoplastic. Immunohistochemistry was used to confirm biomarker expression. Results: The predicted margin from both contrast agents matches the pathology map well. Not only can invasive cancer be detected, but difficult to identify regions of mild dysplasia are also recognized as abnormal. Varying the threshold adjusts the sensitivity and specificity of this margin, which can be calculated based on the area of the region correctly diagnosed or not. The objective classification algorithm created from high-resolution fluorescence images of the nucleic acid intercalator performed with 85% sensitivity and 78% specificity. IHC confirms expression level of epidermal growth factor receptors and GLUT transporters. Conclusions: We have demonstrated non-invasive topical delivery of contrast agents that may help identify oral neoplasia. Contrast agents can be multiplexed to label many biomarkers associated with oncogenesis. Detection using both widefield and high-resolution imaging permits rapid, objective assessment of biomarker expression at the point of care. This may assist a clinician in choosing a region to biopsy for initial diagnosis or in determining an appropriate surgical margin during treatment. The technique can also be applied to other epithelial cancers, thus impacting a large range of clinical cases. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A223.

Biomarkers of cancer can indicate the presence of disease and serve as therapeutic targets. Our goal is to develop an optical imaging approach using molecularly targeted contrast agents to assess several centimeters of mucosal surface for mapping expression of multiple biomarkers simultaneously with high spatial resolution. The ability to image biomarker expression level and heterogeneity in vivo would be extremely useful for clinical cancer research, patient selection of personalized medicine, and monitoring therapy. In this proof-of-concept ex vivo study, we examined correlation of neoplasia with two clinically relevant biomarkers: epidermal growth factor receptor (EGFR) and metabolic activity. Two hundred eighty-six unique locations in nine samples of freshly resected oral mucosa were imaged after topically applying optical imaging agents EGF-Alexa 647 (to target EGFR) and 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (to target metabolic activity). Quantitative features were calculated from resulting fluorescence images and compared with tissue histopathology maps. The EGF-Alexa 647 signal correlated well with EGFR expression as indicated by immunohistochemistry. A classification algorithm for presence of neoplasia based on the signal from both contrast agents resulted in an area under the curve of 0.83. Regions with a posterior probability from 0.80 to 1.00 contained more than 50% neoplasia 99% (84/85) of the time. This study demonstrates a proof-of-concept of how noninvasive optical imaging can be used as a tool to study expression levels of multiple biomarkers and their heterogeneity across a large mucosal surface and how biomarker characteristics correlate with presence of neoplasia. Applications of this approach include predicting regions with the highest likelihood of disease, elucidating the role of biomarker heterogeneity in cancer biology, and identifying patients who will respond to targeted therapy.

Current procedures for oral cancer screening typically involve visual inspection of the entire tissue surface at risk under white light illumination. However, pre-cancerous lesions can be difficult to distinguish from many benign conditions when viewed under these conditions. We have developed wide-field (macroscopic) imaging system which additionally images in cross-polarized white light, narrowband reflectance, and fluorescence imaging modes to reduce specular glare, enhance vascular contrast, and detect disease-related alterations in tissue autofluorescence. We have also developed a portable system to enable high-resolution (microscopic) evaluation of cellular features within the oral mucosa in situ. This system is a wide-field epi-fluorescence microscope coupled to a 1 mm diameter, flexible fiber-optic imaging bundle. Proflavine solution was used to specifically label cell nuclei, enabling the characteristic differences in N/C ratio and nuclear distribution between normal, dysplastic, and cancerous oral mucosa to be quantified. This paper discusses the technical design and performance characteristics of these complementary imaging systems. We will also present data from ongoing clinical studies aimed at evaluating diagnostic performance of these systems for detection of oral neoplasia.