Evaluation of the role of EGFR, SOX2 and PAX9 in oral carcinogenesis

PhD ThesisOral squamous cell carcinoma (OSCC) is a major global healthcare problem. OSCC has devastating consequences for many patients diagnosed with the disease. Outcomes may be improved if the disease is identified in its precursor stages, termed oral potentially malignant disorders (OPMD). Unfortunately, histological assessment of OPMD does not reliably predict which cases will progress to OSCC. Several candidate biomarkers have emerged in recent decades. To date, however, none have been validated for use in clinical practice. This study sought to address the continuing need for biomarkers that stratify OPMD according to their risk of malignant transformation. Our data show that EGFR gene copy number abnormalities correlate with malignant transformation in OPMD. EGFR genomic gain was also present in a quarter of early-stage OSCC. SOX2 had a heterogeneous expression profile in both OPMD and OSCC, limiting its clinical utility. Nevertheless, the pattern of SOX2 expression suggests it may be a marker of OSCC stem cells and consequently represent a potential chemotherapeutic target. PAX9 is down-regulated in OPMD and early-stage OSCC. Following a course of chemical induction, Pax9-deficient mice were more likely to develop OPMD and OSCC than controls. These findings support the hypothesis that PAX9 has a tumour-suppressor function. In addition to enhanced local sensitivity to chemical induction, Pax9-deficient mice were more susceptible to the toxic systemic effects of treatment. A modified protocol for chemical induction in Pax9-deficient mice is recommended. Paradoxically, our analysis of human tissues showed increased PAX9 expression in OPMD that underwent malignant transformation, suggesting that, in some circumstances, PAX9 may have a tumour-promoting effect. Finally, we summarise the generation of stably transfected cell lines in which PAX9 and SOX2 expression may be manipulated by tetracycline administration. These cell lines will facilitate future studies of the functional role of PAX9 and SOX2 in oral carcinogenesis

Multimodal Multispectral Optical Endoscopic Imaging for Biomedical Applications

Optical imaging is an emerging field of clinical diagnostics that can address the growing medical need for early cancer detection and diagnosis. Various human cancers are amenable to better prognosis and patient survival if found and treated during early disease onset. Besides providing wide-field, macroscopic diagnostic information similar to existing clinical imaging techniques, optical imaging modalities have the added advantage of microscopic, high resolution cellular-level imaging from in vivo tissues in real time. This comprehensive imaging approach to cancer detection and the possibility of performing an ‘optical biopsy’ without tissue removal has led to growing interest in the field with numerous techniques under investigation. Three optical techniques are discussed in this thesis, namely multispectral fluorescence imaging (MFI), hyperspectral reflectance imaging (HRI) and fluorescence confocal endomicroscopy (FCE). MFI and HRI are novel endoscopic imaging-based extensions of single point detection techniques, such as laser induced fluorescence spectroscopy and diffuse reflectance spectroscopy. This results in the acquisition of spectral data in an intuitive imaging format that allows for quantitative evaluation of tissue disease states. We demonstrate MFI and HRI on fluorophores, tissue phantoms and ex vivo tissues and present the results as an RGB colour image for more intuitive assessment. This follows dimensionality reduction of the acquired spectral data with a fixed-reference isomap diagnostic algorithm to extract only the most meaningful data parameters. FCE is a probe-based point imaging technique offering confocal detection in vivo with almost histology-grade images. We perform FCE imaging on chemotherapy-treated in vitro human ovarian cancer cells, ex vivo human cancer tissues and photosensitiser-treated in vivo murine tumours to show the enhanced detection capabilities of the technique. Finally, the three modalities are applied in combination to demonstrate an optical viewfinder approach as a possible minimally-invasive imaging method for early cancer detection and diagnosis

Objective localisation of oral mucosal lesions using optical coherence tomography.

PhDIdentification of the most representative location for biopsy is critical in establishing the definitive diagnosis of oral mucosal lesions. Currently, this process involves visual evaluation of the colour characteristics of tissue aided by topical application of contrast enhancing agents. Although, this approach is widely practiced, it remains limited by its lack of objectivity in identifying and delineating suspicious areas for biopsy. To overcome this drawback there is a need to introduce a technique that would provide macroscopic guidance based on microscopic imaging and analysis. Optical Coherence Tomography is an emerging high resolution biomedical imaging modality that can potentially be used as an in vivo tool for selection of the most appropriate site for biopsy. This thesis investigates the use of OCT for qualitative and quantitative mapping of oral mucosal lesions. Feasibility studies were performed on patient biopsy samples prior to histopathological processing using a commercial OCT microscope. Qualitative imaging results examining a variety of normal, benign, inflammatory and premalignant lesions of the oral mucosa will be presented. Furthermore, the identification and utilisation of a common quantifiable parameter in OCT and histology of images of normal and dysplastic oral epithelium will be explored thus ensuring objective and reproducible mapping of the progression of oral carcinogenesis. Finally, the selection of the most representative biopsy site of oral epithelial dysplasia would be investigated using a novel approach, scattering attenuation microscopy. It is hoped this approach may help convey more clinical meaning than the conventional visualisation of OCT images

Evaluation of anti-metastatic therapeutics targeting SEC62 in head and neck cancer using newly established murine in vivo metastasis models and functional characterization of stable SEC62 knock-out head and neck cancer cell lines generated by CRISPR/Cas9

For several years, research has been concerned with the clinical and molecular role of SEC62, a protein located in the ER membrane, which has been considered as a potential oncogene. Over time, significant overexpression of the SEC62 protein has been shown in various human tumour entities, including squamous cell carcinoma of the head and neck (HNSCC) which display a SEC62 overexpression in approximately 86% of cases. In general, head and neck tumours are among the seventh most common tumour entities worldwide with a mostly consistent and poor 5-year survival rate for affected patients of about 50 to 60 %. Head and neck tumours are often diagnosed at an advanced stage of disease and about 50 % of all patients already display lymph node metastases of the neck at the time of diagnosis. About 10% of patients even have distant metastases. Studies have shown that the ER membrane protein SEC62 is not only overexpressed in the majority of HNSCC patients but is also associated with a significantly worse prognosis and an advanced TNM status. In functional in vitro studies of the SEC62 protein, significant changes in migration were found, depending on an artificially altered SEC62 expression. Thus, plasmid-mediated overexpression of SEC62 led to a significantly increased migration. A transient knock-down of SEC62 via siRNA significantly decreased the ability of migration in HNSCC cells. Based on these properties, SEC62 was considered as a potential new target for directed therapy of SEC62-overexpressing HNSCC tumours. Since a direct transfer from those in vitro experiments to human is not possible, this work aimed at a functional knock-down of SEC62 by the administration of TFP and TG. TFP leads to an increased cytosolic Ca2+ content by blocking CaM, which would close the protein translocation channel SEC61 in the ER membrane. A strong concentration gradient causes a flow of Ca2+ from the ER Ca2+-store into the cytosol. Moreover, the simultaneous administration of TG irreversibly inhibits SERCA so that no Ca2+ can be actively transported back into the ER. These two compounds in combination are thought to significantly upset Ca2+ homeostasis in SEC62-overexpressing cancer cells, causing the cells to undergo apoptosis. Since these effects could be proven in vitro, two murine xenograft metastasis models were established within this thesis in order to be able to test the efficacy of both substances in vivo on the metastasis of HNSCC cells. Therefore, on the one hand tumour cells were injected orthotopically into the tongue of mice to test the anti-metastatic ability of TFP and TG. Here, TFP treatment resulted in a reduction of the number of cervical lymph node metastases, whereas TG treatment was able to reduce the size of detectable metastases. On the other hand, tumour cells were injected i.v. into the tail vein of the animals to investigate the impact of both substances on haematogenous metastasis. Here, the evaluation of the combinatory treatment on haematogenous metastasis achieved a reduction in metastatic burden in the lung. In addition to the main aspect of the effects of TFP and TG on metastasis, various imaging techniques and their evaluations including MRI, micro-CT, and histology were also established for monitoring the emerging metastases. In a second part of this work stable SEC62-knockout clones of an immortalized HNSCC cell line were generated using CRISPR-Cas9 technology, which should provide deeper insights into the role of SEC62 and the SEC62-associated processes inside the cell. These SEC62-ko clones were validated using various methods such as immunofluorescence, Western blot and next generation sequencing (NGS), and were also characterised based on whole-RNA-sequencing. Furthermore, first functional studies with SEC62-ko clones and their tolerance to TFP and TG were applied. In summary, two murine xenograft models for lymphatic and haematogenous metastasis of head and neck cancer were successfully established, on which first investigations on the efficacy of two potential new compounds for a targeted therapy of metastatic SEC62-overexpressing head and neck tumours could be performed. Herein, TG and TFP showed promising effects on lymphatic and haematogenous metastasis in vivo motivating for further investigations. Furthermore, two stable SEC62-ko HNSCC cell lines could be generated, validated and characterised using CRISPR-Cas9. Further analyses to understand the influence and function of SEC62 in more detail are the subject of future research.Die Forschung beschäftigt sich bereits seit mehreren Jahren mit der klinischen als auch molekularen Rolle von SEC62, einem Protein, dass sich in der ER Membran befindet und als potentielles Onkogen gehandelt wird. In den letzten ca. 15 Jahren konnte in verschiedenen Tumorentitäten eine signifikante Überexpression des SEC62-Proteins nachgewiesen werden, so auch in ca. 86% aller Plattenepithelkarzinome des Kopf-Hals-Bereiches (head and neck squamous cell carinoma, HNSCC). Im Allgemeinen zählen Kopf-Hals Tumore zu den sieben häufigsten Tumorerkrankungen weltweit mit einer weitestgehend gleichbleibenden, schlechten 5-Jahres Überlebensrate für betroffene Patienten von etwa 50-60%. In der Mehrzahl der Fälle werden Kopf-Hals-Tumore erst in einem späten Stadium der Erkrankung diagnostiziert. Daher weisen zum Diagnosezeitpunkt bereits ca. 50% aller HNSCC-Patienten Hals-Lymphknotenmetastasen auf, ca. 10% der Patienten zeigen sogar bereits Fernmetastasen. In Studien konnte nachgewiesen werden, dass das ER-membranständige Protein SEC62 nicht nur von einem Großteil aller HNSCC Patienten überexprimiert wird, sondern auch mit einer deutlich schlechteren Prognose, sowie mit einem fortgeschrittenen TNM-Stadium assoziiert ist. In funktionellen in vitro Untersuchungen des SEC62 Proteins konnten signifikante Veränderungen in der Migrationsfähigkeit festgestellt werden, je nach artifiziell veränderter SEC62-Expression. So führte eine Plasmid-vermittelte Überexpression von SEC62 zu einer deutlich gesteigerten Migration, bzw. ein transienter knock-down zu einer signifikanten Abnahme der Migrationsfähigkeit in HNSCC Zellen. Aufgrund dieser Eigenschaften bietet sich SEC62 als mögliches neues Target für eine zielgerichtete Therapie von SEC62-überexprimierenden HNSCCs an. Da ein direkter Transfer der genannten in vitro Experimente auf den Menschen nicht möglich ist, wurde in dieser Arbeit ein funktioneller knock-down von SEC62 durch die Gabe von TFP und TG angestrebt. TFP führt zu einem gesteigerten zytosolischen Ca2+-Gehalt durch die Blockade von CaM, das normalerweise den Proteintranslokationskanal SEC61 in der ER-Membran verschließen würde. Durch ein starkes Konzentrationsgefälle von Ca2+ strömen die Ionen aus dem ER Ca2+-Speicher ins Zytosol. Durch die gleichzeitige Gabe von TG wird zudem auch die SERCA irreversible inhibiert, sodass kein Ca2+ energieabhängig ins ER zurückgepumpt werden kann. Diese beiden Substanzen in Kombination bringen die Ca2+-Homöostase in SEC62-überexprimierenden Krebszellen signifikant aus dem Gleichgewicht, sodass eine Apoptose der Tumorzellen erreicht werden kann. Da in vitro diese Effekte gezeigt werden konnten, wurden in dieser Arbeit zwei murine Xenograft Metastasierungs-Modell etabliert, um die Wirksamkeit der beiden Substanzen auch in vivo auf die Metastasierung von HNSCC Zellen testen zu können. Eine TFP-Behandlung orthothop injizierter Tiere zur Analyse der Auswirkungen beider Substanzen auf die lymphogene Metastasierung, führte zu einer Reduktion der Metastasenanzahl. Eine Behandlung mit TG konnte eine Verkleinerung der detektierten Metastasen erzeugen. Die Auswertung der Kombinationsbehandlung auf die hämatogene Metastasierung im zweiten etablierten Tiermodell erzielte eine Reduktion der Metastasenlast in der Lunge. Neben dem Hauptaspekt der Auswirkungen von TFP und TG auf die Metastasierung wurden auch verschiedene Bildgebungsverfahren und deren Auswertungen (Kleintier-MRT, micro-CT, Histologie) zum Monitoring der entstehenden Metastasen etabliert. In einem zweiten Teil dieser Arbeit wurden stabile SEC62-knockout Klone einer immortalisierten HNSCC-Zelllinie mittels CRISPR-Cas9 generiert, die tiefere Erkenntnisse über Rolle von SEC62 und die mit SEC62-assoziierten Prozesse in der Zelle liefern sollten. Diese SEC62-ko Klone wurden mittels verschiedenster Methoden wie Immunfluoreszenz, Western Blot und next generation sequencing (NGS) validiert, sowie basierend auf einer whole-RNA-Sequenzierung charakterisiert. Des Weiteren wurden erste funktionelle Untersuchungen mit den SEC62-ko Klonen und ihre Toleranz gegenüber TFP und TG durchgeführt. Zusammenfassend konnten zwei murine Xenograft Modelle zur lymphogenen und hämatogenen Metastasierung von Kopf-Hals-Tumoren erfolgreich etabliert werden, an denen erste Untersuchungen zur Wirksamkeit zweier potentieller neuer Substanzen zur Therapie von metastasierenden, SEC62-überexpreimierenden Kopf-Hals-Tumoren durchgeführt werden konnten. Hierbei zeigten sich die beiden Substanzen TG und TFP als vielversprechende metastasierungshemmende Wirkstoffe, was zu weiteren Untersuchungen motiviert. Zudem konnten zwei stabile SEC62-ko Zelllinien mittels CRISPR-Cas9 generiert, validiert und charakterisiert werden. Weitere Analysen, um den Einfluss und die Funktion von SEC62 genauer verstehen zu können sind Gegenstand zukünftiger Forschung.Else-Kröner-Fresenius Stiftung, HOMFO

Head and Neck Cancer Invasion: Contributions of Actin Regulatory Proteins and the Microenvironment

Metastasis of primary tumor lesions is the leading cause of cancer-related death. In head and neck cancer, a local-regional disease, metastasis is achieved mainly through invasion into surrounding tissue and spreads to cervical lymph nodes. Movement from the initial tumor site requires dynamic reorganization of the actin cytoskeleton, which utilizes the coordinated action of many actin regulatory proteins. However, there is increasing evidence that the tumor microenvironment is also a driver of invasion. This work aims to determine the contributions of proteins which regulate the actin cytoskeleton during head and neck cancer invasion both in vitro and in vivo, and provide details on how the HNSCC tumor microenvironment influences progression. This was accomplished, by the following Studies. In Study one, the actin binding protein coronin 1B is found to be amplified and overexpressed in invasive HNSCC patient samples, and a novel function in the regulation of protrusive membrane structures called invadopodia is described. Study two defines an in vivo role for the actin regulatory protein cortactin, which has been previously associated with more aggressive cancers in vitro and in patients. This work finds that cortactin expression is dispensable for tongue tumor invasion in a transgenic model of oral cancer, implicating the tumor microenvironment as being the major contributor to driving oral cancer invasion. Study three describes a technique for monitoring and biopsying cervical lymph nodes of mice using high frequency ultrasound. By using this technique, alterations in cervical lymph node size and blood flow were discovered in mice given the carcinogen 4-NQO to induce oral carcinogenesis. Collectively, these studies shed light on the importance of choosing comprehensive model systems for studying roles of actin binding proteins in cancer invasion

Framework for Hyperspectral Image Processing and Quantification for Cancer Detection During Animal Tumor Surgery

Hyperspectral imaging (HSI) is an imaging modality that holds strong potential for rapid cancer detection during image-guided surgery. But the data from HSI often needs to be processed appropriately in order to extract the maximum useful information that differentiates cancer from normal tissue. We proposed a framework for hyperspectral image processing and quantification, which includes a set of steps including image preprocessing, glare removal, feature extraction, and ultimately image classification. The framework has been tested on images from mice with head and neck cancer, using spectra from 450- to 900-nm wavelength. The image analysis computed Fourier coefficients, normalized reflectance, mean, and spectral derivatives for improved accuracy. The experimental results demonstrated the feasibility of the hyperspectral image processing and quantification framework for cancer detection during animal tumor surgery, in a challenging setting where sensitivity can be low due to a modest number of features present, but potential for fast image classification can be high. This HSI approach may have potential application in tumor margin assessment during image-guided surgery, where speed of assessment may be the dominant factor

Genomic landscape and clonal architecture of mouse oral squamous cell carcinomas dictate tumour ecology.

To establish whether 4-nitroquinoline N-oxide-induced carcinogenesis mirrors the heterogeneity of human oral squamous cell carcinoma (OSCC), we have performed genomic analysis of mouse tongue lesions. The mutational signatures of human and mouse OSCC overlap extensively. Mutational burden is higher in moderate dysplasias and invasive SCCs than in hyperplasias and mild dysplasias, although mutations in p53, Notch1 and Fat1 occur in early lesions. Laminin-α3 mutations are associated with tumour invasiveness and Notch1 mutant tumours have an increased immune infiltrate. Computational modelling of clonal dynamics indicates that high genetic heterogeneity may be a feature of those mild dysplasias that are likely to progress to more aggressive tumours. These studies provide a foundation for exploring OSCC evolution, heterogeneity and progression