Despite the advances of therapeutic approaches, morbidity and mortality rate of oral squamous cell carcinoma (OSCC) have not improved significantly during the last 30 years. Photodynamic therapy (PDT) represents a potential treatment modality for locally recurrent OSCC. However, treatment resistance attributed to PDT-induced epidermal growth factor receptor (EGFR) up-regulation was reported. Here, we showed that vandetanib, a multi-target tyrosine kinase inhibitor that inhibits EGFR and vascular endothelial growth factor receptor-2 (VEGFR-2) enhance the efficacy of PDT. First, we observed increased cytotoxicity in the combinatorial treatment that is attributed to impaired DNA double strand break (DSB) repair. We revealed significant down-regulation DNA-dependent protein kinase catalytic subunit (DNA-PKcs) expression to be the downstream target following inhibition of nuclear EGFR accumulation, therefore impairing non-homologous end-joining (NHEJ) following PDT. Next, the combinatorial treatment-mediated tumour vasculature shutdown and normalisation, coupled with reduction of PDT-induced EGFR activation corresponded to the most pronounced tumour growth delay in vivo. Interestingly, we observed the restoration of tumour cell proliferation and vascularisation, coupled with ERK1/2 activation in the recurrent tumours post-vandetanib+PDT treatment. This corroborated the importance of the modulation of the tumour microenvironment for the observed synergistic efficacy of vandetanib+PDT combinatorial treatment. Collectively, our data suggests that vandetanib works synergistically with PDT through the impairment of EGFR-dependent DNA repair machinery and modulation of tumour microenvironment.
Photodynamic therapy (PDT) of cancer works via direct cytotoxicity, causing damage to tumor vasculature and
stimulating the body’s anti-tumor immune response. PDT outcome depends on the parameters used; therefore an in vivo
tumor response monitoring system is useful for optimization of the treatment protocol. The combined use of diffuse
optical spectroscopy and diffuse correlation spectroscopy allows us to measure the tissue oxygen saturation (StO2) and
relative blood flow (rBF) in tumors. These parameters were measured before and after PDT in mouse tumor models and
were calculated as ratios relative to the baseline in each tumor (rStO2 and rBF). Readings were also measured in drugonly
control tumors. In responders (mice with tumor eradication), significant PDT-induced decreases in both rStO2 and
rBF levels were observed at 3h post-PDT. The rStO2 and rBF readings in these mice remained low until 48h post-PDT,
with recovery of these parameters to baseline values observed 2 weeks after PDT. In non-responders (mice with partial
or no response), the rStO2 and rBF levels decreased less sharply at 3h post-PDT, and the rBF values returned toward
baseline values at 48h post-PDT. By comparison, the rStO2 and rBF readings in drug-only control tumors showed only
fluctuations about the baseline values. Thus tumor response can be predicted as early as 3h post-PDT. Recovery or
sustained decreases in rStO2 and rBF up till 48h post-PDT were correlated to long-term tumor control. Diffuse optical
measurements can thus facilitate early assessment of tumor response to PDT to aid in treatment planning.
Diffuse correlation spectroscopy (DCS) is an emerging noninvasive technique that probes the deep tissue blood flow, by using the time-averaged intensity autocorrelation function of the fluctuating diffuse reflectance signal. We present a fast Fourier transform (FFT)-based software autocorrelator that utilizes the graphical programming language LabVIEW (National Instruments) to complete data acquisition, recording, and processing tasks. The validation and evaluation experiments were conducted on an in-house flow phantom, human forearm, and photodynamic therapy (PDT) on mouse tumors under the acquisition rate of ∼400 kHz . The software autocorrelator in general has certain advantages, such as flexibility in raw photon count data preprocessing and low cost. In addition to that, our FFT-based software autocorrelator offers smoother starting and ending plateaus when compared to a hardware correlator, which could directly benefit the fitting results without too much sacrifice in speed. We show that the blood flow index (BFI) obtained by using a software autocorrelator exhibits better linear behavior in a phantom control experiment when compared to a hardware one. The results indicate that an FFT-based software autocorrelator can be an alternative solution to the conventional hardware ones in DCS systems with considerable benefits.
Oral lesions are conventionally diagnosed using white light endoscopy and histopathology. This can pose a challenge because the lesions may be difficult to visualise under white light illumination. Confocal laser endomicroscopy can be used for confocal fluorescence imaging of surface and subsurface cellular and tissue structures. To move toward real-time "virtual" biopsy of oral lesions, we interfaced an embedded computing system to a confocal laser endomicroscope to achieve a prototype three-dimensional (3-D) fluorescence imaging system. A field-programmable gated array computing platform was programmed to enable synchronization of cross-sectional image grabbing and Z-depth scanning, automate the acquisition of confocal image stacks and perform volume rendering. Fluorescence imaging of the human and murine oral cavities was carried out using the fluorescent dyes fluorescein sodium and hypericin. Volume rendering of cellular and tissue structures from the oral cavity demonstrate the potential of the system for 3-D fluorescence visualization of the oral cavity in real-time. We aim toward achieving a real-time virtual biopsy technique that can complement current diagnostic techniques and aid in targeted biopsy for better clinical outcomes.
Oral lesions are conventionally diagnosed using white light endoscopy and histopathology of biopsy samples. Oral
lesions are often flat and difficult to visualize under white light illumination. Moreover, histopathology is timeconsuming
and there is a need to develop minimally invasive optical biopsy techniques to complement current
techniques. Confocal laser endomicroscopy holds promise for virtual biopsy in disease diagnosis. This technique enables
fluorescence imaging of tissue structures at microscopic resolution. We have developed a prototype real-time 3-
dimensional (3D) imaging system using a laser endomicroscope interfaced with embedded computing. A Field-
Programmable Gate Array computing platform has been programmed to synchronize cross-sectional image grabbing and
Z-depth scanning, as well as automate acquisition of confocal image stacks. A PC was used for real-time volume
rendering of the confocal image stacks. We conducted pre-clinical and pilot clinical studies to image the murine and
human oral cavity. High quality volume renderings of the confocal image stacks were generated using 3D texture slicing.
Tissue morphology and 3D structures could be visualized. The results demonstrate the potential of the system for
diagnostic imaging of the oral cavity. This paves the way toward real-time virtual biopsy of oral lesions, with the aim to
achieve same-day diagnosis in a clinical setting.
Photodynamic therapy (PDT) is an alternative cancer treatment modality that offers localized treatment using a photosensitizer and light. However, tumor angiogenesis is a major concern following PDT-induced hypoxia as it promotes recurrence. Bevacizumab is a monoclonal antibody that targets vascular endothelial growth factor (VEGF), thus preventing angiogenesis. The combination of PDT with antiangiogenic agents such as bevacizumab has shown promise in preclinical studies. We use confocal endomicroscopy to study the antiangiogenic effects of PDT in combination with bevacizumab. This technique offers in vivo surface and subsurface fluorescence imaging of tissue. Mice bearing xenograft bladder carcinoma tumors were treated with PDT, bevacizumab, or PDT and bevacizumab combination therapy. In tumor regression experiments, combination therapy treated tumors show the most regression. Confocal fluorescence endomicroscopy enables visualization of tumor blood vessels following treatment. Combination therapy treated tumors show the most posttreatment damage with reduced cross-sectional area of vessels. Immunohistochemistry and immunofluorescence studies show that VEGF expression is significantly downregulated in the tumors treated by combination therapy. Overall, combining PDT and bevacizumab is a promising cancer treatment approach. We also demonstrate that confocal endomicroscopy is useful for visualization of vasculature and evaluation of angiogenic response following therapeutic intervention.
Oral cancers are currently diagnosed using white light endoscopy and histopathology. However, oral tumours are mostly
superficial and can be difficult to visualise. Here we present the use of hypericin with fluorescence endoscopy and laser
confocal fluorescence endomicroscopy interfaced with embedded computing for the diagnosis of oral cancers.
Fluorescence imaging of oral lesions was carried out in the clinic using a fluorescence endoscope. The images were
analyzed to extract the red to blue (R/B) ratios to discriminate between tissue types. The results showed that the R/B
ratio is a good image parameter to discriminate between normal, hyperplastic and malignant oral tissue. We are also
developing an embedded, real-time computing system interfaced to a fluorescence endomicroscope for 3D visualization
of tumors, where synchronization of cross-sectional image grabbing and Z-depth scanning is realized through
programming a Field-Programmable Gate Array. In addition to the programming task, a proprietary control circuit has
been developed for the automated 3D reconstruction of fluorescence sections; and preliminary results from fluorescent
samples have demonstrated the potential of this system for real-time in vivo 3D visualization of tumours. This will
ultimately enable same-day clinical diagnosis to be achieved and further enhance the clinical usefulness of fluorescence
diagnostic imaging.
Photodynamic therapy (PDT) is a standard treatment for various malignant and non-malignant conditions. Though
therapeutic responses are encouraging, recurrences have been noted, as one of the limitations of PDT is treatment-induced
hypoxia that triggers angiogenesis. The present study evaluates the use of angiogenic inhibitors Avastin, that
targets vascular endothelial growth factor (VEGF) and Erbitux that targets epidermal growth factor receptor (EGFR)
with PDT in an in vivo bladder carcinoma xenograft. Tumor bearing mice were assigned to 6 different categories:
control, PDT only, Avastin + Erbitux, PDT + Avastin, PDT + Erbitux and PDT + Avastin and Erbitux. Treated and
control tumors were monitored for recurrence for up to 90 days. VEGF and EGFR expression was detected in the tumor
tissue. Migratory assay was performed to establish the inhibitory effect of the angiogenesis agents. Using confocal laser
endomicroscopy, the tumor microvasculature was assessed. Tumors treated with the combination therapy of PDT +
inhibitors showed significantly greater response compared to control and PDT only treated group. Combination therapy
treated tumors also showed the most post-treatment damage with reduced tumor vasculature. These results demonstrate
that the combination of PDT with inhibitors that target different angiogenesis pathways can improve tumor control.
Malignancies of the oral cavity are conventionally diagnosed by white light endoscopy, biopsy, and histopathology. However, it is often difficult to distinguish between benign and premalignant or early lesions. A laser confocal endomicroscope (LCE) offers noninvasive, in vivo surface and subsurface fluorescence imaging of tissue. We investigate the use of an LCE with a rigid probe for diagnostic imaging of the oral cavity. Fluorescein and 5-aminolevulinic acid (ALA) were used to carry out fluorescence imaging in vivo and on resected tissue samples of the oral cavity. In human subjects, ALA-induced protoporphyrin IX (PpIX) fluorescence images from the normal tongue were compared to images obtained from patients with squamous cell carcinoma (SCC) of the tongue. Using rat models, images from normal rat tongues were compared to those from carcinogen-induced models of SCC. Good structural images of the oral cavity were obtained using ALA and fluorescein, and morphological differences between normal and lesion tissue can be distinguished. The use of a pharmaceutical-grade solvent Pharmasolve® enhanced the subsurface depth from which images can be obtained. Our initial results show that laser confocal fluorescence endomicroscopy has potential as a noninvasive optical imaging method for the diagnosis of oral cavity malignancies.
Confocal endomicroscopy is a novel, noninvasive microscopic technique that enables surface and subsurface imaging of living tissues or cells in vivo. This study was to explore the possibility of utilizing a novel rigid confocal endomicroscope (RCE) system for detecting morphological changes in living normal and neoplastic human and murine tongue tissue in combination with different photosensitizers, i.e. hypericin and 5-aminolevulinic acid (ALA) induced endogenous protoporphyrin IX (PPIX) fluorescence. Subjects were topically or systemically applied photosensitizer to the oral mucosa, and then fluorescence confocal endomicroscopy was performed on the tongue using the RCE system with the laser excitation wavelength at 488 nm. The preliminary results showed that confocal fluorescence images of the tongue can be acquired in real-time with well-defined micro-morphological structures, and changes of tissue structures associated with cancer transformation can also be identified. This study suggests that photosensitizer-mediated confocal endomicroscopy have a significant potential for rapid, non-invasive detection of early oral cancers in vivo.
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