In-vivo optical detection of cancer using chlorin e6--polyvinylpyrrolidone induced fluorescence imaging and spectroscopy.

BACKGROUND: Photosensitizer based fluorescence imaging and spectroscopy is fast becoming a promising approach for cancer detection. The purpose of this study was to examine the use of the photosensitizer chlorin e6 (Ce6) formulated in polyvinylpyrrolidone (PVP) as a potential exogenous fluorophore for fluorescence imaging and spectroscopic detection of human cancer tissue xenografted in preclinical models as well as in a patient. METHODS: Fluorescence imaging was performed on MGH human bladder tumor xenografted on both the chick chorioallantoic membrane (CAM) and the murine model using a fluorescence endoscopy imaging system. In addition, fiber optic based fluorescence spectroscopy was performed on tumors and various normal organs in the same mice to validate the macroscopic images. In one patient, fluorescence imaging was performed on angiosarcoma lesions and normal skin in conjunction with fluorescence spectroscopy to validate Ce6-PVP induced fluorescence visual assessment of the lesions. RESULTS: Margins of tumor xenografts in the CAM model were clearly outlined under fluorescence imaging. Ce6-PVP-induced fluorescence imaging yielded a specificity of 83% on the CAM model. In mice, fluorescence intensity of Ce6-PVP was higher in bladder tumor compared to adjacent muscle and normal bladder. Clinical results confirmed that fluorescence imaging clearly captured the fluorescence of Ce6-PVP in angiosarcoma lesions and good correlation was found between fluorescence imaging and spectral measurement in the patient. CONCLUSION: Combination of Ce6-PVP induced fluorescence imaging and spectroscopy could allow for optical detection and discrimination between cancer and the surrounding normal tissues. Ce6-PVP seems to be a promising fluorophore for fluorescence diagnosis of cancer.

Correction: Early assessment of tumor response to photodynamic therapy using combined diffuse optical and diffuse correlation spectroscopy to predict treatment outcome.

[This corrects the article DOI: 10.18632/oncotarget.15720.].

Vandetanib sensitizes head and neck squamous cell carcinoma to photodynamic therapy through modulation of EGFR-dependent DNA repair and the tumour microenvironment.

BACKGROUND: Epidermal growth factor receptor (EGFR) overexpression is characteristic in head and neck cancers and is associated with tumour regrowth following photodynamic therapy (PDT). PURPOSE: We investigated vandetanib, which selectively blocks EGFR and vascular endothelial growth factor receptor-2 (VEGFR-2), to enhance the efficacy of PDT. METHODS: We assessed the in vitro therapeutic efficacy of: 1) vandetanib; 2) PDT with the photosensitizer Chlorin e6 (Fotolon®); and 3) combined PDT + vadetanib treatment in CAL-27 oral squamous cell carcinoma (OSCC) cell line by cell viability, γH2AX foci immunostaining, cell cycle arrest and western blot. We also performed in vivo tumour regression study and immunohistochemical staining of formalin-fixed paraffin-embedded (FFPE) regressed and regrown tumour tissues. RESULTS: First, we observed significantly higher cytotoxicity and residual DNA damage in vandetanib + PDT-treated CAL-27 OSCC cells than tumour cells treated with PDT alone. This is due to impaired DNA DSB repair caused by downregulation of EGFR-mediated DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activation. Next, combined vandetanib and PDT resulted in significant tumour growth delay in vivo that is linked to reduction of PDT-induced EGFR phosphorylation and cellular proliferation, along with loss of tumour vasculature. In particular, we observed significant revascularisation of the microenvironment that is associated with upregulated ERK1/2 phosphorylation in regrown tumours post-vandetanib + PDT, thereby corroborating the importance of microenvironmental modification for the observed drug-PDT synergistic interaction. CONCLUSION: Taken together, our data suggests that vandetanib enhances the efficacy of PDT through both direct and indirect effects on the cellular DNA repair machinery and tumour microenvironment, respectively.

Immune response against angiosarcoma following lower fluence rate clinical photodynamic therapy.

Tumor response to photodynamic therapy (PDT) is dependent on treatment parameters used. In particular, the light fluence rate may be an important determinant of the treatment outcome. In this clinical case report, we describe the response of angiosarcoma to PDT carried out using different fluence rates and drug and light doses. A patient with recurrent multifocal angiosarcoma of the head and neck was recruited for PDT. A new generation chlorin-based photosensitizer, Fotolon, was administered at a dose of 2.0 to 5.7 mg/kg. The lesions were irradiated with 665 nm laser light for a light dose of 65 to 200 J/cm2 delivered at a fluence rate of 80 or 150 mW/cm2. High dose PDT carried out at a high fluence rate resulted in local control of the disease for up to a year; however, the disease recurred and PDT had to be repeated. PDT of new lesions carried out at a lower fluence rate resulted in tumor eradication. More significantly, it also resulted in spontaneous remission of neighboring and distant untreated lesions. Repeat PDT carried out on a recurrent lesion at a lower fluence rate resulted in eradication of both treated and untreated lesions despite the lower total light dose delivered. Immunohistochemical examination of biopsy samples implies that PDT could have activated a cell-mediated immune response against untreated lesions. Subsequent histopathological examination of the lesion sites showed negative for disease. Our clinical observations show that lower fluence rate PDT results in better outcome and also indicate that the fluence rate, rather than the total light dose, is a more crucial determinant of the treatment outcome. Specifically, lower fluence rate PDT appears to activate the body's immune response against untreated lesions.

Early assessment of tumor response to photodynamic therapy using combined diffuse optical and diffuse correlation spectroscopy to predict treatment outcome.

Photodynamic therapy (PDT) of cancer involves the use of a photosensitizer that can be light-activated to eradicate tumors via direct cytotoxicity, damage to tumor vasculature and stimulating the body's immune system. Treatment outcome may vary between individuals even under the same regime; therefore a non-invasive tumor response monitoring system will be useful for personalization of the treatment protocol. We present the combined use of diffuse optical spectroscopy (DOS) and diffuse correlation spectroscopy (DCS) to provide early assessment of tumor response. The relative tissue oxygen saturation (rStO2) and relative blood flow (rBF) in tumors were measured using DOS and DCS respectively before and after PDT with reference to baseline values in a mouse model. In complete responders, PDT-induced decreases in both rStO2 and rBF levels were observed at 3 h post-PDT and the rBF remained low until 48 h post-PDT. Recovery of these parameters to baseline values was observed around 2 weeks after PDT. In partial responders, the rStO2 and rBF levels also decreased at 3 h post PDT, however the rBF values returned toward baseline values earlier at 24 h post-PDT. In contrast, the rStO2 and rBF readings in control tumors showed fluctuations above the baseline values within the first 48 h. Therefore tumor response can be predicted at 3 to 48 h post-PDT. Recovery or sustained decreases in the rBF at 48 h post-PDT corresponded to long-term tumor control. Diffuse optical measurements can thus facilitate early assessment of tumor response. This approach can enable physicians to personalize PDT treatment regimens for best outcomes.

Novel delivery of Chlorin e6 using anti-EGFR antibody tagged virosomes for fluorescence diagnosis of oral cancer in a hamster cheek pouch model.

PURPOSE: Overexpression of epidermal growth factor receptor (EGFR) is observed in oral squamous cell carcinoma (OSCC) and is associated with increased proliferation, metastasis and therapeutic resistance. We aim to develop a novel drug delivery system comprised of a photosensitizer Chlorin e6 (Ce6) that is encapsulated in a viral envelope and tagged with anti-EGFR antibody to target OSCC. METHODS: Ce6 was encapsulated in both virosomes (Ce6-Vir) and virosomes tagged with anti-EGFR antibody (Ce6-Vir-EGFR'). In vitro studies were conducted to assess the cellular uptake and bioavailability of the photosensitizer in OSCC cells. Ce6 alone or in constructs was then administered in a hamster cheek pouch model and fluorescence imaging and spectroscopy was performed. RESULTS: In vitro results showed that the uptake of Ce6-Vir-EGFR' was lower than that for Ce6-Vir and Ce6 possibly due to its large size. Nevertheless, in vivo results showed significant tumor specificity of Ce6-Vir-EGFR' compared to Ce6. The tumor to normal mucosa ratio showed that Ce6-Vir-EGFR' can successfully target OSCC lesions and therefore shows potential for use in fluorescence diagnosis of OSCC. CONCLUSIONS: Both the virosome-Ce6 constructs were internalized by OSCC cells and successfully used for fluorescence imaging. Tagging with anti-EGFR antibody further improved the targeting ability toward OSCC.

Nimotuzumab increases the anti-tumor effect of photodynamic therapy in an oral tumor model.

Oral squamous cell carcinoma (OSCC) represents 90% of all oral cancers and is characterized with poor prognosis and low survival rate. Epidermal growth factor receptor (EGFR) is highly expressed in oral cancer and is a target for cancer therapy and prevention. In this present work, we evaluate the efficacy of photodynamic therapy (PDT) in combination with an EGFR inhibitor, nimotuzumab in oral cancer cell lines and OSCC xenograft tumor model. PDT is a promising and minimally invasive treatment modality that involves the interaction of a photosensitizer, molecular oxygen and light to destroy tumors. We demonstrated that EGFR inhibitors nimotuzumab and cetuximab exhibits anti-angiogenic properties by inhibiting the migration and invasion of oral cancer cell lines and human endothelial cells. The EGFR inhibitors also significantly reduced tube formation of endothelial cells. Chlorin e6-PDT in combination with nimotuzumab and cetuximab reduced cell proliferation in different oral cancer and endothelial cells. Furthermore, our in vivo studies showed that the combination therapy of PDT and nimotuzumab synergistically delayed tumor growth when compared with control and PDT treated tumors. Downregulation of EGFR, Ki-67 and CD31 was observed in the tumors treated with combination therapy. Analysis of the liver and kidney function markers showed no treatment related toxicity. In conclusion, PDT outcome of oral cancer can be improved when combined with EGFR inhibitor nimotuzumab.

Dopaminergic cell death precedes iron elevation in MPTP-injected monkeys.

Though increasing lines of evidence suggest that iron accumulation and iron-induced oxidative stress might be important pathological factors responsible for substantia nigra (SN) cell death in Parkinson's disease (PD), it is still unknown whether iron accumulation is a primary cause or consequence of nigral cell death. Using nuclear microscopy, iron histochemistry, TUNEL method for apoptosis detection, and tyrosine hydroxylase (TH) immunohistochemistry, the present study investigated possible changes in iron contents in the SN and correlations of dopaminergic cell death progression with the process of iron accumulation in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced parkinsonian monkey from 1 d to 18 months after MPTP administration. Our study demonstrated that apoptosis occurred in the ipsilateral SN at 1 d after MPTP injection and the number of TH-positive cells decreased significantly from 1 week onward. However, iron content was significantly increased in the ipsilateral SN from 4.5 months to 18 months after MPTP injection, and the iron increase was significantly correlated to the extent of dopaminergic cell death. These results suggest that dopaminergic cell death induced by MPTP administration might lead to iron accumulation in the monkey SN, and increased iron might contribute to the progression of nigral degeneration.

Confocal endomicroscopic imaging of normal and neoplastic human tongue tissue using ALA-induced-PPIX fluorescence: a preliminary study.

Confocal endomicroscopy is a novel and non-invasive microscopic technique that enables surface and subsurface imaging of living tissues or cells in vivo. The purpose of this study was to assess the possibility of utilizing a rigid confocal endomicroscope (RCE) system developed for detecting morphological changes in living normal and neoplastic human tongue tissue in combination with 5-aminolevulinic acid (ALA)-induced endogenous protoporphyrin IX (PPIX) fluorescence. Three patients with squamous cell carcinoma (SCC) of the tongue were examined using the novel RCE system with the excitation wavelength at 488 nm from an argon-ion laser and the detection wavelengths of the tissue fluorescence above 515 nm. Patients were topically applied with 0.4% of 5-ALA rinsing solution to the oral mucosa for approximately 15 min, and then the confocal endomicroscopic imaging of tissue PPIX fluorescence was performed on the lesion sites of the tongue after an optimal incubation period of 90-120 min. For comparison purposes, ALA-PPIX fluorescence confocal endomicroscopic imaging was also carried out on the normal sites of the tongue in vivo from two healthy volunteers. Image distortions due to tissue motion can be minimized using a specially designed tissue stabilizer attached to the RCE probe. Good quality ALA-mediated confocal fluorescence images of the tongue can be acquired in real-time, providing well-defined micro-morphological structures (e.g., filiform papillae, keratinized epithelium and fungiform papillae) of the tongue in vivo. Changes of tissue structures in oral tissue associated with cancer transformation can also be clearly identified using the RCE imaging. Preliminary results obtained in this study suggest that ALA-mediated rigid confocal endomicroscopy may have a significant potential for the rapid, non-invasive diagnosis and evaluation of early oral cancers in vivo.

Hemodynamic monitoring of Chlorin e6-mediated photodynamic therapy using diffuse optical measurements.

Tumor response during photodynamic therapy (PDT) is heavily dependent on treatment parameters such as light dose, photosensitizer concentration, and tissue oxygenation. Therefore, it is desirable to have a real-time hemodynamic monitoring device in order to fine-tune the parameters and improve PDT efficacy. In this paper, such a tumor response monitoring system was built incorporating both frequency domain diffuse optical spectroscopy (FD-DOS) and diffuse correlation spectroscopy (DCS), which enables concurrent monitoring of tissue oxygenation (StO2), total hemoglobin concentration (THC) and relative blood flow (rBF). The tumor metabolic rate of oxygen (TMRO2) was calculated by using the hemodynamic parameters. Mouse models bearing xenograft tumors were subjected to chlorin e6 (Ce6)-mediated PDT, and the four parameters were monitored with varying treatment conditions. The results show (1) At 3 h post-PDT, rStO2, rBF and rTMRO2 exhibited sharp PDT-induced decreases in responders (>40% reduction in tumor volume). Statistically significant difference between responders and non-responders were observed in rStO2 and rBF, but not in rTMRO2. (2) Non-responders show gradual recovery of rStO2, rBF and rTMRO2 from ∼24 h post-PDT, while responder group did not show recovery up until 48 h post-PDT. Long-term study results up to 2 weeks are also shown. It suggests the hybrid diffuse optical system is not only capable of real-time treatment monitoring, but also able to extract tumor metabolic rate of oxygen to provide more insights about therapy mechanism. Translation of this technique to the clinic will make a quick prognosis feasible and help with treatment optimization.

Diffuse correlation spectroscopy with a fast Fourier transform-based software autocorrelator.

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.

Toward real-time virtual biopsy of oral lesions using confocal laser endomicroscopy interfaced with embedded computing.

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.

Evaluation of hypericin-mediated photodynamic therapy in combination with angiogenesis inhibitor bevacizumab using in vivo fluorescence confocal endomicroscopy.

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.