Fluorescence imaging and spectroscopy of ethyl nile blue A in animal models of (pre)malignancies.

Discrimination between normal and premalignant tissues by fluorescence imaging and/or spectroscopy may be enhanced by a tumor-localizing fluorescent drug. Ethyl Nile Blue A (EtNBA), a dye with no phototoxic activity, was investigated for this purpose. The pharmacokinetics and tissue-localizing properties were investigated in a rat palate model with chemically induced premalignant mucosal lesions (0.5 mg/kg EtNBA intravenous [i.v.]), a hairless mouse model with UVB-induced premalignant skin lesions (1 mg/kg EtNBA intraperitoneal) and in a rat skin-fold observation chamber model on the back of a rat with a transplanted solid tumor (2.5 mg/kg EtNBA i.v.). Fluorescence images and spectra were recorded in vivo (600 nm excitation, 665-900 nm detection) and in frozen tissue sections at several time points after EtNBA administration. In the rat palate the EtNBA fluorescence was maximum almost immediately after injection, whereas in the mouse skin and the observation chamber the fluorescence maximum was reached between 2 and 3 h after injection. EtNBA cleared from tissues after 8-24 h. EtNBA localizes in the transplantable solid tumor, but is not targeted specifically to the dysplastic location in the rat palate and mouse skin. However, in the rat palate the EtNBA fluorescence increased significantly with increasing dysplasia, apparently due to the increasing thickness of the upper keratinized layer of the epithelium where the dye was found to localize. Localization in this layer occurred both in the rat palate and in hairless mouse skin.

Localisation and accumulation of a new carotenoporphyrin in two primary tumour models.

We have investigated the tumour-localising properties and in vivo fluorescence kinetics of a hexamethoxylated carotenqporphyrin (CP6) in two primary tumour models: UV-B-induced early skin cancer in hairless mice and chemically induced mucosal dysplasia in the rat palate. CP6 fluorescence kinetics are investigated by measuring in vivo fluorescence spectra and images of the mouse skin and the rat palate at different time points after injection. For the tumour-localising properties, microscopic phase-contrast and fluorescence images are recorded. The in vivo fluorescence kinetics in the mouse skin show localization of CP6 in the tumours. However, fluorescence microscopy images show that CP6 localises in the dermis and structures that are not related to the malignant transformation of the mouse skin. The fluorescence kinetics in the rat palate show a significant correlation between the degree of malignancy and the CP6 fluorescence build-up time in the palate. The microscopic images show that CP6 fluorescence localises in the connective tissue and not in the dysplastic epithelium. In conclusion, CP6 does not localise preferentially in (pre-) cancerous tissue in the two primary tumour models studied here, in contrast to reports about localisation of carotenoporphyrins in transplanted tumours. However, the CP6 build-up time in rat palates correlates with the degree of malignancy and this might possibly be a useful parameter in tumour detection.

Classification of clinical autofluorescence spectra of oral leukoplakia using an artificial neural network: a pilot study.

The performance of an artificial neural network was evaluated as an alternative classification technique of autofluorescence spectra of oral leukoplakia, which may reflect the grade of tissue dysplasia. Twenty-two visible lesions of 21 patients suffering from oral leukoplakia and six locations on normal oral mucosa of volunteers were investigated with autofluorescence spectroscopy (420 nm excitation, 465-650 nm emission). Pre-scaled spectra were combined with the corresponding visual and histopathological classifications in order to train artificial neural networks. A trained network is mapping input spectra to tissue characteristics, which was evaluated using a blind set of spectra. Abnormal tissue could be distinguished from normal tissue by a neural network with a sensitivity of 86% and a specificity of 100%. Also, classifying either homogeneous or non-homogeneous tissue performed reasonably well. Weak or no correlation existed between spectral patterns and verrucous or erosive tissue or the grade of dysplasia, hyperplasia and hyperkeratosis.

In vivo detection of dysplastic tissue by Raman spectroscopy.

The detection of dysplasia and early cancer is important because of the improved survival rates associated with early treatment of cancer. Raman spectroscopy is sensitive to the changes in molecular composition and molecular conformation that occur in tissue during carcinogenesis, and recent developments in fiber-optic probe technology enable its application as an in vivo technique. In this study, the potential of Raman spectroscopy for in vivo classification of normal and dysplastic tissue was investigated. A rat model was used for this purpose, in which dysplasia in the epithelium of the palate was induced by topical application of the carcinogen 4-nitroquinoline 1-oxide. High quality in vivo spectra of normal and dysplastic rat palate tissue, obtained using signal integration times of 100 s were used to create tissue classification models based on multivariate statistical analysis methods. These were tested with an independent set of in vivo spectra, obtained using signal collection times of 10 s. The best performing model, in which signal variance due to signal contributions of the palatal bone was eliminated, was able to distinguish between normal tissue, low-grade dysplasia, and high-grade dysplasia/carcinoma in situ with a selectivity of 0.93 and a sensitivity of 0.78 for detecting low-grade dysplasia and a specificity of 1 and a sensitivity of 1 for detecting high-grade dysplasia/ carcinoma in situ.

In vivo photo-detection of chemically induced premalignant lesions and squamous cell carcinoma of the rat palatal mucosa.

Photo-detection using in vivo fluorescence was studied for different stages of chemically induced premalignant lesions and squamous cell carcinoma (SCC) of the Wistar rat palatal mucosa. It was found that the epithelial dysplasia (numerically expressed in the epithelial atypia index (EAI) of the rat palate, induced by repeated application of the carcinogen 4-nitroquinoline 1-oxide (4NQO), showed an increase approximately proportional to the duration of the application period. Photo-detection of the lesions using Photofrin-induced fluorescence was studied with dual-wavelength excitation and the subtraction of images, in an attempt to reduce the autofluorescence. The Photofrin dose was 2.5 mg kg-1. This was based on a dose-response study for normal tissue damage by photodynamic therapy (PDT) in this animal model, because the underlying rationale was to study photo-detection as a method of locating additional (early) malignancies in patients treated by PDT. Fluorescence intensities 24 and 48 h after injection of Photofrin were shown to increase with the duration of 4NQO application and with increasing EAI. For an EAI greater than 15, there was a statistically significant difference (p < 0.01) between the fluorescence signals obtained with and without the injection of Photofrin. Fluorescence signals of these lesions without the use of Photofrin (autofluorescence) also showed an increase with increasing stages of epithelial dysplasia of the rat palate. However, the fluorescence signals obtained with Photofrin were always higher than those of the autofluorescence. From this study, we conclude that photo-detection with Photofrin has potential in distinguishing chemically induced premalignant lesions and squamous cell carcinomas from the normal rat palatal mucosa. Photofrin (2.5 mg per kg of body weight) certainly adds to the sensitivity of photo-detection, but autofluorescence alone also has promising features for detecting premalignant and malignant lesions of the oral mucosa.

Distribution of aluminum phthalocyanine disulfonate in an oral squamous cell carcinoma model. In vivo fluorescence imaging compared with ex vivo analytical methods.

Photosensitizer-induced fluorescence is studied as a technique for the detection of cancer. Therefore we investigated the ability of a photosensitizer, aluminum phthalocyanine disulfonate (AIPcS2), to localize in tumor tissue. In vivo endoscopic fluorescence imaging, fluorescence microscopy, conventional spectrofluorometry and high performance liquid chromatograpy combined with diode laser-induced fluorescence (HPLC-Dio-LIF) were used. Squamous cell carcinomas were induced with 4-nitro-quinoline-1-oxide (4NQO) in the mucosa of the palate of the rat. In vivo fluorescence images, taken after injection of 1.5 mumol/kg AIPcS2 intravenously, showed that 4NQO-treated palates had higher fluorescence signals than normal palates. Areas displaying locally high amounts of AIPcS2 fluorescence (hot spots) were present only in 4NQO-treated rats 2-8 h but had disappeared 24 h after injection. However, HPLC-Dio-LIF showed that the relative AIPcS2 content was highest at 24/48 h in biopsies taken in the areas of the hot spots. Fluorescence microscopy revealed that AIPcS2 was present only between 2 and 8 h in the epithelial layer, while in biopsies the connective tissue contained large quantities of AIPcS2 at 24/48 h. In vivo fluorescence imaging appears to show mainly fluorescence from the epithelial layer and the ex vivo analytical techniques mainly show the connective tissue fluorescence. Care should be taken when interpreting data using one technique only.

In vivo fluorescence kinetics and localisation of aluminum phthalocyanine disulphonate in an autologous tumour model.

Sulphonated phthalocyanines are studied as photosensitizers for photodynamic therapy of cancer. Their strong fluorescence and tumour-localising properties make them also potentially useful for detection of cancer by fluorescence. For this purpose, we have studied the fluorescence kinetics and localisation of aluminum phthalocyanine disulphonate (AlPcS2) in 4-nitroquinoline 1-oxide (4NQO)-induced dysplasia and invasive cancer of the oral mucosa of the hard palate in Wistar albino rats. Twenty-two rats were divided into six groups. Five groups were subjected to a 4NQO application period of 8, 12, 16, 20 or 26 weeks and one was a control group. The dysplasia varied from slight to severe and was correlated with the duration of the application period. All animals received a dose of 1 micromol/kg AlPcS2 i.v. Fluorescence images were recorded via a specially designed 'palatoscope' with excitation at 460 +/- 20 nm for autofluorescence, 610 +/- 15 nm for AlPcS2 fluorescence and detection of emission at 675 +/- 15 nm. After subtraction of the two images the specific AlPcS2 fluorescence remained. AlPcS2-mediated fluorescence increased significantly when the severity of dysplasia increased (P<0.04). Also the phenomenon of strong fluorescent spots on the fluorescence images was observed. This always occurred within the first 10 h after injection of AlPcS2. Histological analysis showed a local alteration to a mucosa in 67% of these spots, which was either invasive cancer (29%) or inflammation (38%). These results suggest two different mechanisms of AlPcS2 uptake in tissue, one associated with the presence of generalised dysplasia and another associated with local changes of the epithelial/connective tissue, which is not necessarily specific for tumours.