Uptake and localisation of mTHPC (Foscan) and its 14C-labelled form in normal and tumour tissues of the hamster squamous cell carcinoma model: a comparative study

Abstract

The aim of this study was to evaluate the pharmacokinetics of meta(tetrahydroxyphenyl)chlorin (mTHPC) on different tissues of interest in a hamster tumour model and to confirm our earlier animal studies on semi-quantitative fluorescence microscopy. The results obtained by three different evaluation methods were compared: in vivo spectrofluorometry, ex vivo fluorescence microscopy and chemical extraction of (14)C-labelled mTHPC. Following intracardiac injection of 0.5 mg kg(-1) mTHPC, groups of five tumour-bearing animals were used for in situ light-induced fluorescence spectroscopy. Afterwards, the biopsies were taken and snap frozen for fluorescence microscopy. The presence of radioactivity in serum and tissues was determined after chemical digestion in scintillation fluid using a scintillation counter. For each analysed tissue, a good correlation was observed between the three evaluation methods. The highest fluorescence intensity and quantities of mTHPC were observed between 12 and 24 h in liver, kidney, serum, vascular endothelium and advanced neoplasia. The majority of mTHPC was found at around 48 h in smooth muscle and at 96 h in healthy cheek pouch mucosa and early malignant lesions. The lowest level of mTHPC was noted in striated muscle at all times. No selectivity in dye localisation was observed between early squamous cell carcinoma and healthy mucosa. Soon after the injection, a significant selectivity was noted for advanced squamous cell carcinoma as compared to healthy cheek pouch mucosa or striated muscle. A significant difference in mTHPC localisation and quantity was also observed between striated and smooth muscle during the first 48 h following the injection. Finally, this study demonstrated the usefulness of non-invasive in situ spectroscopic measurements to be performed systematically prior to photodynamic therapy as a real-time monitoring for each treated patient in order to individualise and adapt the light dosimetry and avoid over or under treatments.

Figure 1

Macroscopic appearance on the left side and histology on the right side of the hamster cheek pouch during various steps of squamous cell carcinogenesis. (A) Smooth and uniform surface of the healthy cheek pouch with thin mucosa; corresponding histology showing the top 3–4 layers of squamous epithelial cells (E) including well-delimited basal membrane (BM); lamina propria (LP) containing stromal cells, fibro-connective tissue and blood vessels and underlying striated muscle (SM). (B) Early dysplastic changes varying from low- to high-grade dysplasia appear between 6 and 9 weeks after DMBA application. Macroscopically, the cheek pouch mucosa is more-or-less thick, showing erythroleukoplastic changes. Histologically, this corresponds to an abnormal proliferation of epithelial cells with large nuclei, abundant cytoplasm and frequent mitotic figures. BM is less well delimited. No isolated tumour cells are visible in the lamina propria. (C) About 10 weeks after DMBA application, erythroleucoplastic changes, irregularity and thickening of the mucosa become much more severe. Histologically, besides the cytonuclear abnormalities, a small island of isolated tumour cells appears in the lamina propria. This stage corresponds to μ-invasive carcinoma often associated with slight peritumoural inflammation. (D) At 16 weeks following DMBA application, the bulky formation appears on the painted cheek pouch surface, corresponding to more-or-less well-differentiated SCC. At this stage, a tumour shows a highly infiltrative pattern and large tumour nests invade the lamina propria and striated muscle. The lesions are highly vascularised and tumour-associate inflammation is abundant. Original magnification ×10 (A,D) and ×20 (B,C).

Figure 2

The comparative pharmacokinetics of mTHPC in serum and vascular endothelium by means of in situ LIFS (A), extraction of ¹⁴C-labelled mTHPC (B) and ex vivo FM (C). The fluorescence photomicrograph and corresponding HE staining of the blood vessel wall 48 h after mTHPC administration shows a much higher fluorescence intensity in blood vessel (V) endothelium as in adjacent structures such as fibro-connective tissue (FC) of lamina propria and striated muscle (SM) (D). Scale: the bar in the right bottom corner represents 20 μm.

Figure 3

The comparative pharmacokinetics of mTHPC in the liver and kidney using in situ LIFS (A), extraction of ¹⁴C-labelled mTHPC (B) and ex vivo FM (C). Fluorescence photomicrographs and corresponding HE stains show the high fluorescence intensity, uniform in liver parenchyma (D) and kidney glomerulus (white areas), while a low dye amount was observed in proximal and distal tubes of kidney parenchyma (dark area) soon (24 h) after mTHPC administration (E). Scale: the bar in the right bottom corner represents 20 μm.

Figure 4

The comparative pharmacokinetics of mTHPC in healthy mucosa, early and advanced SCC observed by in situ LIFS (A), extraction of ¹⁴C-labelled mTHPC (B) and ex vivo FM (C). The photomicrographs and corresponding HE stains of healthy hamster mucosa (D), early (E) and advanced SCC (F) show high fluorescence intensity in the epithelial layers in both normal or tumoural epithelium (E), while adjacent structures such as underlying lamina propria (LP) and striated muscle (SM) show a weak fluorescence intensity. Scale: the bar in the right bottom corner represents 20 μm.

Figure 5

The comparative time-dependent fluorescence and quantity of mTHPC in striated and smooth muscle observed by in situ LIFS (A), extraction of ¹⁴C-labelled mTHPC (B) and ex vivo FM (C). Fluorescence photomicrographs and corresponding HE stains of striated (D) and smooth muscles (E) at 72 h after dye administration show a weak and negligible fluorescence intensity in striated muscle, while a very high dye level was noted in the smooth muscle. Scale: the bar in the right bottom corner represents 20 μm.