Analysis of differential PDT effect in rat bladder tumor models according to concentrations of intravesical hexyl-aminolevulinate

The hexylester of 5-aminolevulinic acid (HAL) is a very efficient precursor of the photosensitizer protoporphyrin IX (PpIX) for photodynamic therapy (PDT). Our previous study, performed in rat orthotopic bladder tumors, indicated an opposite effect of HAL/PpIX-PDT according to HAL concentration. The present study investigated possible reasons for this differential effect considering the impact of extracted amounts of PpIX in normal and tumor bearing bladders along with PpIX distribution in distinctive histopathological layers. High performance liquid chromatography (HPLC) analysis of tumor and normal bladder tissues after 8 mM and 16 mM HAL instillation showed that PpIX was the main porphyrin species. The PpIX production in tumor bladders instilled with 8 mM HAL was significantly higher than after 16 mM HAL. Fluorescence confocal microscopy demonstrated a punctuate bright fluorescence pattern in tumor zones of bladders instilled with 8 mM HAL, whereas a more diffuse cytoplasmatic fluorescence distribution was observed after 16 mM HAL instillation. Immunofluorescence staining together with transmission electron microscopy showed severe mitochondrial damage in tumor zones of bladders treated with 8 mM HAL/PpIX PDT, with intact mitochondria in tumor zones of bladders treated with 16 mM HAL/PpIX PDT. We conclude that the differential response to HAL/PpIX PDT in function of HAL concentrations could be attributed to diminished PpIX synthesis and differential intracellular localisation of PpIX. Mitochondria were shown to be the critical photodamaged sites of HAL/PpIX PDT and as such tissue sensitivity to treatment can be estimated through investigation of intracellular PpIX distribution

Multimodality point spectroscopy : interests, development and application to in vivo diagnosis of bladder and skin cancers

International audienceCombining autofluorescence (AF) and Diffuse Reflectance (DR) spectroscopies is supposed to improve diagnosispsila accuracy of early stages of cancer (as well as precancerous stages) which is of great clinical importance. For the present study, we developed a bimodal instrumentation combining spatially resolved AF and DR spectroscopies, and evaluated its ability to distinguish between healthy, inflammatory and early stages of cancers in vivo. In order to get such tissue types, we used 2 animal models: a rat bladder orthotopic cancer model and a mice UV-irradiated skin model. The first study shows that combining AF and DR improves both sensitivity and specificity of the diagnosis compared to one modality used alone: Se = 67% (DR alone), 72% (AF alone) increases up to 78% when combining the two modalities. Preliminary results of the second study reveal that some spectroscopic criteria may help quantitative histological analysis in making the difference between acute and precancerous hyperplasia

Mechanisms of the selective photodynamic efficiency of the hexyl-aminolevulinate (h-ALA)-protoporphyrine IX (PpIX) in the treatment of bladder cancer

The goal of this work was first to study the efficiency of the photodynamic therapy in the treatment of the bladder cancer and to explain the mechanisms that govern this efficiency. In our study we have used Hexvix® or hexylaminolevulinate (hALA), that inside the cells induced PpIX formation. The tumor model orthotopic and syngenic, developed in Female Fischer Rats 344. We have previously shown that rat tumor bladders instilled with 8 mM Hexvix® induced tumor necrosis with intact bladder wall but instillation with 16 mM Hexvix® showed total wall necrosis without impact on the tumor. We have investigated fluorescence spectroscopy in situ, photobleanching kinetics in vivo, the quantity of PpIX and the formation of different fluorescent porphyrins by HPLC, after both hALA instillation. We have also studied mitochondial damage after PDT by immunofluorescence of beta ATP synthase (a mitochondrial marker) and electronic microscopy on bladder tumors sections at both hALA concentrations. The same initial fluorescence intensities were achieved at both hALA concentrations. The photobleaching of PpIX in bladder cancer follows mono-exponential kinetics with similar decay constants at 8 mM (K = 0,296 , Chi 2 = 0,98) and 16 mM (K = 0,27 , Chi 2 = 0,74). One photoproduct, photoprotoporphyrin IX was observed with a fluorescence peak around 670 nm at both concentrations. HPLC indicates that 90 % of the total amounts of fluorescing porphyrin produced was protoporphyrin IX. However the quantity of PpIX formed at 16 mM hALA is 3 times lowers than at 8 mM d'hALA. The results suggest that there are modifications of the metabolic way of PpIX at large Hexvix concentrations. The mitochondrial damage was evaluated by immunohistochemistry at two end points after PDT (0h and 4h). At 0 h the same punctiform labelling was observed at both hALA concentrations, similar to controls. However at 4 h the labelling was diffuse cytoplasmic at 8mM hALA contrarily to 16 mM d'hALA where the marking comparable to controls. We suppose that this difference is due to damage of the mitochondria caused by PDT for 8 mM hALA. Further electron microscopic studies of these damages are in progress