Temporally and spatially heterogeneous distribution of mTHPC in a murine tumor observed by two-color confocal fluorescence imaging and spectroscopy in a whole-mount model.

Efficient intratumor delivery of anticancer drugs and photosensitizers is an important factor in the success of chemotherapy and photodynamic therapy, respectively. Unfortunately, their adequate and uniform intratumor distribution is impeded by several physiological barriers and by binding to tissue components. Measurement of gross tumor drug accumulation is a routine method of investigating the uptake and clearance of chemotherapy agents and photosensitizers but tells little about their extravascular spatial distribution. We use whole-mount two-color confocal fluorescence imaging and imaging spectroscopy of unprocessed excised murine tumor fragments to investigate the intratumor distribution of the photosensitizer meso-tetrahydroxyphenyl chlorin (mTHPC) as a function of distance from blood vessels perfused with 0.2 mum diameter fluorescent microspheres. Significant mismatches between drug and perfused vasculature are caused by heterogeneities in tumor blood supply. We describe complex microscopic mTHPC gradients that reverse dramatically relative to the perfused vasculature with time after injection. This imaging technique can be applied to screen the dynamic intratumor distribution of other fluorescent photosensitizers and anticancer drugs.

Necrotic and apoptotic features of cell death in response to Foscan photosensitization of HT29 monolayer and multicell spheroids.

Photodynamic therapy (PDT) is an approved anticancer treatment modality that eliminates unwanted cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which is selectively taken up by tumor cells. Present study reports the modalities of cell death after photosensitization of human adenocarcinoma HT29 monolayer and spheroid cells with a second generation photosensitizer Foscan. Kinetics of apoptosis and necrosis after Foscan-PDT in monolayer cells determined by flow cytometry using labeling of cleaved poly(ADP-ribose) polymerase (PARP) and staining with propidium iodide (PI) demonstrated that Foscan was not a strong inducer of apoptosis and necrosis was a prevailing mode of cell death. Cytochrome c release (cyt c) and mitochondrial membrane potential (Deltapsim) addressed by flow cytometry technique at different time points post-Foscan-PDT demonstrated that cell photoinactivation was governed by these mitochondrial events. Foscan-loaded HT29 multicell spheroids, subjected to irradiation with different fluence rates and equivalent light doses, displayed much better tumoricidal activity at the lowest fluence rate used. Apoptosis, measured by caspase-3 activation was evidenced only in spheroids irradiated with the lowest fluence rate and moderate fluence inducing 65% of cell death. Application of higher fluence rates for the same level of photocytotoxicity did not result in caspase-3 activation. The observation of the fluence rate-dependent modulation of caspase-3 activity in spheroids offers the possibility of regulating the mechanism of direct cell photodamage and could be of great potential in the clinical context.

Foscan-based photodynamic treatment in vivo: correlation between efficacy and Foscan accumulation in tumor, plasma and leukocytes.

The tumoricidal effect of Foscan-mediated photodynamic therapy may involve both vessel and tumor cell destruction. The relevant importance of each mechanism seems to be defined by the time interval between photosensitizer administration and illumination (drug-light interval, DLI). Short drug-light intervals favor vascular damage due to the preferential photosensitizer accumulation in the tumor vasculature, whereas long drug-light intervals trigger direct tumor cell damage due to the dye localization in the tumor. The purpose of this study was to investigate the influence of tumor, plasma and leukocyte concentrations of Foscan at different times after photosensitizer delivery on PDT response. Both pharmacokinetic and tumor-response studies were carried out in nude mice bearing s.c. Colo26 tumors. One to 96 h after i.v. injection of 0.5 mg/kg Foscan, animals were exposed to 10 J/cm(2) 652-nm light delivered at 30 mW/cm(2). Mean tumor regrowth time was determined for each schedule of treatment and correlated to Foscan distribution in the compartments of interest at the time of illumination. PDT efficacy was greatest for irradiations performed at 6 and 12 h post Foscan injection and limited at 96 h. Unlike tumor and plasma Foscan concentrations, photosensitizer accumulation in leukocytes exhibited a good correlation with PDT efficacy. The results suggest that leukocytes could play an important role in the mechanism of PDT-induced vascular damage either by being one of the main effector compartments or by better reflecting Foscan accumulation in endothelial cells compared to plasma. The prevalence of indirect damage was highlighted by the fact that PDT efficacy was not modified by the use of a higher fluence rate of irradiation (160 mW/cm(2)), which depleted intratumor oxygen and did not restrain PDT-induced cell toxicity.