5.02] Preliminary results: In vitro and in vivo evaluation of photodynamic techniques for the experimental treatment of human hepatoblastoma and neuroblastoma

Abstract

Objective Hepatoblastoma and neuroblastoma are cancers which occur in early childhood. Intraoperative fluorescence guidance or post-surgical photodynamic therapy (PDT) might improve treatment outcome. In this study, human cell lines derived from these cancers were studied in vitro and in an animal model for their responsiveness to photodynamic methods using 5-aminolevulinic acid (5-ALA) as a precursor of the fluorescent photosensitizer protoporphyrin IX (PpIX). Material and methods Cell cultures of human hepatoblastoma (HuH6) and neuroblastoma (MHH-NB-11) were incubated in 96-well plates with 5-ALA (medac, Wedel, Germany) at increasing concentrations and incubation times to determine optimal incubation parameters by measurement of PpIX-fluorescence. Human fibroblast cells (N1) served as controls. Cell survival following PDT with increasing light doses was tested with the CTB assay (Promega, Madison, WI, USA) at 150 μg/ml 5-ALA for 4 h. HuH6-cells were also studied in vivo after laparoscopic implantation of 3×106 cells between peritoneum and abdominal wall of immunoincompetent nude rats. After 7 weeks of tumor growth, fluorescence kinetics (tumor, adjacent peritoneum, liver, colon) was assessed by fiber-based measurements following i.p. injection of 5-ALA at 500 mg/kg bodyweight (10 animals) and on further 24 animals, laser irradiation was performed with a light dose of 50 J/cm2 at 25 or 100 mW/cm2. Two days later, rats were again injected with 5-ALA and irradiated tissues were excised and studied by conventional and fluorescence microscopy. Results In vitro, PpIX fluorescence approached saturation at 150 μg/ml 5-ALA-concentration, whereas there was no saturation effect observed with respect to incubation time, 4 h was set as practically feasible. HuH6 and NB-11-cells showed comparable fluorescence intensities, whereas N1 fluorescence was low. After PDT, cell survival was lowest for HuH6-cells (LD50 at 1.5 J/cm2) compared to MHH-NB-11 (LD50 at 6 J/cm2) and N1-cells (LD50>20 J/cm2). In vivo, tumor fluorescence was highest at around 200 min post injection with a maximum contrast to the surrounding peritoneum at approx. 150 min. The mean contrast of tumor fluorescence versus fluorescence measured in peritoneum and liver was 22 at this time (not corrected for optical parameters), whereas versus colon it was 5. PDT response was visible in irradiated tumor and liver tissue. The depth of necrosis could be assessed by observation of fluorescence on cryosections of excised tissue. While peritoneum exposed to PDT remained unaffected, irradiation of the liver led to superficial necrosis and sensitized tumor showed deep necrosis. Conclusion The human hepatoblastoma and neuroblastoma cells were susceptible to selective PpIX accumulation and PDT effect. In vivo, HuH6-tumors showed high and selective PpIX accumulation and extended necrosis after PDT. The possibility of a PDT-induced necrosis of normal liver tissue must be considered for clinical trials.