A new method of implanting orthotopic rat bladder tumor for experimental therapies.

We developed a model of orthotopic transplantation of bladder tumor cells in female Fischer rats using a new reproducible technique. After first performing the mechanical abrasion of a portion of the bladder urothelium with an Abrader inserted transurethrally via a catheter, we administered a suspension of 5-40 x 10(6) viable AY-27 tumor cells in sterile phosphate-buffered saline to the bladder cavity. This rapidly led to a tumor growth incidence of approximately 100%. The induced bladder tumors grew expansively into the bladder cavity from the surface (mucosa) and gradually invaded the submucosa, muscles, serosa and surrounding tissue (high-stage invasive transitional cell carcinoma). Size and staging were related to the quantity of tumor cells instilled into the bladder cavity. This model matches the characteristics of human bladder tumor more closely than other bladder cancer models induced with tumor cells. Moreover, it presents many advantages: the method is reproducible, tumors grow rapidly, they are directly attached to the bladder surface and they are always located on the bladder wall, in line with the urethra. This proves especially helpful for evaluating chemotherapeutic agents by different means such as in vivo fluorescence spectroscopy, a noninvasive method used in photodynamic therapy, or other methods designed to detect and treat transitional cell carcinoma.

Determination of the maximal tumor:normal bladder ratio after i.p. or bladder administration of 5-aminolevulinic acid in Fischer 344 rats by fluorescence spectroscopy in situ.

The two major steps in our study on the treatment of bladder tumors by photodynamic therapy (PDT) were the development of a new bladder tumor model in Fischer rats by implantation of tumor cells and the use of fluorescence spectroscopy, a semi-quantitative and non-invasive method, in order to determine the time after general or local administration of a photosensitizer when the tumor:normal bladder ratio was at its highest. 5-Aminolevulinic acid (5-ALA) (250 mg/kg body weight) was injected i.p. or instilled directly into the bladder cavity for 1, 2 or 4 h and fluorescence was measured on normal and bladder tumor tissues every 30 min for 8-10 h after administration, with a special miniaturized optical-fiber captor. The better tumor:normal bladder ratios were 2.85+/-1.2 at 3.5 h after i.p. administration and 3.96+/-1.04 after bladder instillation for 4 h, respectively. These results were confirmed by fluorescence microscopy. PDT with the same dose of 5-ALA as in this pharmacokinetic study must also be carried out in order to compare the toxicity of the two administration routes of the photosensitizer and to determine which one is the better for this bladder tumor model.

Biodistribution of hypericin in orthotopic transitional cell carcinoma bladder tumors: implication for whole bladder wall photodynamic therapy.

In a recent clinical study, we reported a selective uptake of hypericin in superficial bladder tumors. The results suggested that hypericin, a potent photosensitizer, could be used not only for diagnosis but also for photodynamic therapy (PDT) of superficial bladder tumors. In the present study, we investigated the biodistribution of hypericin in an orthotopic rat bladder tumor model by assessing the extent of hypericin penetration and the kinetics of accumulation into rat bladder tumors and normal bladder wall. Hypericin (8 or 30 microM) was instilled into the bladder via the catheter for 1, 2 or 4 hr. The fluorescence of hypericin in the bladder tumors and normal bladder was documented using fluorescence microscopy. In situ quantification of hypericin fluorescence in the tumor or normal bladder was performed using the laser-induced fluorescence technique. There was much more hypericin fluorescence in the tumor than in the normal bladder, with the tumor-to-normal-bladder ratio mounting to 12:1 after 4 hr of hypericin (30 microM) instillation. Moreover, hypericin was retained in the tumor for at least 1 hr before it was gradually lost from the tissue. Microscopically, the fluorescence of hypericin was restricted to the urothelial tumor and normal urothelium without fluorescence in the submucosa and the muscle layers. Subsequently no hypericin was detected in plasma, indicating that under these conditions systemic side effects should not be expected. Because the conditions used in this study were similar to those used in our previous clinical study, it is therefore likely that whole bladder wall PDT in the clinic under these conditions will produce selective urothelial tumor destruction without causing damage to the underlying muscle layers.

Induction of syngeneic intradermal and orthotopic epidermal carcinomas in hairless Skh-1 mice as a reproducible model for experiments.

Epidermoid carcinomas, clinically and histologically similar to human squamous cell carcinomas (SCC), were obtained in hairless Skh-1 mice. Tumor cells originated from chemically-induced skin cancers. We developed three models of orthotopic skin tumors: (1) intradermal injection of a tumor cell suspension, (2) superficial abrasion of the skin, cell grafting and application of a hydrocolloid dressing, (3) skin incision, seeding and application of a hydrocolloid dressing. Intradermal injection was 100% successful. Skin incision, displaying histological evidence of rapid invasive tumor growth, was 75% successful. Though skin tumor growth after abrasion was only 20% successful, the tumor histogenesis exactly imitated human SCC development. These carcinomas provide research models for further experiments such as photodynamic therapy or antiangiogenesis therapy.