Immunohistochemistry

Frozen sections were used to detect transferrin receptors in tumor tissue and nude mouse brain. Subcutaneously grown glioblastomas were excised when the tumor growth reached an area of about 150 mm² in nude mice (see chapter 3). The tumor tissue was embedded in frozen tissue matrix (Tissue-Tek^®O.C.T.™ Compound, Sakura Finetek, Giessen, Ger-many) to ensure freezing without trapped air and stored in liquid nitrogen. The sections were cut on a freezing microtome (FrigocutE 2800, Reichert-Jung, Nussloch-Heidelberg, Germany) with a thickness of 5 to 8 µm, and two sections each were mounted on Super-frost Plus (Fisher Scientific, Pittsburgh, PA) glass slides. The sections were immersed immediately in acetone for 15 min to fix the sections on the slides. After fixation, the slides were rinsed for 5 min with phosphate-buffered saline (PBS), pH 7.4, and subjected to immunostaining.

Endogenous peroxidase was blocked by incubation in PBS containing 3 % H₂O₂ and 10 % methanol for 30 min, followed by two incubations in PBS with 0.04 % Triton-X-100 (PBST) for 5 min. Nonspecific binding was prevented by incubation for 30 min in 10 % natural donkey serum (NDS, Sigma, München, Germany), 2.5 % skimmed milk powder (Milupa, Friedrichsdorf, Germany), and 0.3 % Triton-X-100 in PBS. Subsequently, cells were washed three times with PBST. Primary mouse anti-human CD71 antibody (M 0734, clone Ber-T9, anti-human CD71, DAKO, Hamburg, Germany) was diluted 1:5 with wash-ing buffer consistwash-ing of 10 ml of milk powder solution (2.5 % skimmed milk powder in PBST), 100 µl 1 % NDS, and 0.3 % Triton-X-100. After incubation with the primary antibody for 48 h at 4 ℃, the slides were washed three times with PBST and treated with biotinylated donkey anti-mouse antibody as secondary antibody (1:100, Dianova, Hamburg, Germany) for 2 hours at room temperature. All incubations with antibody

180 The transferrin receptor - a possible loophole at the blood-brain-barrier were performed in humid chambers. Afterwards, slides were washed three times in PBST and incubated for 1 hour with horseradish-conjugated avidin-biotin complex (Vectastain Elite Kit ABC, Vectorlabs, Burlingame, United Kingdom) in PBST. Slides were washed for 5 min in PBST before incubation in a diaminobenzidine (DAB) solution for 10 min (0.05 % DAB, 0.02 % ammonium nickel sulfate in PBST). H₂O₂ (10 %) was added to a final concentration of 0.006 %, and slides were incubated for 1.5 min. Afterwards, they were washed three times in PBS and counterstained with Harris’ haematoxylin. Then the slides were placed in PBS and demineralized water, processed in an ascending alcohol series and mounted in DePex. All stained sections were evaluated with a BH2 microscope (Olympus, Hamburg, Germany) mounted with a CCD-camera for digital imaging.

8.2.4 Chemosensitivity assay

The chemosensitivity assay was performed according to the procedure described by Bern-hardt et al. (1992). Details are given in chapter 3.2.6.

8.2.5 In vivo experiments

Rowett(rnu/rnu) nude rats and NMRI(nu/nu) mice obtained from the nude mice labo-ratory of the department were used for the in vivo growth studies. The animals were allowed to take water and food, a combined breed and maintenance nutrition (Altromin), ad libitum. The water was supplemented with 1.33 g/l of potassium sorbate, 2 g/l of chloramphenicol and 1 g/l of hydrochloric acid yielding a pH value of 2.5. The animals were housed under specified pathogen free conditions at a 12 h light/dark cycle at a tem-perature of 25 ℃ and a relative humidity of 70 %. At an age of 6 weeks the rats were used for studies.

The subcutaneous tumor model is discussed in chapter 3.

The intracerebral model has been described by Altenschöpfer (1998). In brief: the parietal bone of anaesthetized nude rats was drilled with a 1-mm-diameter bit 3 mm to the right-hand side of the sagittal line and 3 mm rostral of the coronal line. The rats were anesthesized with sevoflurane (Abbott, Wiesbaden, Germany). Human glioblastoma cells (3·10⁵ cells) suspended in 5 µl of serum-free EMEM were injected 5 mm deep into

8.3 Results and discussion 181 the brain tissue, and the wound was closed with a surgical clamp. The body weight was observed to assess the tumor burden and the rats were killed when 20 % weight reduction occurred.

For histological staining of the tumor bearing brains, the nude rat brains were removed and fixed for at least three days in Bouin’s solution. The tissues were embedded in paraffin by a standard procedure in a Histokinette (Shandon, Frankfurt/Main, Germany), cut by a Microtome Leitz 1516 (Leitz, Wetzlar, Germany) in sections with a thickness of 6 µm and put on object slides prepared with poly-L-lysin. Prior to each staining procedure, the sections were deparaffinized with xylol and various ethanol solutions with descending concentrations. The sections were stained according to the Masson-Goldner (MG) procedure which is described in detail in chapter 3.2.3. All stained sections were evaluated with a BH2 microscope (Olympus, Hamburg, Germany) mounted with a CCD-camera for digital imaging.

8.3 Results and discussion

The aim of the study was the examination of several human brain tumor cell lines with re-spect to their suitability for a targeted treatment with an immunoliposome based delivery system. The selected cell lines have to fullfill to the following requirements. The tumor cells should express sufficient quantities of TfR to target the immunoliposomes directly to the tumor cells after they have crossed the BBB. This was investigated in vitro and the TfR expression was confirmed in vivo in tumors growing subcutaneously in nude mice.

As a second prerequisite, the cells have to be chemosensitive to low concentrations of the cytostatic drug that is incorporated in the immunoliposome. In addition, the tumor cells have to be tumorigenic in the brains of nude rats. These animals were used for the experiments, because of the possibility to overcome the BBB by means of transcytosis mediated by the OX-26 antibody which is targeted to the rat TfR.

182 The transferrin receptor - a possible loophole at the blood-brain-barrier