Ex vivo/ in vivo experiments 27

Male SCID mice (CB17/lcr-PrkdcSCID/Crl) aged 8-10 weeks were housed in individually vented cages under specific pathogen free conditions with a 12h day/night cycle and with food and water ad libitum.

2.4.2. Tumor models

PC3 cells were cultured as described above. Cells were cultured without antibiotics for at least 3-4 passages, before tumor implementation and were harvested when reaching approx. 70% confluence. After washing with PBS 106 PC3 cells in 100µl PBS were injected subcutaneously with a 25G needle (Braun, Melsungen, Germany) into the flank of the mice.

2.4.3. Tumor Measurement

Animals were controlled regularly for tumor progression. When the tumor volume reached a size of at least 10mm3_{, tumor progression was monitored with a digital} measuring slide (Digi-Met, Preisser, Gammertingen). Each measurement consisted of three diameters each 90° apart and tumor volume was calculated by the formula axbxcx0.4 (with a, b and c indicating the three diameters).

All animal procedures were approved and controlled by the local ethics committee and carried out according to the guidelines of the German law of protection of animal life.

2.4.4. Chemotherapy

The chemotherapeutical drug CPA (Cyclophosphamide, Sigma, Taufkirchen) was solved in PBS at a concentration of 10mg/ml followed by sterile filtration (0.22 µm sterile filter, Millex-GV, Millipore Carrigtwohill, Ireland). Application was performed intraperitoneally. The CPA solution was administered with a 25 G needle (Braun, Melsungen). The application of the CPA solution was carried out every 6th day. The single dose of each application was based on animal body weight. Toleration of the treatment with CPA was monitored by regular measurement of body weight. The vehicle group (PBS) and the drug treatment group (CPA dissolved in PBS) were housed separately.

2.4.5. Tumor Histology

2.4.5.1. HE stain

Cryosections of the tissue was fixed with 4% paraformaldehyde and stained with Haematoxilin (Sigma, St. Loius, USA) for 30 min. After a washing step with PBS and aqua dest., sections were incubated with a 1:100 dilution of Eosin (Sigma, St. Louis, USA) for 4 min. Afterwards, sections were washed with aqua dest., embedded with PBS and analyzed by transmission light microscopy. Analysis was performed with an Anxiovert 200 microscope (Zeiss, Jena, Germany).

2.4.5.2. Immunohistochemistry

Cryosections (5µm) were transferred to a microscope slide and fixed with 4% paraformaldehyde (in PBS) for 5 min. Afterwards, tissue sections were rehydrated and washed with the blocking solution (PBS containing 5% FBS) prior to antibody incubation.

Simultaneous staining for laminin and endothelial marker CD31:

Staining was performed with the rabbit-anti-laminin antibody (Chemicon Europe, Hampshire, UK) and simultaneous with the rat-anti-mouse CD31 (CALTAG, Burlingame, USA) antibody; both antibodies were used in a 1:200 dilution (in the blocking solution). After an incubation time of 12h at 4ºC in a humidified atmosphere, sections were washed repeatedly with the blocking solution followed by secondary

Materials and Methods 29

antibody staining. Therefore the sections were incubated with the Texasred labelled goat-anti-rabbit antibody (Vector, Burlingame, UK) and the ALEXA488 labelled goat- anti-rat antibody (Invitrogen, Oregon, USA); the staining was performed with a 1:200 dilution (in blocking solution) of both antibodies for 2h at room temperature in a humidified atmosphere. Before analysis by fluorescence microscopy, sections were washed with the blocking solution repeatedly. Analysis was performed with a Anxiovert 200 microscope (Zeiss, Jena, Germany) using appropriate filter sets.

2.4.6. Isolation of tumor/organs

2.4.6.1. For Histology

Tumors were dissected from the flank of sacrificed mice and embedded into cryo conservation medium (OCT tissue Tak, Sakura, USA) previous to freezing at -20°C , -80°C for long term storage.

2.4.6.2. For Microarray analysis

PC3-wt, PC3-A3, PC3-D3 and PC3-D4 were implanted subcutaneously and were grown as Xenografts for 10 days for the reisolated cells (PC3-A3, PC3-D3 and PC3- wt) respectively for 17 days for the parental PC3-wt cells until they reached a mean volume of 35 mm³. At day 9 respectively 16 one group of each subline was treated with 120mg/kg CPA and the other group was used as untreated control. 24h after CPA application tumors were dissected from the flank of sacrificed mice and were divided into two parts. One half was embedded for histology as described in 2.4.6.1 and the other half was cut into small pieces (Ø 0.5 mm). Randomly half of the pieces were collected and embedded in RNAlater (Ambion, Darmstadt, Germany) for RNA conservation. The rest of the pieces were prepared for FACS analysis as described in 2.3.3.

2.4.7. Reisolation of tumor cells

2.4.7.1. Isolation of tumor cells for cell culture

For the reisolation of tumor cells, mice were sacrificed with CO2. The skin was cleaned and sanitized by isopropanol (70% in water v/v) followed by drying under sterile conditions. Tumors were collected and immediately inserted in the indicated Penstrep (Biochrome, Germany) containing RPMI medium. Tumor tissue was reduced to small pieces under sterile conditions. This procedure was repeated until

tumor tissue was homogenized. The obtained homogenized cell suspension was diluted with fresh medium, followed a centrifugation step (150g/5min). The supernatant was removed and the tumor cells were resuspended in fresh, Penstrep containing medium. The tumor cell containing suspension was transferred to collagen coated (Collagen G, Biochrom) tissue flasks (TPP, Switzerland) and incubated under standard conditions (37ºC, 5% CO2) in a humidified atmosphere for 2-3 days. When cells were attached to the bottom of the flasks, medium was replaced every second day, until cells reached a confluence of about 70%. Obtained cells were cultured for 2 passages before harvested by tyrpsin/EDTA treatment. Reimplantation studies were performed by injection of 106 _{PC3 tumor cells at a passage number of 4-5.}

Results 31