3.2.1 Drugs and chemicals
Cisplatin, doxorubicin, etoposide, paclitaxel and vinblastine were purchased from Sigma (München, Germany). Docetaxel (Taxotere®) was obtained from Aventis (Bad Soden, Germany), topotecan (Hycamtin®) from GlaxoSmithKline (München, Germany), vin-cristine (Vinvin-cristine 1mg/ml®) from Rhone-Poulenc (Strasbourg, France) and vinorelbin (Navelbine®) from Pierre Fabre Pharma (Freiburg, Germany). Stock solutions of all drugs were prepared in 70 % ethanol with exception of cisplatin (DMSO), docetaxel (pure ethanol) and vinorelbin (sterile deionized water). The stock solutions of topote-can and vinorelbin were stored at 4 ℃, all other stock solutions were stored at -80 ℃.
Fetal calf serum (FCS) was obtained from Biochrom. All other chemicals and reagents were procured either from Merck or from Serva. Water was de-ionized by Milli Q
wa-3.2 Materials and methods 39 ter system (Millipore). Phosphate buffered saline (PBS) contained 8.0 g/l NaCl, 1.0 g/l Na2HPO4x2H2O, 0.2 g/l KCl, 0.2 g/l KH2PO4 and 0.15 g/l NaH2PO4xH2O. Bouin’s solution consisted of 300 ml of aqueous saturated picric acid, 100 ml of formaldehyde and 20 ml of glacial acetic acid.
3.2.2 Culture conditions
Lung cancer cell lines were obtained from the ATCC (Rockville, USA). The tumor cells were cultured as "monolayer cultures" in 75 cm2 flasks (Nunc, Wiesbaden, Germany).
The cell cultures were maintained at 37 ℃/5 % CO2 in different culture media. For the NCI-H460 cell line RPMI 1640 culture medium (Sigma) was used containing 10 mM HEPES, 1 mM sodium pyruvate, 2.5 g/l glucose, 1.5 g/l sodium hydrogen carbonate and 10 % FCS. The small cell lung cancer cell lines DMS 53, DMS 114 and DMS 153 were cultivated first in Waymouth’s MB 752/1 culture medium (Sigma, München, Germany) supplemented with 2.24 g/l sodium hydrogen carbonate and 10 % FCS, and then adapted to EMEM culture medium (Sigma, München, Germany) supplemented with 110 mg/l sodium pyruvate, 2.2 g/l sodium hydrogen carbonate and 5 % FCS. All culture media were adjusted to pH 7.4. Subculturing was carried out with 0.2 % trypsin/EDTA (Viralex, Paa Laboratories, Pasching, Austria) in PBS once a week. Mycoplasm contamination was monitored by a mycoplasma PCR detection kit (Venor Dem, Minerva Labs, Worcester, USA) periodically. A tumor bank according to the seed stock concept (Hay 1988) was created to have suitable cell material available at any time. For that purpose, the cells were expanded in lower passages and frozen in culture medium with an addition of 10 % DMSO in an ampule for storage in liquid nitrogen. To thaw a sample the ampule was put into water (40 ℃) and afterwards, the cells were cultivated in the appropriate culture medium.
3.2.3 Cytological and histological staining
For cytological staining cells were cultivated on microscopic slides that were prepared in accordance to the chromosome preparation. The cells were fixed for 30 min with Carnoy’s solution (ethanol:chloroform:glacial acetic acid 6:3:1) and stained by the method
40 Characterization of human lung cancer cell lines of Papanicolaou (Takahashi 1987). Harris’ haematoxylin, orange G6 and eosin azure 31 were utilized as dye reagents to stain plasma turquoise, nuclei blue and nucleoli deep blue.
For histological sections of the tumor tissue NMRI(nu/nu) nude mice with subcutaneous solid tumors were killed, and the tumor and part of the dermal tissue 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. For the haematoxylin-eosin (HE) staining the method described elsewhere (Romeis 1989) was used with Mayer’s haematoxylin and eosin. With these dye reagents the nuclei appear blue, whereas the rest of the section is stained pink or red. The Masson-Goldner (MG) staining was also carried out according to Romeis in consideration of the modification of Jerusalem. Here, Weigert’s haematoxylin, acid fuchsin-ponceau, orange G and light green were used to stain nuclei violet, erythrocytes red and connective tissue green. All stained sections were evaluated with a BH2 microscope (Olympus, Hamburg, Germany) mounted with a CCD-camera for digital imaging.
3.2.4 In vitro growth determination
For the in vitro growth characterization the lung cancer cells were cultured in 96 well (100 µl/well) flat-bottomed microtitration plates (Greiner, Frickenhausen, Germany) at an appropriate density of 10 to 20 cells per microscopic field (320x, Diavert microscope, Leitz, Wetzlar, Germany) depending on the cell line. After an incubation at 37 ℃/5 % CO2 for 24 h another 100µl/well culture medium were added. Cell growth was stopped at various periods of incubation time and the cell density was determined by a crystal violet staining as described in chapter 3.2.6. The absorbance of the cells was measured by a BioTek EL 309 Autoreader (Bad Friedrichshall, Germany) and the average and standard deviation values were calculated. Absorbance values outside of the confidence interval (95 %) were not considered for the calculation. The incubation time was plotted against the absorbance. The doubling time was computed using a computer analysis program (Reile et al. 1990) and plotted against the incubation time.
3.2 Materials and methods 41
3.2.5 Metaphase chromosomes preparation
Metaphase chromosomes of the tumor cell lines were prepared by the following procedure.
Microscopic slides were prepared according to the method of Rooney and Czepulkowski (1986). For that purpose the slides were degreased for 24 h in ethanol hydrochloric acid (6 ml of concentrated hydrochloric acid in 200 ml 70 % ethanol), rinsed with de-ionized water and dried. Before culturing the cells the slides were autoclaved. Cells were cultured on the prepared slides in Quadriperm 4-well lux-multiplates up to a confluence of 50 %.
Metaphase cells were arrested with colcemide (0.04 mg/ml). After an incubation time of 3 h at 37 ℃/5 % CO2, the culture medium was removed by suction and replaced with 37 ℃ warm hypotonic potassium chloride solution (75 mM) for 30 min to lyse the cells.
Afterwards, an equal volume of icecold fixative (methanol/glacial acetic acid 3:1) was added and removed at once. This step was repeated twice and after the last fixation the slides were stored at 4 ℃ for 10 min. The fixative was removed and the slides were dried.
Chromosomes were stained with a Giemsa dye solution (10 ml filtered Giemsa solution + 90 ml 0.025 M KH2PO4 , pH 6.8) for 8 min. After washing and drying the slides were mounted with DePex. The chromosome numbers were determined by an Olympus BH-2 microscope (Olympus, Hamburg, Germany) with 60x and 100x SPlanApo oil immersion objective. For each cell line 50 metaphases were counted. In addition, digital imaging was performed by means of an Axiovent 200 M microscope (Zeiss, Jena, Germany) and LSM 510 imaging software.
3.2.6 Chemosensitivity assay
The chemosensitivity of the tumor cells was quantified by the crystal violet assay described elsewhere (Bernhardt et al. 1992, Reile et al. 1990). In brief: tumor cells were seeded (100µl/well) in 96 well flat-bottomed microtitration plates (Nunc, Wiesbaden, Germany) at an appropriate density of 10 to 20 cells per microscopic field (320x, Diavert microscope, Leitz, Wetzlar, Germany) depending on the cell line. After incubation at 37 ℃/5 % CO2 for 48 h to 72 h according to the cell growth characteristics, the medium was carefully removed by suction and replaced with fresh medium (200µl/well) containing either drug or pure solvent. The drug solutions were diluted 1:1,000 with culture medium. In 16
42 Characterization of human lung cancer cell lines wells on every plate culture medium with vehicle was given, so that these wells served as control. For every drug concentration 16 wells were used. After various periods of incubation at 37 ℃/5 % CO2 the culture medium was shaken off and the cells were fixed with 100 µl 1 % glutardialdehyde in PBS per well for 30 min. The fixative was replaced by 180 µl PBS per well, and the plates were stored at 4 ℃. At the end of the experiment the cells were simultaneously stained with 100 µl of 0.02 % crystal violet solution (N-hexamethylpararosanilin · HCl in water) per well for 20 min. To remove excess dye the plates were rinsed with demineralized water for 20 min and dried. The stain bound by the cells was redissolved in 70 % ethanol (180µl/well) while shaking the microplates for about 3 h on a Köttermann 4010 shaker. Absorbance was measured at 578 nm using a BioTek EL 309 Autoreader (Bad Friedrichshall, Germany) and the average and standard deviation values were calculated. Absorbance values outside of the confidence interval (95 %) were not considered for the calculations. The drug effects were expressed as corrected T/C values according to
T /Ccorr.[%] = T −C0 C−C0
·100%
where T is the mean absorbance of the treated cells, C the mean absorbance of the control and C0 the mean absorbance of the cells at the time when the drug was added (t=0). When the absorbance of the treated cells T is less than that of the culture at t=0 (C0) the extent of cell killing must be calculated as
cytocidal effect[%] = T −C0
C0 ·100%
3.2.7 In vivo experiments
For the characterization of the in vivo growth of the lung cancer cells NMRI(nu/nu) nude mice obtained from the nude mice laboratory of the department were used. The mice 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 leading in a pH value of 2.5. The animals were housed under specific pathogen free conditions at a 12 h light/dark cycle at a
3.2 Materials and methods 43 temperature of 25 ℃ and a relative humidity of 70 %. At an age of 6 weeks the mice were used for studies. For the subcutaneous injection cell suspensions of the corresponding cells were prepared in vitro. Cells were detached from the culture flask with trypsin/EDTA and FCS free medium and washed twice with FCS free medium. To adjust the appropriate cell quantity (2 mill. cells/100 µl in case of the NCI-H460 cell line and 4 mill. cells/100 µl for DMS 53 and DMS 114 cell lines, respectively) cells were resuspended in FCS free medium. Under aseptic conditions 100 µl of the cell suspensions were injected under the thoracic dermis. To maintain the subcutaneous tumors in the mice the solid tumor was excised when the tumor growth reached an area of about 150 mm2. Pieces of 2 mm3 were prepared in sterile PBS and transplanted into new mice. The NCI-H460 tumor was grafted every three weeks whereas the other tumors were transplanted at an average period of seven weeks.
44 Characterization of human lung cancer cell lines