Individual mammary tumor-derived cell cultures
Small tissue pieces from 8 different breast cancer patients were collected during surgery and pathologically characterized as ductal carcinomas, respectively. Informed written consent was obtained from each patient for the use of individual biopsy material and the study has been approved by the Institutional Review Board, Project #3916 on June 15th, 2005. The tissue samples were cut into small blocks of approximately 1 mm3 and washed extensively in PBS to remove blood cells and cell debris. After negative testing for HIV-1, hepatitis B & C, bacteria, yeast and fungi, respectively, the tissue pieces of the mammary tumors were incubated using plain uncoated plastic dishes (Nunc GmbH, Langenselbold, Germany) in serum-free mammary epithelial cell growth medium (MEBM) (PromoCell GmbH, Heidelberg, Germany), supplemented with 52 μg/ml of bovine pituary extract, 0.5 μg/ml of hydrocortisone, 10 ng/ml of human recombinant epidermal growth factor and 5 μg/ml of human recombinant insulin in a humidified atmosphere at 37°C. Half of the cell culture medium was replaced about every fourth day and the other half was used as conditioned medium. Under these conditions, an outgrowth of primary tumor-derived cells was observed, which were adherent to the tumor tissue blocks and to each other. In the subconfluent growth phase the tumor tissue pieces were separated from the culture and placed into a separate culture dish to allow further outgrowth of primary tumor cells. The remaining tumor-derived cells were used for the appropriate assays.
Normal human mammary epithelial cell cultures
Primary cultures of normal human mammary epithelial cells (HMEC) were isolated from a 50 year old caucasian female and commercially provided by BioWhittaker Inc. (Walkersviell, MD, USA) as culture passage 7 (Lot #1F1012). HMEC were tested positive for cytokeratins 14 and 18 and negative for cytokeratin 19, respectively. They were performance tested and tested negative for HIV-1, hepatitis B & C, mycoplasma, bacteria, yeast and fungi. HMEC were seeded at 4,500 cells/cm2, cultured in MEBM (PromoCell) and the appropriate medium of each culture was replaced every two to three days. At subconfluent conditions the cells were subcultured by incubation with 0.025%/0.01% trypsin/EDTA (PromoCell) for about 6 min/37°C until the cells detached. Thereafter, immediate addition of trypsin neutralization solution (TNS) from soybean was required to inactivate the trypsin followed by subsequent centrifugation (220 g/6 min). The pelleted cells were resuspended in new medium at about 4,500 cells/cm2 and cultured further on in the next passage number. Subcultured cells required about 24 h to recover and resume growth.
MCF-7 cell line
Human MCF-7 mammary gland adenocarcinoma cells originally isolated from a 69 year old caucasian woman with several characteristics of differentiated mammary epithelium were derived from the American Type Culture Collection (ATCC #HTB-22) as passage 146 or earlier and cultured inititally at about 1,500 cells/cm2 in DMEM-medium (Invitrogen GmbH, Karlsruhe), including 10% (v/v) heat-inactivated fetal calf serum (FCS) (Biochrom KG), 2 mM L-Glutamin (Invitrogen), 1 mM Na-Pyruvat (Invitrogen) and 1 mM Penicillin/Streptomycin (Invitrogen).
MDA-MB-231 cell line
Human MDA-MB-231 mammary gland adenocarcinoma cells isolated as one of a series of breast tumor lines from pleural effusions of a 47 year old caucasian female were derived from the ATCC (#HTB-26) and cultivated inititally at about 1,500 cells/cm2 in Leibovitz's L-15-medium (Invitrogen) with 10% (v/v) FCS, 2 mM L-Glutamin and 1 mM Penicillin/Streptomycin.
The mammary tumor tissues were cultured on appropriate microscope slides for scanning (SEM) and transmission electron microscopy (TEM), respectively. Following ex vivo outgrowth of tumor-derived cells, the individual cultures were fixed on these slides in a solution containing 3% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4 for at least 24 h. Thereafter, the samples were postfixed in 1% OsO4 in H2O before being dehydrated in an ethanol gradient. For SEM, critical point-dried specimen were coated with gold-palladium (SEM coating system E5400, Polaron, Watford, UK) and examined in a JEOL SSM-35CF scanning electron microscope at 15 kV.
For TEM, the ethanol dried mammary tumor tissues were embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead acetate and examined in a Philips CM10 electron microscope, operated at 80 kV.
Mammary tumor-derived cells were cultured onto microscope slides, washed 3× with PBS/Tween-20 for 5 min, and air-dried for 60 min. Thereafter, the samples were fixed with ice-cold acetone for 10 min and rehydrated in PBS for 5 min. After treatment with PBS/5% (w/v) BSA for 10 min to block non-specific binding-sites, the samples were incubated with a mouse anti-vimentin antibody (cloneV9 (1:100); Dako, Hamburg, Germany) for 30 min. Following three washes with PBS/Tween-20 for 5 min, respectively, the samples were incubated with a TRITC-labelled anti-mouse secondary antibody ((1:40); Dako) for 90 min. Another 3 washes with PBS/Tween-20 were performed for 5 min, and after blocking with a mouse serum ((1:40); Dako), the samples were incubated with a FITC-conjugated monoclonal anti-pan cytokeratin antibody (clone MNF116 (1:20); Dako) for 90 min. After further three washes with PBS/Tween-20 for 5 min, the samples were incubated with a DAPI-containing medium (Dako), which simultaneously preserve the samples for subsequent immunofluorescence microscopy. For background and control staining, the tumor-derived cell passages were incubated with mouse sera of the appropriate IgG subclass instead of using the primary antibodies. Fluorescence microscopy was performed with an Olympus SIS F-View II CCD-camera associated with an Olympus IX-50 fluorescence microscope (Olympus, Hamburg, Germany). The fluorescence image analysis and the fluorescence overlay image was obtained with the SIS bundle analySIS'B image software (Olympus). Accordingly, cytokeratin filaments demonstrated green, vimentin filaments red, and DNA within the cell nuclei blue fluorescence, respectively.
Cytokeratin and vimentin quantification by flow cytometry
About 5 × 105 mammary tumor-derived cells were fixed by consecutive addition of ice-cold ethanol to a final concentration of 70% (v/v). Thereafter, the cells were stored at 4°C for at least 24 h. Following 2× washes with PBS, the cells were incubated with a monoclonal anti-pan-cytokeratin (clone MNF116; Dako), anti-vimentin antibody (clone V9; Dako) and anti-desmin antibody (clone D33; DakoCytomation), respectively, for 30 min at 4°C. After washing with PBS the samples were incubated with a RPE-conjugated F(ab')2 fragment of goat anti-mouse immunoglobulin (1:10 (v/v); Dako) for 30 min at 4°C in the dark. Incubation of the cells with the secondary antibodies alone was used as a negative control and background staining. Following three washes with PBS the samples were analyzed in a Galaxy FACScan (Dako) using FloMax analysis software (Partec GmbH, Münster).
Flow cytometry analysis of surface marker expression
Tumor-derived HBCEC obtained from the same tumor piece after tissue culture for 176d and for 462d, respectively, were trypsinized and fixed in 70% ice-cold ethanol at 4°C for 24 h. Thereafter, the cells were washed twice with PBS and incubated with the FITC-conjugated CD24, CD44, and CD227 antibodies (all from BD Biosciences, Heidelberg, Germany, according to the manufacturer's protocol) and the isotype-specific negative controls (Dako), for 30 min at room temperature. After two additional washing steps, the cells were measured with a Galaxy FACScan (Dako) using FloMax analysis software (Partec).
The mammary tumor-derived cells after 722d of tumor tissue culture were compared to normal HMEC in passage 16 after 32d. The cells were fixed and stained against the senescence-associated β-galactosidase (SA-β-gal) for 24 h/37°C in the dark according to the manufacturers protocol and recommendations (Cell Signaling Technology, Danvers, MA, USA). Following two washes with PBS the differentially-stained cell cultures were documented by phase contrast microscopy with an Olympus IX50 microscope using the Olympus imaging software cellB (Olympus).
The TRAPEZE® Gel-Based Telomerase Detection assay (Chemicon International, Temecula, CA, USA) was performed according to the manufacturer's protocol using the isotopic detection. HBCEC populations from two different patients were tested, whereby one was obtained after 308d of tumor tissue culture. HBCEC from the other patient were collected after 152d of tumor tissue culture both, by trysinization or by scraping with a rubber policeman. The human embryonic kidney (HEK) cell line 293T was obtained by trypsinization of a steady state culture and used as a positive control. Briefly, HBCEC and 293T control cells were washed with ice-cold PBS and homogenized in 100 μl ice-cold 1× CHAPS lysis buffer (Chemicon). After incubation for 30 min on ice, the homogenates were centrifuged (12000 g/30 min/4°C) and the supernatants were transferred to a new tube and subjected to a protein quantification measurement using the BCA protein assay. According to the Chemicon protocol, the TS primer were radioactively end-labeled with γ-32P-ATP before the telomeric repeat amplification reaction was set up to allow the isotopic detection (see Chemicon protocol). Each assay included an internal standard (36 bp band) to control the amplification efficiency. A primer-dimer and PCR contamination control was performed by substituting the cell extract with 1× CHAPS lysis buffer. For data analysis, 25 μl of the amplified product were loaded on a 12.5% non-denaturating PAGE in 0.5× TBE buffer and eventually visualized using a PhosphorImager (GE Healthcare, Freiburg, Germany).
ATP release assay following treatment with chemotherapeutic compounds
The effects of chemotherapeutic reagents on two different primary HBCEC were analyzed using the luciferin-luciferase-based ATP tumor chemosensitivity assay (ATP-TCA). Cytotoxicity was determined by measuring the luminescence of luciferin that is proportional to the ATP-release of intact cells. Triplicates of about 1.5 × 104 HBCEC were incubated with different concentrations of chemotherapeutic compounds (Taxol (Bristol-Myers-Squibb); Epothilone A and B (kind gift from Prof. G. Höfle, Helmholtz Center for Infection Research, Braunschweig, Germany); Epirubicin (Pharmacia&Upjohn); Doxorubicin (Sigma)) in a 96-well plate for 6d at 37°C, 5% CO2. The ATP-TCA assay was performed according to the manufacturer's protocol (DCS Diagnostica GmbH, Hamburg, Germany) using non-treated cells and cells incubated with the Maximum ATP-inhibitor Solution (DCS) as controls together with an ATP standard. Following lysis of the tumor cells with an extraction buffer (DCS), the luminescence was measured in a fluoro/luminometer (Fluoroskan Ascent FL Labsystems, Thermo Scientific, Dreieich, Germany) after addition of the luciferin-luciferase reagent and the percentage of intact (viable) cells was calculated using the Ascent software (Thermo Scientific).