Colorectal (HT-29-ATCC: HTB-38; CaCo-2-ATCC: HTB-37; HCT116-ATCC: CCL-247) and mammary (MCF-7-ATCC: HTB-22; SKBR3-ATCC: HTB-30; MDA-MB-231-ATCC: TB-26) adenocarcinoma cell lines were used for the optimization of the CK19-EPISPOT. Head and neck squamous cancer cell lines (SCC-14C/SCC-22A) were kindly provided by Prof. Brakenhoff (Department Otolaryngology, Amsterdam, The Netherlands). ML-1 and C643 thyroïd cancer cell lines were kindly provided by Dr. Grimm (Department of Nuclear Medicine, Regensburg University, Germany) and Dr. Heldin (Department of Genetics and Pathology, Uppsala University Hospital, Uppsala, Sweden), respectively.
HT-29 and HCT 116 cells were maintained in McCoy's 5A medium (Invitrogen, Leiden, The Netherlands); CaCo-2 and C643 cells, in minimal essential medium (Eurobio); and MCF-7, SKBR3, MDA-MB-231, ML-1, SCC-14C, and SCC-22A cells, in Dulbecco's modified Eagle's medium (DMEM; Biochrom KG, Berlin, Germany). These media were supplemented with 1% L-glutamine (GlutaMax; Life Technologies, Paisley, Scotland), 10% fetal calf serum (FCS; Life Technologies), 500 U/ml penicillin and 500 μg/ml streptomycin (Life Technologies).
Patients and healthy controls
As shown in Table , 45 breast cancer patients (median age, 49 years; range, 31–66 years) at different stages (20 stage IV patients with overt metastases and 25 patients with apparently localized disease: stage I, one patient; stage III, 19 patients; inflammatory cancer, 5 patients) were included at the Val d'Aurelle Centre (Montpellier), as well as 11 patients with a lymphoma as the negative control group. All breast cancer patients included in this study (from 1996 through 1998) were participating in a therapeutic trial with the agreement of the local bioethics committee, and BM aspirates were taken at the time of diagnosis of their breast cancer. Concerning the control group, the BM aspirates were taken for the diagnosis of their lymphoma. Written informed consent was obtained from all patients. All 25 breast cancer patients with apparently localized disease were included after surgery but before any systemic treatment (e.g., radiotherapy/chemotherapy). BM aspiration in the 20 patients with metastatic cancer was performed before and after initiation of systemic therapy (9 and 11 patients, respectively).
Preparation of BM
BM was aspirated from the upper iliac crest, and mononuclear cells were isolated with Ficoll-density centrifugation, as described previously [12
]. Cells derived from BM samples were then cryopreserved in liquid nitrogen before the CK19-EPISPOT.
CYFR 21-1 measurement
CYFRA 21-1 was measured with the CYFRA 21-1 immunometric assay (B.R.A.H.M.S., Hennigsdorf, Germany) in culture supernatants (Tables and ).
Detection of CK19 in cancer cell lines by different methods
Comparative sensitivity of two immunometric assays for detection of released CK19 in cancer cells
The EPISPOT was performed as previously described [13
]. For the CK19-EPISPOT, the anti-CK19 Ks19.1 (6 μg/ml) and the Alexa488
-conjugated anti-CK19 Ks19.2 (3 μg/ml) mAbs (Progen Biotechnik GMBH, Heidelberg, PA) and/or and the Alexa555
-conjugated anti-CK19 mAb AE1 (3 μg/ml) (Chemicon International, Temecula, CA) were used.
For the dual-fluorescent CK19/MUC1-EPISPOT, a mixture of anti-CK19 mAb Ks19.1 and anti-MUC1 mAb 115D8 (5 μg/ml) (Centocor, Malvern, PA) with a mixture of Alexa488-conjugated anti-CK19 mAb Ks19.2 and Alexa555-conjugated anti-MUC1 mAb DF3 (1:3,000) (Centocor) were used.
CK19-EPISPOT with Brefeldin A was performed to study the CK19 intracellular transport. Brefeldin A (3 μg/ml) was added in the culture medium of MCF-7 cells during the 24-hour cell-incubation step of the EPISPOT.
The expression of CK19 in cancer cell lines (HT-29, HCT-116, CaCo2, MCF-7, SKBR3, MDA-MB-231, SSC-14C, SSC-22A, ML1, and C643) was determined with flow cytometry (FC 500 apparatus; Beckman-Coulter, Villepinte, France) (Table ). Intracytoplasmic CK19 staining was performed by using the Alexa488- or Alexa555-conjugated anti-human CK19-Ks19.2 mAb (Progen Biotechnik), and the IntraPrep permeabilization reagent kit (Beckman-Coulter).
Cell lines were immunostained with Alexa488-conjugated anti-CK19 mAb, as described earlier for the flow cytometry (Table ). Then cells were seeded on glass slides, which were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen) and analyzed (Axio Imager M1, Carl Zeiss Vision, Halbermoos, Germany).
MCF-7 cells were incubated with vincristine, 20 μmol/l (Sigma-Aldrich, Steinheim, Germany), an inducer of apoptosis, and immunostained by using the M30 CytoDEATH Fluorescein (Peviva, Bromma, Sweden) and the Alexa555-conjugated anti-CK19 mAb Ks19.2.
A dual immunostaining was performed on MCF-7 cells by using the Alexa488-conjugated anti-CK19 mAb Ks19.2 and the Alexa555-conjugated anti-MUC1 mAb DF3. Nuclear counterstaining was performed with DAPI.
To detect the expression of CK19 protein in the cell lines (MCF-7, SSC-14C, SSC-12A, and C643; Table ), cells were lysed in Triton-DOC lysis buffer. After centrifugation, supernatants were collected and analyzed with Western blotting. Samples were mixed with an equal volume of 2 × Laemmli buffer, boiled (5 min), and then loaded onto a 12% polyacrylamide gel. Proteins were electroblotted onto Immobilon membranes, and CK19 was detected by using the Alexa488-conjugated anti-CK19 KS 19.2 mAb. The Immobilon membrane was scanned by using a Typhoon model 8600 phosphorimager with ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Mass spectrometry and protein identification
MCF-7 and C643 cells were cultured in FCS-free medium and tested with the EPISPOT to confirm that full-length CK19 still could be detected. FCS-free medium was used as negative control. Five milliliters of each cell-culture supernatant was concentrated by using 5-kDa cut-off centrifugal filter units (Millipore). The concentrated supernatants were diluted with 50 mmol/l ammonium bicarbonate in a volume of 50 μl and digested with 1 μg trypsin at 37°C. Hydrophobic proteins attached to the centrifugal filter unit were digested with 1 μg trypsin in 50 μl 10% (vol/vol) acetonitril in 50 mmol/l ammonium bicarbonate at 37°C. Samples were cleaned of salts by using STAGE tips [14
Nanoscale LC-MS/MS was performed by coupling an Agilent 1100 Series LC system to a LTQ XL quadrupole ion-trap mass spectrometer (Finnigan, San Jose, CA), as described earlier [15
Kaplan-Meier life-table curves were used to visualize for overall survival. The log-rank test was used to compare significance of differences between the curves.
Cox's proportional hazards regression model with a stepwise selection procedure was applied to investigate main prognostic factors. Hazards ratios with 95% CIs were presented to display modifications in the risk of death.
All statistical tests were two-sided, and a P value of less than 0.05 was considered statistically significant. Statistical analyses were performed by using the SAS v9 software (SAS Institute, Cary, NC).