Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFβ1 from Breast Cancer Patients

Patients and ALN Assessment

Twelve consecutive female BC patients, attending the hospitals within 6 weeks (from August 24, to October 04, 2007), underwent extirpation of the entire axillary region. Out of this fresh tissue, 12-15 nodes per patient have been screened histopathologically and the study cohort of 6 ALN-positive patients was obtained and presented in Table 1. None of these patients were previously treated. The proposed prospective study had received the Institutional Review Board approval and a written informed consent was obtained from each woman according to the National Health Regulation. For each BC patient, a fresh tissue of single metastatic ALN with maximal size of tumor deposits was divided in half. The first half underwent routine histopathological examination using cryotome-cut frozen sections stained by H&E. After confirmation of the presence of metastatic deposits, the second half of fresh tissue, selected for this research, was immediately snap-frozen and stored at − 70 °C, within the shelf time of 2 months. Simultaneously with mALN, autologous, histopathologically verified, normal ALN control (nALN) was collected. These pairs of tissue samples, after defrosting, were subjected to disaggregation protocol (see below) to establish respective ALN Cell Suspensions and further fractionation to obtain FNCS specimens for subsequent cFOS and TGFβ1 determination.

Protocol for Disaggregation of mALN Tissue and Establishment of mALN Cell Suspension Specimen

Previous mechanical disaggregation procedures of ALN tissue involved chopping with scalpel blade and multiple injections [19], use of rotating knifes [15] and filtering through 100 μm cell strainer [16]. In our study, frozen ALN tissue specimens were processed for fractionation on ice in the following manner:

a. The ALN tissue samples were quickly thawed, the weight measured and then chopped with scalpel in a small glass petri dish; b. The pieces of ALN tissue were resuspended with PBS (4 × 1 mL) and simultaneously transferred on an INOX 18/10 sieve (mesh size 100 × 100 μm, net diameter ⌀ 20 mm) obtained from Fasil A.D. Arilje, Serbia, (www.fasil.co.rs) which is placed above a new petri dish (Fig. 6); c. Macerated tissue was pulverized on the sieve surface with rubber piston to separate fat and connective tissue from intact cells which are filtered through a mesh into a petri dish; d. The cell suspension was then transferred into an Ependorf tube, centrifuged (850 g, 15 min, 4 °C), and the cell pellet packed volume (Vpack) estimated; e. The supernatant was decanted and the obtained cell pellet diluted with 5 x Vpack of ice-cold hypotonic lysis buffer and processed as described below (see Section Protocol for fractionation of HeLa cells...); f. An aliquot (50 μL) of pooled cell pellet was taken for viable cell counting with heamocytometer (Neubauer chamber) by Trypan blue exclusion. Further steps of fractionation of mALN cell suspension were identical as performed for HeLa cells (see below under: Steps 1-11)

Fig. 6figure 6

Image of the sieving device (100 μm cell strainer) for pulverization and filtration of ALN tissue to obtain cell suspension free from fat and connective tissue (for details see Methods)

Hela Cells

HeLa cells were grown in RPMI 1640 medium supplemented with 10% Fetal Calf Serum (serum) in the CO2 incubator. For the time course experiment, 6 medium T flasks (dimensions: 75 cm2) were grown until 80-90% confluency. Then cell monolayers, in all flasks, were washed with PBS followed by addition of serum free RPMI which was left to incubate with the cells for 12 hour. At the time point “zero”, a moment after a 12-hour incubation ended, serum free RPMI was replaced by the working medium (RPMI plus 10% FCS) to initiate serum-treatment. Cell suspension from the “zero” flask was fractionated immediately and successive fractionations followed at the incubation time points of 2, 4, 5, 6 and 7 hours.

Protocol for Fractionation of HeLa Cells and/or mALN Cell Suspension

Fractionation into nuclear and cytosolic extracts of HeLa cells and/or mALN Cell Suspension was prepared by the modifications of the protocols of Prusty et al. [9] and Riol et al. [32]. Considering the facts that the quoted references were used on primary tumor tissue and lymphocytes, respectively, we here describe the full fractionation protocol, adopted from Chemicon Inc. [21], which was used in this study:

1. After trypinization of the HeLa cell monolayer and determination of the cell pellet packed volume (Vpack), the cell pellet was resuspended in 5xVpack of ice-cold hypotonic lyses buffer (see below under Extraction Solutions); 2. For the purpose of cell swelling, the lysate was incubated on ice for 15 min, centrifuged at 850 g for 15 min at 4 °C, and supernatant discarded; 3 The cell pellet, from multiple ependorf tubes was again resuspended in 5xVpack, pooled in one tube and recentrifuged at 850 g for 15 min at 4 °C, and supernatant discarded; 4. Subsequent cell pellet was resuspended in 2xVpack of ice-cold hypotonic lyses buffer, and homogenized by drawing and ejecting the cell suspension content with a syringe/No 27 gauge needle; 5. Homogenate was centrifuged at 8.000 g for 20 min at 4 °C, and supernatant saved (containing cytosolic extract) and an aliquot (50 μL) was used to measure total cytosolic protein; 6. Remaining cytosolic extract was immediately aliquoted, snap frozen with liquid nitrogen and stored at − 70 °C for TGFβ1 ELISA analysis, within the shelf time of 4 months; 7. Remaining pellet (which contains the nuclear portion of the cell lysate) was resuspended on ice in 1x Vpack (the original cell pellet volume) in the nuclear extraction buffer (see below, under Extraction Solutions); 8. Nuclei were lysed (disrupted) by drawing and ejecting the content with a syringe/No 27 gauge needle, with addition of 1% Igepal CA-630 when necessary; 9. Homogenate was gently agitated for 30 min on ice and centrifuged at 18.000 g for 10 min at 4 °C; 10. Supernatant (which contains nuclear extract) was saved and an aliquot taken for total nuclear protein determination; 11 Remaining nuclear extract was immeditelly aliquoted, snap frozen with liquid nitrogen and stored at –70 °C for cFOS determination.

Solutions and Reagents

Fractionation of both HeLa cells and ALN cell suspensions included the use of following buffers: A. Hypotonic cell lysis buffer: 10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 2.5 mM DTT, 0.1% Triton X-100, plus PKIC (Protein Kinase Inhibitor Cocktail); and B. Nuclear extraction buffer: 20 mM HEPES pH 7.9, 1.5 mM MgCl2, 420 mMNaCl, 0.2 mM EDTA, 2.5 mM DTT, 1% Igepal CA-630, 25% (v/v) glycerol plus PKIC. In order to reduce proteolysis, dephosphorylation and denaturation of proteins, related inhibitors were added into the lysing buffers prepared as stock solutions of inhibitor cocktails: I. 50 x PIC (Protease Inhibitor Cocktail) in ethanol as solvent containing: 0.5 mg/mL leupeptin; 0.5 mg/mL pepstatin; 0.8 mg/mL benzamidine hydrochloride hydrate; and 0.1 M PMSF. The stock was aliquoted in 50 μL aliquots and kept at −20o C. II. 100 x KIC (Kinase Inhibitor Cocktail) in water as solvent, contained: 0.5 M sodium fluoride (NaF); and 0.1 M sodium orthovanadate. The stock was aliquoted in 60 μL aliquots and kept at -20 °C. III. 40 x DTT (0.1 M DTT solution is used to reduce disulphide bridges in proteins. The stock was aliquoted in 100 μL aliquots and kept at -20 °C.

Determination of Protein Yields and cFOS / TGF-β1 Protein Levels

Protein determination of nuclear and cytosolic protein yields (presented in Table 2) was performed in microplates by a micro Lowry assay [33] with the absorbance read at 650 nm (Bio Tek Instruments, Inc., Winooski, Vermont, USA). Final concentration of total proteins for both cytoplasmic and nuclear ALN tissue extracts covered the range from 3.2-9.9 mg/mL. However, the optimal total protein concentration of 2 mg/mL for TGF-β1 and 3 mg/mL for cFOS was used for comparative analysis within the single plate/assay (Fig. 5). The level of cFOS protein was determined by the Transcription Factor Assay Colorimetric kit according to the manufacturer’s instructions [21]. The results are presented as percentage of the absorbance at 450 nm compared to simultaneously analyzed cFOS positive control (4 hr. time point of serum treated HeLa nuclear extracts) considered as 100%. The TGFβ1 protein concentration was determined as described before [7]. The immunoreactive TGF-β1, obtained by acid-activation of latent TGF-β1, was analyzed by the Quantikine TGF-β1 ELISA kit according to the manufacturer’s protocol for the cell culture/Urine with RD1-21 diluting agent/ dilution factor: 14-24. (R&D Systems Inc. Minneapolis, MN, USA). Taken together, ELISA spectrophotometric analysis for the measurements of cFOS and TGF-β1, with the absorbances at 450 nm and 650 nm, was performed on the Microplate ELISA reader Wallac 1420 (PerkinElmer, Inc., Waltham, Massachusetts, USA). The cFOS / TGF-β1 levels in ALN extracts are presented in Table 2 and Fig. 5.

Statistics

For statistical analysis, the Stat Soft (Hamburg, Germany) statistical package was used. The median values and one-tailed test were used, for ALN tissue extracts measurements, since the number of ALN samples was small [34]. The significance levels between the medians for the subgroup distributions of cFOS and TGFβ1 points in control versus metastatic nuclear and cytosolic ALN tissue extracts, respectively was calculated via the Chi-sqare, one-tailed test. Level of significance for statistical tests was set at p < 0.05. All experimental points in Figs. 2, 3, 5B and C were performed in duplicates. The variation between the duplicates in a single assay did not exceed 20%. The coefficients of variation (CV) for the cFOS and TGF β1 groups ranged from 22.74 to 70.21%.

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