The present study was conducted on 124 IDC tissue specimens obtained from patients treated at the Lower Silesian Oncology Centre in Wroclaw, Poland. All patients were treated by mastectomy, quadrantectomy and/or axillary lymph node resection. In total, 11 (8.9%) patients received neoadjuvant chemotherapy and 93 (75%) of patients received adjuvant chemotherapy. A total of 58 (46.8%) patients were treated with radiotherapy. The patients were followed for 64 months (range, 1–80 months). The median follow-up was 80 months. The clinical and pathological data were obtained from the archives of the hospital. Tumour histological type and malignancy grade (G) were determined according to the World Health Organisation (WHO) criteria (27). The patient and tumour characteristics are listed in Table I. The study was approved by the Bioethics Committee of the Wroclaw Medical University and was conducted according to GCP guidelines. Written informed consent was obtained from all participants.

The human BC cell lines, MCF-7, MDA-MB-231 (both from ATCC, Washington, DC, USA), SK-BR-3, (from the Cell Line Collection of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, Wroclaw, Poland) and BO2 (derivative of the MDA-MB-231 cell line isolated from bone metastasis; courtesy of Dr Philippe Clezardin, INSERM U664, Lyon, France), were selected to determine the expression levels of nestin. The BC cell lines were cultured in α-MEM supplemented with 10% fetal calf serum (FCS; Invitrogen, Carlsbad, CA, USA), 2 mM L-glutamine and 1% penicillin-streptomycin solution (Gibco, Carlsbad, CA, USA). The normal mammary epithelial cell line, hTERT-HME1 (from ATCC), was cultured in MEGM Bullet kit medium (Lonza, Basel, Switzerland).

The tissue samples were fixed in 4% buffered formalin, dehydrated and embedded in paraffin. The IHC reactions were examined on the 4-µm-thick paraffin sections in Autostainer Link48 (Dako, Glostrup, Denmark). The sections were boiled in EnVision FLEX Target Retrieval Solution (pH 9.0, 97°C, 20 min) using Pre-Treatment Link Platform (both from Dako). To inactivate endogenous peroxidase, the sections were incubated for 5 min with EnVision FLEX Peroxidase-Blocking Reagent (Dako). As primary antibodies, we used a panel of mouse-anti human monoclonal antibodies against the following: nestin (1:100 dilution, OBT1610, Bio-Rad, Hercules, CA, USA), Ki-67 (ready-to-use, IR610, Dako), CD31 (ready-to-use, IR610, Dako), CD34 (ready-to-use, IR632, Dako), SOX-18 (1:25 dilution, sc-166025, Santa Cruz, CA, USA), ER clone 1D5 (ready-to-use, IR654; Dako) and PR clone 636 (ready-to-use, IR068; Dako). The samples were incubated with the primary antibodies for 20 min at room temperature and then incubated with EnVision FLEX/HRP for 20 min (Dako). The sections were then incubated for 10 min with 3,3′-diaminobenzidine (DAB, Dako) as the peroxidase substrate. All slides were counterstained with the EnVision FLEX Hematoxylin (Dako) and closed with coverslips in Dako Mounting Medium (Dako). The expression status of HER2 was determined using the HercepTest and the HER2 FISH pharmDx kit (both from Dako) following the procedure recommended by the manufacturer.

The IHC reactions were evaluated using a BX-41 light microscope (Olympus, Tokyo, Japan). The reactions were evaluated simultaneously by two observers in order to achieve a consensus score. Nestin expression in tumour cells was evaluated using the semi-quantitative immunoreactive score (IRS) of Remmele and Stegner (28). The Chalkley count, reflecting an area and number of vessels (29), was assessed on anti-nestin, anti-CD31, anti-CD34 and anti-SOX18-stained slides. Firstly, the slides were briefly examined under low magnification (×100) to identify three areas with the highest vascular density (hot spots). A 25-point Chalkley eyepiece graticule (Pyser Sgi., Edenbridge, UK) was applied to each hot spot at a higher magnification (×200), and oriented to permit the maximum number of points to hit on or within the immunohistochemically stained microvessels (30). The final score for each slide was presented as a mean value from the three hot-spots. The status of ER and PR receptors was scored from 0 to 3 points, depending on the percentage of positive cells (0 points, no reaction; 1 point, 1–10%; 2 points, 11–50%; 3 points, 51–100% stained cells) (31). HER2 expression was determined using a scale considering the percentage of positive tumour cells and the intensity of the membrane reaction (32). The nuclear expression of Ki-67 antigen was evaluated using a semi-quantitative 5-grade scale as follows: 0 points (0% of cells stained), 1 point (1–10% of cells stained), 2 points (11–25% of cells stained), 3 points (26–50% of cells stained), or 4 points (51–100% of cells stained).

The investigated tumour cell lines were grown on glass coverslips for 24 h at 37°C. The cells were then washed with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde. After washing, the cells were permeabilised with 0.2% Triton-X. The ICC reactions in Dako Autostainer Link48 (Dako) according to the procedure described above. The slides were incubated with anti-nestin antibody for 20 min at room temperature, and then incubated with EnVision FLEX (Dako) to visualize the antigens. For IF reactions, the tumour cell lines were cultured and fixed as stated above. All slides were incubated at 4°C overnight with mouse monoclonal antibody against nestin (1:100, OBT1610, Bio-Rad) solved in antibody diluent (Dako). Donkey anti-mouse antibody conjugated with rhodamine (1:2,000, polyclonal; 715-025-151 Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) was applied as a secondary antibody. The slides were covered with ProLong^® Gold Antifade Mountant mounting medium (Molecular Probes, Eugene, OR, USA) with 4′,6-diamidino-2-phenylindole (DAPI) counterstaining. The reactions were viewed using a BX51 fluorescence microscope (Olympus, with DP72 camera and CellF software).

Total RNA from was isolated from the hTERT-HME1, MCF-7, SK-BR-3, MDA-MB-231 and BO2 cells using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. To eliminate genomic DNA, we applied on-column DNase digestion. The quantity and the purity of the RNA were assessed by measuring the absorbance at 260 and 280 nm using a NanoDrop-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). First-strand cDNA was synthesised using High-Capacity cDNA Reverse Transcription kits (Applied Biosystems, Foster City, CA, USA). The relative nestin gene (NES) expression level was determined in relation to the expression of the normal mammary epithelial cell line, hTERT-HME1, by quantitative (real-time) PCR with the 7500 Real-Time PCR System and the iTaq Universal Probes Supermix (Bio-Rad), according to the manufacturer's instructions. For the reactions, we applied the following human TaqMan probes: NES Hs04187831_g1 for nestin and ACTB Hs99999903_m1 for β-actin (both Applied Biosystems). β-actin was used as a reference gene for determining relative NES expression in the analysed tumour cell lines. The reactions were carried out in triplicate under the following conditions: initial denaturation at 95°C for 10 min, 45 cycles of denaturation at 95°C for 15 sec, followed by annealing and elongation at 60°C for 60 sec. The relative mRNA expression level of the NES gene was calculated using the ΔΔCq method (33).

The cells from the cell culture were trypsinised, washed in PBS and lysed with lysis buffer (50 mM Tris HCl, 150 mM NaCl, 0.1% SDS, 1% Igepal CA-630, 0.5% sodium deoxycholate) containing 1X protease inhibitor cocktails (Pierce, Rockford, IL, USA) and 0.5 mM PMSF. Following 20 min of incubation on ice, the contents were centrifuged at 12,000 × g at 4°C for 10 min. The supernatants were collected and whole cell protein concentrations were quantified using the BCA protein assay (Pierce). The protein (30 µg) was processed for 6% SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon; Millipore, Bedford, MA, USA) and incubated with monoclonal mouse antibody against nestin (1:1,000, OBT1610, Bio-Rad) overnight at 4°C. The membranes were then incubated with a secondary anti-mouse antibody conjugated with horseradish peroxidase (1:3,000, polyclonal; 715-035-150, Jackson Immunoresearch, Mill Valley, CA, USA) for 1 h at room temperature, rinsed, and treated with the Immun-Star-HRP Chemiluminescent Kit (Bio-Rad). β-tubulin, detected with anti-β-tubulin antibody (rabbit anti-human, 1:1,000, poly-clonal; ab6046, Abcam, Cambridge, UK), was used as an internal control to normalize the amounts of nestin at the same membrane after stripping. The protein bands were visualized and quantified with densitometry using Image Lab 3.0 on a Molecular Imager ChemiDoc XRS imaging system (Bio-Rad).

For flow cytometric immunophenotyping of intercellular nestin, the cells were fixed and permeabilized with BD Cytofix/Cytoperm™ (Becton-Dickinson, San Jose, CA, USA) according to the manufacturer's instructions. Subsequently, the cells were stained with mouse monoclonal antibody against human nestin (1:100; OBT1610, Bio-Rad) for 30 min at room temperature and then labelled with donkey anti-mouse antibody conjugated with fluorescein (1:1,000, polyclonal; 715-096-151 Jackson ImmunoResearch Laboratories). The cells were then analysed by flow cytometry using BD FACSCanto™ II flow cytometer (Becton-Dickinson) equipped with three lasers: blue (488 nm, air-cooled, 20 mW solid state), red (633 nm, 17 mW HeNe) and violet (405 nm, 30 mW solid state). Data were recorded for 20,000 events using BD FACSDiva™ software (Becton-Dickinson), analysed on the ungated population and presented as histograms using WinMDI2.7 software (Scripps Research Institute, La Jolla, CA, USA).

The data were analysed using Prism 5.0 (GraphPad, La Jolla, CA, USA) software. Analysed results are presented as the mean values ± standard deviation (SD). The associations between the clinicopathological characteristics and the expression of nestin were analysed by the non-parametric Mann-Whitney U test. The differences between nestin expression levels in tumour cells were analysed by one-way analysis of variance with a post hoc Tukey's test. The correlations between the scores of the examined antigens were tested using the Spearman rank correlation test. The Kaplan-Meier method and the Mantel-Cox test were used to determine the significance of patient overall survival (OS). In addition, for the analysis of survival, the Cox's univariate and multivariate proportional hazard model was used. Differences were considered statistically significant with a value of P<0.05.

A positive nestin expression was observed in 39 (31.5%) of investigated tumour cases. Nestin expression was observed in the cytoplasm of tumour cells (Fig. 1A–C), tumour vessels (Fig. 1A and B) and in myoepithelial cells of the breast ducts (data not shown). A significantly higher nestin expression in tumour cells was noted in G3 compared to G2 cases (Fig. 2A; P= 0.024; Mann-Whitney U test). Moreover, our results revealed a significantly higher nestin expression in TNBC (Fig. 2B; P<0.0001; Mann-Whitney U test). Of note, nestin expression in tumour cells negatively correlated (Spearman rank correlation test) with ER (Fig. 3G; n=124, r=−0.39, P<0.0001) and PR (Fig. 3H; n=124, r=−0.23, P=0.012), but not with HER2 expression (Fig. 3I; n=124, r=−0.11, P=0.26). A correlation was also noted between nestin expression in tumour cells and Ki-67 expression in the cell nuclei of tumour cells (Fig. 3F; n=112, r=0.25, P=0.0062; Spearman rank test). Survival data analysis indicated that a postive nestin expression (IRS ≥1) in tumour cells was associated with a shorter patient overall survival (Fig. 2C; P=0.02; Mantel-Cox test). However, multivariate analysis revealed that nestin was not an independent prognostic factor (Table III). We also found a correlation (Spearman rank correlation test) between nestin expression in tumour cells and the area and number of vessels immunolabelled with antibodies against CD31 (Fig. 3A; n=105, r=0.24, P=0.01), CD34 (Fig. 3B; n=90, r=0.38, P=0.0002), nestin (Fig. 3C; n=112, r=0.19, P=0.04) and SOX-18 (Fig. 3D; n=101, r=0.27, P=0.005). A strong correlation was also observed between the area and number of nestin⁺ vessels and SOX-18⁺ vessels (Fig. 3E; n=101, r=0.34, P=0.0004; Spearman rank test).

The NES mRNA expression level in BC cell lines (MCF-7, SK-BR-3, MDA-MB-231, BO2) was determined by real-time PCR. The relative NES expression was assessed in relation to the expression of the normal mammary epithelial cell line, hTERT-HME1. The highest NES expression was noted in the metastatic BO2 cell line when compared to the less aggressive MCF-7, SK-BR-3 cells and MDA-MB-231 cells (Fig. 2D; all P<0.001; ANOVA, Tukey's post hoc test). Of note, at the mRNA level, NES expression was higher in the MCF-7 cells than in the more aggressive SK-BR-3 and MDA-MB-231 cells (Fig. 2D; both P<0.001, ANOVA, Tukey's post hoc test).

The results of ICC and IF reactions revealed the cytoplasmic expression of nestin in all investigated BC cell lines. In both experiments, we observed more intense reactions in metastatic MDA-MB-231 (Fig. 1G and K) and BO2 (Fig. 1H and L) cell lines than in the less aggressive MCF-7 (Fig. 1E and I) and SK-BR-3 cell lines (Fig. 1F and J). The analysis of nestin protein levels by western blot analysis in the BC cell lines revealed an increased expression in the metastatic MDA-MB-231 and BO2 cells (Fig. 1D). Weaker bands were detected in the MCF-7 and SK-BR-3 cell lines (Fig. 1D). Flow cytometric analysis was performed to determine the expression level of nestin in the investigated BC cell lines. The mean fluorescence intensity (MFI) for nestin was shown as MFI for samples incubated with primary and secondary antibodies minus MFI for the isotype control (Table II). Measurements of the MFI revealed a higher fluorescence intensity in the metastatic MDA-MB-231 and BO2 cells (Table II; MFI=20.43 and MFI=26.48) than in the less aggressive MCF-7 and SK-BR-3 cells (Table II; MFI=17.2 and MFI=9.45).