The cell lines used for this study have previously been given an account of Król et al.
[23–25]. Two adenocarcinoma cell lines isolated from the canine mammary gland tumors (CMT-W1 and CMT-W2) and two cell lines isolated from their lung metastases (CMT-W1M and CMT-W2M) were kindly donated by Prof. Dr. Maciej Ugorski and Dr. Joanna Polanska from Wroclaw University (Poland). The cell lines were cultivated in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 50 U/ml penicillin, 50 μg/ml streptomycin, 2,5 μg/ml amphotericin B (reagents obtained from Sigma Aldrich Chemical Co., USA) and grown in tissue culture flasks (Nunc™, Denmark) in an atmosphere of 5% CO2 and 95% humidified air at 37°C, and routinely subcultured every second day.
Canine mammary tumor tissue sections were derived from the archives of the Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences –WULS (Poland). The samples were surgically obtained during the mastectomy from 50 female dogs of various breeds. The tissue samples were fixed in 8% neutral buffered formalin and routinely embedded in paraffin. Eighteen canine mammary tumors were snap frozen using liquid nitrogen and archived at -80°C.
Most of the cases’ information about the presence/absence of metastases was known. The tumors that gave metastases were surgically removed together with the metastatic site. The presence of neoplastic cells on the metastatic site was histologically confirmed. The immunohistochemical examination of cytokeratin, vimentin, smooth muscle actin, s100 protein and p63 protein expression was performed (data not shown). Histological diagnoses were achieved on haematoxylin and eosin (HE) stained slides according to the World Health Organization (WHO) Histological Classification and Mammary Tumors of the Dog and Cat classification
. Tumor grading was based on the Misdorp
 classification and was evaluated in respect to tubule formation, degree of differentiation and mitotic index as: the 1st, the 2nd and the 3rd grade of malignancy. The tumors were classified as benign adenomas or malignant carcinomas (simple carcinomas or complex carcinoma). The tumors were divided into five categories: benign adenomas (n = 10), malignant adenocarcinomas: of the 1st grade of malignancy (n = 10), of the 2nd grade of malignancy (n = 10), of the 3rd grade of malignancy (n = 10) and metastatic (n = 10) (which gave local or distant metastases).
Four micrometer (4 μm) sections from paraffin blocks containing tumor tissue were baked in 37°C overnight. After dewaxing in xylene and rehydration in ethanol, for antigen retrieval, the slides were placed in 0.02 M citrate buffer, pH 6.0 and boiled in the decloaking chamber.
The cell lines were cultured on Lab-Tek (Nunc Inc. USA) 4-chamber culture slides for 24 hrs and subjected to the immunohistochemical analysis after ethanol (70%) fixation (10 min).
The samples were incubated in the Peroxidase Blocking Reagent (Dako, Denmark) for 10 min at room temperature prior to the antibody incubation. After 30 min incubation in 5% bovine serum albumin (Sigma Aldrich, Germany), the following primary antibodies were used (diluted in 1% bovine serum): rabbit polyclonal anti-ghrelin (Abcam, United Kingdom) diluted 1:50 and incubated one hour at room temperature; rabbit polyclonal anti-Growth Hormone Secretagogue Receptor (Novus Biologicals, USA) diluted 3 μg/ml and incubated overnight at +4°C. For the staining the EnVision kit (Labelled Polymers consist of secondary anti-rabbit antibodies conjugated with the HRP enzyme complex obtained from Dako) was used. To develop a colored product, the 3,3′-Diaminobenzidine (DAB) substrate was used (Dako). Finally, the hematoxyline was used for nuclei counterstaining.
For each immunohistochemical experiment of paraffin slides, positive controls were set aside: canine stomach (for ghrelin) and canine pituitary (for GHS-R). For each experiment of paraffin slides and cell lines the negative control was stained without use of primary antibodies.
Three consecutive tissue sections and four slides of each cell line were analyzed. Ten to 20 pictures of each slide were taken (depending on the sample size) using Olympus microscopy BX60. Only the regions of cancer cells and stroma were analyzed in the primary tumors whereas the necrotic regions were avoided. The number of ghrelin positive cells (brown color) and the colorimetric intensity of the GHS-R expression reflected as IHC-stained antigen spots (brown color) were counted by a computer-assisted image analyzer (Olympus Microimage™ Image Analysis, software version 4.0 for Windows, USA). The antigen spot color intensity is expressed as a mean pixel integrated optical density (IOD) on a 1-40,000 scale.
Total RNA from the frozen canine mammary tissues and cell lines suspensions was isolated using a Total RNA kit (A&A Biotechnology, Poland) according to the manufacturer’s protocol. Isolated RNA samples were dissolved in RNase-free water. The quantity of isolated RNA was measured using NanoDrop (NanoDrop Technologies, USA). The samples with adequate amounts of RNA were treated with DNaseI to eliminate DNA contamination. The samples were subsequently purified using RNeasy MiniElute Cleanup Kit (Qiagen). Finally RNA samples were analyzed on a BioAnalyzer (Agilent, California, USA) to measure final RNA quality and integrity.
The ghsr and ghrl primers were designed using PRIMER3 software (free on-line access) and checked using Oligo Calculator (free on-line access) and Primer-Blast (NCBI database). The used sequences for ghsr were as follow: 1) TTCCACGTGGGCGATATTTATT and TGGCAGCACTGAGGTAGAAGAGG (to assess ghsr expression at mRNA level in examined cell lines and tissues) and CCTGGTATCCTTTGTCCTCTTCT and CTTTCATCCTTCAGAGTGGAGAG (to assess ghsr knockdown effect after siRNA treatment). The optimal annealing time for the first pair was 5 sec, whereas optimal annealing temperature was 59°C. The optimal annealing time for the second pair was 10 sec, whereas optimal annealing temperature was 63°C. The used sequences for ghrl were as follow: TCAGGTGAGTGCTTCTAGGG and CACTCGGGAAGTTTCTTCAA. The optimal annealing time was 5 sec, whereas optimal temperature was 61°C. rps19 and hprt genes were used as a non-regulated reference for the normalization of target gene expression (sequences and reaction conditions has been previously given an account of:
[23–25, 28, 29].
Quantitative RT-PCR was performed using fluorogenic SYBR Green and the Sequence Detection System, Fast 7500 (Applied Biosystems). Data analysis was carried out using the 7500 Fast System SDS Software Version 22.214.171.124 (Applied Biosystems, USA). The results were analyzed using comparative Ct method
. Relative transcript abundance of the gene equals ΔCt values (ΔCt = Ctreference – Cttarget). Relative changes in transcript are expressed as ΔΔCt values (ΔΔCt = ΔCtcontrol – ΔCtexamined). The experiment was conducted three times.
Cell viability assay (MTT-assay)
Cell viability (metabolic activity of viable cells) was quantified by MTT assay. Cells were seeded into 96-well plate (Nunc Inc., Denmark) at the concentration of 6 × 104 cells per well. When cells reached 60-70% confluence, the medium was replaced with medium containing 10% FBS and n-octanoylated ghrelin peptide (Peptides International) at the concentrations of 1, 10, 100 and 1000 nM. To protect ghrelin from inactivation, the culture medium contained also 20 nM serine protease inhibitor, 4-(2-aminoethyl) benzenesulphonyl fluoride hydrochloride (AEBSF, Sigma Aldrich)
. The cultures were incubated for 24 hours.
In order to determine the mechanism by which ghrelin increased proliferation/viability of the cells (to assess if it acts via GHS-R), they were additionally pre-treated for 1 hr with the 100 and 200 μM of GHS-R-specific antagonist [D-Lys3]-GHRP6 (Peptides International). Then, cells were incubated in 0.5 mg/ml tetrazolium salt MTT diluted in phenol red-free RPMI 1640 medium (Sigma Aldrich) for 4 hrs at 37°C. To complete solubilization of the formazan crystals, 100 μl of DMSO (Dimethyl sulfoxide, Sigma Aldrich) was added to each well. Cells viability was quantified by measuring photometric absorbance at 570 nm in multi well plate reader Infinite 200 PRO Tecan™ (TECAN, Mannedorf, Switzerland). All the samples were examined in triplicate, each experiment was conducted five times (n = 15).
Cell proliferation assay (BrdU incorporation assay)
BrdU cell proliferation assay was carried out according to the BrdU labeling protocol (BD Bioscience, USA). Briefly, canine mammary cancer cells where cultivated in culture flask to reach 60-70% confluence. The medium was then removed and replaced medium containing 10% FBS with 20 nM AEBSF and: (1) ghrelin at the concentration of 1, 10, 100 and 1000 nM or (2) GHS-R-specific peptide antagonist [D-Lys3]-GHRP6 (10, 100, 200, 500 and 1000 μM) for 24 hrs. Then, 10 μM of BrdU (BD Bioscience) was added to culture medium for one hour. The cells were washed twice with PBS, harvested by trypsinization, suspended in PBS and fixed in ice-cold ethanol (70%). The cells were then stored at -20°C for 12 hrs and then washed with Wash Buffer (BD Bioscience, USA) and suspended in denaturing solution (2M HCl) for 20 min at room temperature (RT) to denature DNA. The cells were then washed again with Wash Buffer and resuspended in 0.1 M sodium borate (Na2B4O7, Sigma Aldrich, USA) of pH 8.5, for 2 minutes at RT, to neutralize any residual acid. The cells were then washed with Wash Buffer and incubated with FITC-conjugated anti-BrdU antibodies (20 μl) and isotype control (BD Bioscience, USA) for 30 min at RT. Cells were then incubated with 10 μl/ml of propidium iodide (PI) (Sigma Aldrich) for 30 min at RT. All the samples were analyzed using flow cytometry (FACS Aria II, Becton Dickinson, USA). The experiment was conducted in three replicates.
The siRNA transfection procedure on canine mammary cancer cells has been given an account of in our previous paper
. However, the procedure was optimized for CMT-W1M cell line. Thus the cell density, transfection reagent toxicity and transfection efficacy has been assessed (according to the procedure described in our previous manuscript:
). The canine (Canis lupus familiaris) ghsr sequence was obtained from Gene Bank with accession number [EF536345.1]. The siRNA duplexes were designed by
http://www.sigmaaldrich.com/life-science/custom-oligos/sirna-oligos/sirna-design-service.html. The results were confirmed using two independent algorithms: Dharmacon (OligoWalk) and Ambion and at last two duplexes were chosen for further experiments (obtained from Sigma Aldrich) (1st duplex sequences, ghr1, are as follow: CCAUCAAUCCCAUUCUGUAdTdT and UACAGAAUGGGAUUGAUGGdTdT; 2nd duplex sequences, ghr2, are as follow: GCUCUCCACUCUGAAGGAUdTdT and AUCCUUCAGAGUGGAGAGCdTdT). Each duplex was used at 2 different concentrations (ghr1 or ghr2, and ghr1′ or ghr2′ at concentration of 6 or 12 pmol, respectively) the mixture of both duplexes was also used (ghr1 + ghr2: 6 pmol + 6 pmol). The RNA interference has been assessed by Real-time qPCR. Transfected and control CMT-W1M cells (cells cultured according to the transfection procedure with transfection reagent + non-coding RNA (Negative Universal Control, Invitrogen)) were scraped, and the total RNA from the cell suspension samples was isolated. Because during optimization of the siRNA transfection procedure for CMT-W1M cell line any significant changes in cells viability between control cells and cells treated with only transfection reagent has been observed (data not shown), the mock-transfected control has been omitted.
The Annexin V-FITC and propidium iodide (PI) dual staining was applied for apoptosis analysis. Normal cells and cells treated with (1) ghrelin at the concentration of 1, 10, 100 and 1000 nM and 20 nM AEBSF, (2) inhibitor of ghrelin receptor [D-Lys3]-GHRP6 at the concentration of 10 μM, 100 μM, 200 μM, 500 μM and 1000 μM for 24 hrs (3) transfection reagent + non-coding RNA (as a control for transfected cells) and (4) ghsr siRNA, were harvested by trypsinization and together with the cells floated in medium were stained using an Annexin V Kit (Becton Dickinson, USA), according to the manufacturer’s protocol. The cells were immediately (within 1 hr) analyzed by flow cytometry (BD FACS Aria II, Becton Dickinson, USA). Early apoptotic cells with exposed phosphatidylserine but intact cell membranes bound to Annexin V-FITC but excluded PI. Cells in late apoptotic stages were labeled with both Annexin V-FITC and PI, whereas necrotic cells were labeled with PI only. All samples were assayed in triplicate, and each experiment was performed three times (n = 9).
In vitro wound healing assay (scratching test)
Scratch assay was conducted to assess the influence of ghrelin on canine mammary carcinoma cell motility. The cells were seeded in a high density at 600 mm Petri dishes (Corning-Costar, Cambridge, MA, USA). After 24 hrs, the medium was removed and replaced medium containing 10% FBS with 20 nM AEBSF and (1) ghrelin at concentration of 1, 10, 100 and 1000 nM or (2) specific GHS-R inhibitor [D-Lys3-GHRP6 at concentration of 10 uM, 100 μM, 200 uM, 500 μM and 1000 μM for 24 hrs. When the cells reached full confluence, the monolayers were wounded by scratching the surface as uniformly and straight as possible with a pipette tip (1000 μl) at least three times. The images of cells invading the scratch were captured at indicated time points (0, 3, 6, 9, 12 and 24 hrs) using phase contrast microscopy (IX 70 Olympus Optical Co., Germany). The pictures have been analyzed using a computer-assisted image analyzer (Olympus Microimage™ Image Analysis, software version 4.0 for Windows, USA). The migration rate was expressed as percentage of scratch closure and was calculated as follows: % of scratch closure = a-b/a, where (a) is a distance between edges of the wound, and (b) is the distance which remained cell-free during cell migration to close the wound
. The values are the means of three independent wound fields from three independent experiments (n = 9).
Invasion and migration assay
The BD BioCoat™ 24-Multiwell Invasion System (BD Biosciences, USA) pre-coated with BD Matrigel™ Matrix were used according to the manufacturer’s protocol. The insert plates were prepared by rehydrating the BD Matrigel™ Matrix layer with phosphate buffered saline (PBS) for two hours at 37°C. The rehydration solution was then carefully removed, and 500 μl of cell suspension was added to the apical chambers (2.5 × 105 cells). Cell suspension was prepared by trypsinizing cell monolayers (80% confluent) and resuspending the cells in RPMI 1640 medium containing 0.1% FBS, 20 nM of AEBSF and ghrelin at the concentration of 1, 10 and 100 nM. Then 750 μl of chemoattractant (10% FBS) was added to the each of the basal chambers. Assay plates were incubated for 22 hours at standard culturing conditions. Followed incubation medium was carefully removed from apical chamber and insert system was transferred into a second 24-well plate containing 500 μl of 2.5 μg/ml Calcein AM in Hanks’ Balanced Salt solution (HBSS). Plates were incubated one hour at standard culturing conditions. Then the fluorescence of invaded cells was measured at excitation wavelength 485 nm and emission wavelength 530 nm using florescent plate reader with bottom reading capabilities Infinite 200 PRO Tecan™ (TECAN, Switzerland). All samples were assayed in triplicate, and each experiment was conducted three times (n = 9).
To evaluate migratory potential the BD Falcon™ FluoroBlock™ 24-Multiwell Insert Plates (8 micron pore size) (BD Biosciences, USA) was used. The determination protocol for the canine mammary cancer cells migration was the same as the invasion assay, with the exception that no Matrigel was used and rehydrating of the plate was omitted. In order to determine fluorescence of cells invaded through membrane coated by Matrigel fluorescence microscopic analysis using Olympus microscopy BX60 at x4 magnifications was applied.
The statistical analysis was conducted using Prism version 5.00 software (GraphPad Software, California, USA). The one-way ANOVA and Tukey HSD (Honestly Significant Difference) post-hoc test, Dunnett’s test and t-test were applied as well as regression analysis. The p-value < 0.05 was regarded as significant whereas p-value < 0.01 and p-value < 0.001 as highly significant. The data was expressed as means +/- S.E.M. unless otherwise stated. The in vitro wound healing assay was analyzed using two-way RM ANOVA and Bonferroni post-hoc test.