Cell cultures of BCA cells
BCA cell lines, MDA-MB-231, BCA55-121, and MCF-7 were used in the study. MDA-MB-231 is a triple negative BCA cell line while BCA55-121 and MCF-7 are luminal BCA cell lines. The BCA55-121 cell line was the cell line developed from a primary tumor of a female Thai patient by the Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok under agreement of the Siriraj Institutional Review Board No. Si519/2010. It was cultured with Dulbecco Modified Eagle’s Medium/F12 (DMEM/F-12) medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) while the other cell lines were cultured with Dulbecco Modified Eagle’s Medium (DMEM) (Gibco). These media contained 10% fetal bovine serum (FBS) (Gibco), using penicillin/streptomycin (Gibco) as antibiotics and amphotericin B (Gibco) as an antifungal drug with 5% CO2 and 90% humidity at 37oC.
LipofectamineTM 3000 (Invitrogen, Thermo Fisher Scientific) was used to transfect the blank pCDNATM3.1 plasmid (v385-20, Invitrogen) or pCDNATM3.1 PN-plasmid into BCA cell lines. After transfection, the cells were selected by GeneticinTM (Gibco) (up to 1 mg/ml) to create stable cell lines. PN and integrin expressions were tested by reverse transcriptase (RT)-polymerase chain reaction (PCR) using Light Cycler® 480 II system (Roche, Basel, Switzerland) with specific primers (Table 1) [9, 32]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was used as an internal control (Table S1). The cycle threshold (Ct) value was used for calculation of expression folding.
Western blot analysis
To determine the secreted PN amount in conditioned-media of each cell, 3x105 cells were seeded into 6-well plates with 3 ml of complete media. Cell media were removed the next day and washed with phosphate-buffered saline (PBS), and serum free media was added. Media was collected at 24 h, centrifuged and supernatant was taken and concentrated by Vivaspin®6 (VS0691, Sartorius, Goettingen, Germany). Protein concentration was determined by Bradford reagent (#5000006, Bio-Rad Laboratories, Hercules, CA, USA) and the amounts were adjusted to 5 mg per loading. To determine protein expression or phosphorylation in the cellular part, 2x106 cells were lysed with RIPA buffer (sc-24948, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Each sample was separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membrane. The membranes were blocked with 5% bovine serum albumin (BSA) and immunodetection for PN was continued with goat anti-PN polyclonal antibody (sc49480, Santa Cruz Biotechnology) and rabbit anti-goat IgG conjugated with horseradish peroxidase (HRP) (HAF017, R&D Systems, Minneapolis, MN, USA) for PN detection, rabbit anti-Akt polyclonal antibody (#9272, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pAkt polyclonal antibody (#9271, Cell Signaling Technology), rabbit anti-survivin polyclonal antibody (#2803, Cell Signaling Technology) and goat anti-rabbit-HRP (ab6721, Abcam, Cambridge, UK) for AKT, phosphorylated AKT (pAKT) and survivin. β-actin expression was determined as an internal control for the cellular part using mouse anti-β-actin polyclonal antibody (sc47778, Santa Cruz Biotechnology) and horse anti-mouse-HRP (#7076, Cell Signaling Technology). HRP was detected by Pierce™ enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific) and chemiluminescence signal was detected by G:BOX gel documentation system (Syngene, Cambridge, UK). Expression level of PN was determined by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Immunocytochemistry
BCA cells, approximately 1x105 cells, were plated on top of coverslips in 24-well plates and cultured for 24 h. After that, media was removed and cells were washed with PBS, fixed with 4% paraformaldehyde, washed again and blocked with 5% FBS. The primary antibody used was mouse anti-human integrin aVβ5 monoclonal antibody (MAB2019Z, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), at a concentration of 15 mg/ml and incubated with the cells at 37oC for 4 h. After that, cells were washed with PBS and incubated with Cy™3 AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG, Fcγ fragment specific (115-166-071, Jackson Immuno Research Inc., West Grove, PA, USA), at a concentration of 0.5 mg/ml, at room temperature for 1 h. Hoechst 33258 was used for nuclear staining. The fluorescence signal was detected by confocal fluorescence microscope (ZEISS LSM800, Carl Zeiss, Oberkochen, Germany).
Serum and tissue specimens
Serum was obtained from left-over specimens of pre-operative BCA patients and normal females who came for health checks for determination of PN concentrations. Cancer tissues from BCA patients were taken from the remaining tissue after pathological examination. Serum and tissues and clinical information collections were performed under agreement of Siriraj Institutional Review Board No. Si519/2010.
Phage Biopanning
Ph.D.TM-12 phage display peptide library (New England Biolabs, Ipswich, MA, USA) was used for phage biopanning of anti-PN peptide following the manufacturer’s protocol. In summary, a peptide fragment correlated to the integrin binding site of PN [31] conjugated with biotin (Biotin-ERIMGDKVASEALMKYHILN) was added into Pierce™ Streptavidin Coated High Capacity Plates (#15500, Thermo Fisher Scientific). Continuously, 1010 plaque forming units (pfu) of bacteriophage were poured into the wells and incubated at room temperature for 1 h. Unbound phages were then washed off and bound phages were collected. Phage titering was continued using E. coli strain ER2738 (New England Biolabs). The bacteria were centrifuged, and supernatant with virus was kept in a fresh tube. Phage precipitation was performed by adding of 1/6 volume of NaCl/polyethylene glycol solution (20% w/v PEG-8000 with 2.5 M NaCl). After that, phage titering was observed on LB/IPTG/Xgal plates, and the amplified phages were used for next round. In this way, the panning process was repeated seven times. During the biopanning process, a negative selection for phage clones was also performed to exclude streptavidin and plastic binding phage. Twenty phage clones per round from third, fifth and seventh rounds were randomly selected for DNA sequencing. Selection of candidate phage clones was done according to the results of sequencing. The sequence with highest frequency was assumed as the best phage clone to be used for further experiments. The sequences were also checked by online database to target unrelated peptides (http://i.uestc.edu.cn/sarotup3/index.html) [33] and to identify and rule out the peptide sequences which had high probability of binding to streptavidin and plastic more than 0.5. The binding affinity of selected phage clones were confirmed by the dot blot method. Volumes of 1 µl with 500 ng of recombinant PN (rPN) (RD172045025, BioVendor, Brno, Czech Republic) or BSA were spotted on nitrocellulose membranes, dried for 15 min and placed in 96-well plates then blocked with 5% BSA. Membranes were incubated with the selected phage or blank phage clones (1012 pfu in 50 ml) at 4oC overnight. Then membranes were washed and incubated with 50 ml (2 mg/ml) of anti-M13 antibody-HRP (ab50370, Abcam) at room temperature for 1 h and detected by ECL.
Peptide design and synthesis
After selecting the best binding sequence of 12-amino acids peptides, 2 types of peptide would be synthesized, plain peptide and peptide conjugated with fluorescein isothiocyanate (FITC). For the synthesis of the latter, a spacer region (GGGSCK) would be added at the C-terminal end of the peptide and FITC was conjugated with the side chain of lysine. Finally, C-terminal amidation would be performed. The synthesis of plain and FITC-labelled anti PN peptides was ordered from Syn Peptide company (Shanghai, China). FITC-labelled anti-PN peptide tested binding affinity to non-denaturing cell lysate of transfected BCA cells and their parental cells and rPN by dot blot analysis. Briefly, 12.5 μg of cell lysate or 500 ng of rPN in 1 ml was applied onto nitrocellulose membrane. The membrane was blocked with 5% BSA followed by peptide incubation at 4°C overnight and the fluorescent signal was detected the next day using the G:BOX gel documentation system. The checking of anti-PN peptide binding to intact PN-transfected BCA cells was also performed with similar process as immunocytochemistry plus a step of cell membrane permeabilizing after fixation by incubated with 1% Triton X for 1 min at room temperature. The single staining step was done by incubation of the permeabilized cells with 2 µM FITC-labelled anti-PN peptide for 1 h at room temperature, washed and then nuclear stained with Hoechst 33258. The observation was viewed under confocal microscope.
Determination of peptide binding affinity
Binding affinity of anti-PN peptide was determined with isothermal titration calorimetry [34] using the MicroCal PEAQ-ITC Machine (Malvern Panalytical Ltd., Malvern, UK) in which 700 nM of rPN (or BSA as negative control) and either 70 nM of commercial goat anti-PN polyclonal antibody or 100 nM of plain anti-PN peptide in 50 mM Tris and 150 mM NaCl (pH 7.5) buffer were added into syringe and cell compartments of the machine. The procedure followed the instructions for the machine. The results would be determined by the measurement of the exothermic energy after intermittent injection into the cell compartment and reported as the binding affinity constant (KD).
Proliferation assay
The proliferation assay of BCA cells was determined by the cell viability assay using CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS assay) (Promega, Madison, WI, USA). Briefly, 3x103 cells in 100 ml medium was seeded in 96-well plates for 24 h. The day 1 baseline determination used the MTS assay following company instructions. For the experiment, cells were placed into new media containing 2% FBS with or without rPN (100 ng/ml) and anti-PN peptide (1 μM) and cells were cultured for a further 72 h. After treatment, cell viability was measured by the MTS assay. Proliferation rate was calculated as the folding of cell numbers increased from the base line.
Determination of the half-maximal inhibitory concentration (IC50)
Determination the IC50 of BCA cells in response to chemotherapeutic drugs was analyzed by MTS assay. The experiment started from 5x103 of BCA cells in 100 ml medium that were seeded in 96-well plates for 24 h, then media was changed with different concentrations of new chemotherapeutic drugs [doxorubicin (S1208), paclitaxel (S1150), cisplatin (S1166), 5-fluorouracil (5-FU) (S1209) and gemcitabine (S1714) that were purchased from Selleck Chemicals, Houston, TX, USA] with or without rPN and anti-PN peptide for a further 48 h. After that, cell viability was determined by the MTS assay. IC50 was analyzed by GraphPad Prism® software (GraphPad Software, San Diego, CA, USA).
Migration assay
The wound healing assay was performed to determine migration activity of the BCA cells after treatment under various conditions. Briefly, 50,000 cells of parental BCA cells or mock/PN-transfected cells were seeded in 24-well culture plates with their regular media for 24 h and media changed to 1% FBS with or without rPN (100 ng/ml) and anti-PN peptide (1 μM) and culture continued for 1 day until they reached approximately 95% confluency. Wounds were applied by scratching using 200 ml micropipette tip in a single straight line. The media with floating cells would be removed and refilled with the new experimental media. The culture process would be continued for a further 24 h and photos of the area would be taken at 8 h intervals. The migration area would be analyzed from the photos taken using TScratch software (https://github.com/cselab/TScratch) [35] and migration activity calculated as mm2/h. Independent duplicated experiments were performed.
Enzyme-linked immunosorbent assay (ELISA) for serum PN measurement
Periostin ELISA Kit (Human) (Shino-Test Corporation, Tokyo, Japan) was used for measurement of serum PN following the procedure as previously described [14].
Immunohistochemistry
Matching tissues with the serum PN measurement from BCA patients were used for PN staining. An immuno-peroxidase staining procedure was performed using the method in paraffin-embedded BCA tissues as previously described. The area and intensity of expression was estimated and semi-quantitatively graded as 0-3 scores. For area determination, 0 was up to 5%, 1 was 6-25%, 2 was 26-50% and 3 was 51-100% of either the cancer or fibroblast area. For intensity scoring, 0 was negative, 1 was weakly positive, 2 was moderately positive and 3 was strongly positive when compared to positive and negative controls. Final scoring was determined by multiplying the area and intensity scores. For statistical analysis, the scores of 0-4 were categorized as low expression, and 6-9 as high expression.
Statistical analysis
ANOVA test or Student’s t-test were applied for statistical analysis of the experiments. GraphPad Prism® 7.0 software (GraphPad Software Inc., California, U.S.A) was used in calculation of IC50 values of chemotherapeutic drugs and PASW Statistics software version 18 (SPSS, IBM, Armonk, NY, USA) was used for other analysis. Dose-responses were compared by Holm-Sidak’s multiple comparison test. Mann-Whitney ranked-sum test was used for comparisons between normal and patient serum PN. Chi-square or Fisher’s exact test were used to determine the correlation between PN expression and clinical data in BCA patients. Kaplan-Meier Log-rank test was used to determine the correlation of PN expression and BCA patients’ survival. P-values of < 0.05 were used as statistical significance.