Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University. All the animals were kept in an SPF facility with consistent temperature (22℃) and humidity (55%), and a 12-hour light/dark cycle. The facility was operated by experienced zookeepers who were responsible for serving bedding material, food, and sterilized water, under the supervision of a veterinarian. 4 to 5 mice were housed in each cage. All the animals had free access to food and water. All mice were sacrificed by carbon dioxide asphyxiation as approved by the IACUC of Seoul National University. Five-week-old female ICR mice and eight-week-old male BALB/c-nude mice were purchased from OrientBio (Sungnam, Korea). ICR mice were used for bone marrow cell preparation. Bone marrow-derived macrophages were prepared as previously described . Bone-metastatic cancer selection was described in our earlier publication . The bone-metastasis rate of cancer cells by intra cardiac injection was approximately 20 percents in our previous study. Thus we used five mice for bone-metastatic cancer selection. Briefly, left ventricular cardiac injection was performed with 1 x 105 PC3 cells injected into nine-week-old male BALB/c-nude mice (n=5). After 8 weeks, metastasized cells in femurs and tibiae were flushed out from the bone marrow and expanded in culture dishes for 8 weeks. Then, the immortal cells were re-injected into the left ventricle for another round of in vivo selection. We named the cancer cells harvested after the second round the mtPC3 cells. Each purpose of experiment was to obtain primary cells or bone-metastasized cancer cell. No control group was used.
Recombinant M-CSF and RANKL were purchased from PeproTech. Recombinant mouse S100A4 was purchased from Prospec. Recombinant OPG was purchased from R&D Systems. Antibodies against vimentin and c-Fos were purchased from Santa Cruz. Antibodies against N-cadherin and snail2 were obtained from Cell Signaling Technology. Antibodies for RAGE (DD/A11) were from Millipore. Anti-S100A4 antibody was purchased from Abcam. Anti-b-catenin antibody was purchased from Invitrogen. Antibodies for NFATc1 (7A6), E-cadherin, and hHLA were purchased from BD Pharmingen. Anti-β-actin antibody (AC-74) and the leukocyte acid phosphatase kit (for TRAP staining) were purchased from Sigma-Aldrich. Lipofectamine for siRNA transfection was purchased from Life Technologies. siRNA oligonucleotides and shRNA lentiviral particles were purchased from Santa Cruz. Dentin slices were purchased from Immunodiagnostic Systems.
Cell lines and culture conditions
LNCaP and PC3 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Cell lines were validated by STR-PCR analysis by the Korean Cell Line Bank, and the STR profiles of each cell line can be accessed from their website (https://cellbank.snu.ac.kr/main/tmpl/sub_main.php?m_cd=22&m_id=0503). Cells were cultured in high-glucose DMEM (Lonza) supplemented with 10% FBS (Life Technologies) and 1% penicillin and streptomycin (WELGENE, Korea).
mtPC3 cells were authenticated by labelling with PE-conjugated mouse anti-human b2-microglobulin (BD Pharmingen). Human b2-microglobulin is associated with the HLA Class I antigen complex. During flow cytometry analyses, mouse bone marrow cells served as a negative control and human embryonic kidney-293 cells were utilized as a positive control. Briefly, 1 x 105 cells were incubated with the antibody (1:100) for half an hour and softly washed with ice-cold PBS three times before analysis using FACSCalibur (BD Science).
Cells were washed with ice-cold PBS and lysed with RIPA buffer (10 mM Tris pH 7.2, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1% sodium deoxycholate, and 5 mM ethylenediaminetetraacetic acid). A standard protocol for Western blotting was performed with cell lysates.
Cell proliferation assay
Cells were incubated at the indicated time with 10% CCK solution in cell culture medium for 1 hour at 37°C. Optical density was then measured with an ELISA reader (iMARK Microplate Absorbance Reader, Bio-Rad, Hercules, CA, USA) at 450 nm.
Cell migration assay
The cell migration assay was performed using trans-well plates with 8.0-μm polycarbonate membranes (Corning). 1 x 105 cancer cells were seeded onto the upper chamber. The cells were tested for the migration toward the lower chamber during stimulation by serum with or without S100A4 (2 μg/ml) or S100A4 neutralizing antibodies (4A, 30 μg/ml). After 16 hours, migrated cells were fixed with fixing solution, followed by crystal violet staining for visualization.
Conditioned medium preparation
1 x 106 cancer cells were seeded onto a 60-mm culture dish with DMEM and incubated overnight. The next day, the culture medium was exchanged to alpha-MEM (WELGENE) and further incubated for 24 hours. Then, the supernatant was collected and centrifuged at 1200 rpm to remove dead cells. A 3:7 (supernatant to fresh medium) ratio was used unless specifically indicated in the figure legend.
BMMs were prepared from 5-week-old female ICR mice as previously described . Pre-osteoclasts were generated by culturing BMMs with M-CSF (30 ng/mL) and RANKL (50 ng/mL) for 36 to 48 hours. Then, mature osteoclast formation was induced by incubating pre-osteoclasts with M-CSF (30 ng/mL) and conditioned medium from cancer cells. Multinucleated TRAP+ cells usually formed within 24 to 48 hours after conditioned medium treatment. TRAP+ cells with 3 or more nuclei were visualized under a microscope and considered to be mature osteoclasts (Olympus BX51, DP2-BSW software (version 2.2)).
Real-time PCR analysis
A standard protocol for real-time PCR analysis was pursued for the quantification of mRNA levels. Primers for real-time PCR analyses are as follows: human hprt (hypoxanthine-guanine phosphoribosyltransferase) forward, 5’- accccacgaagtgttggata-3’; human hprt reverse, 5’- aagcagatggccacagaact-3’; human s100a4 forward, 5’-gcccagcttcttggggaaaa-3’; human s100a4 reverse, 5’- atggcgatgcaggacaggaa-3’.
Enzyme-linked immunosorbent assay (ELISA)
1 x 105 cancer cells/well were seeded onto a 48-well tissue culture plate and incubated overnight. The culture medium was replaced with 100 μL serum-free medium and further incubated for 24 hours. Supernatant was collected and subjected to ELISA with a human S100A4 ELISA kit (CycLex Co.) according to the manufacturer’s protocol.
Osteoclast resorption assay
A dentin slice was placed into each well of a 48-well tissue culture plate, after which 4 x 104 BMMs were seeded as well. Osteoclast differentiation and bone resorption were induced by supplementing M-CSF (30 ng/mL) and RANKL (50 ng/mL). Dentin slices were washed with distilled water for cell removal and mounted on glass slides. The resorbed depth and area were calculated by inspecting the dentin surface using a Zeiss LSM 5 PASCAL laser-scanning microscope (Carl Zeiss Microimaging GmbH, Goettingen, Germany). The Zeiss LSM Image Browser program was utilized (version 3.0 SP3).
S100A4 and control shRNA lentiviral particles were purchased from Santa Cruz Biotechnology, Inc. mtPC3 cells were transduced with lentiviral particles and incubated for 2 days. The cells were incubated with puromycin (10 μg/mL) for an additional 3 days to sort the successfully transduced cells. For siRNA transfection, BMMs were seeded in the presence of M-CSF (30 ng/mL) and incubated overnight. The next day, Lipofectamine 2000 (Invitrogen) was used for the formation of the liposome complex containing siRNAs. The complex was incubated with BMMs for 6 hours, followed by fresh media replacement.
Data are presented as the mean ± SD of biological replicates. An unpaired two-tailed Student’s t-test was used to define differences between two samples. One-way ANOVA or Two-way ANOVA with a post hoc Bonferroni or Tukey’s test were used for analyses of multiple groups. All statistical tests were performed using SigmaPlot 11.0 (Version 18.104.22.168, Systat software Inc., San Jose, CA, USA). p < 0.05 was considered statistically significant.