Resected specimens and patient characteristics
We collected clinical data and tumor specimens from 136 patients with BTC treated between 2005 and 2020 at Osaka University Hospital or Osaka Cancer Institute. The use of resected samples was approved by the Human Ethics Review Committee of the Graduate School of Medicine, Osaka University (No. 20493). Written informed consent was obtained from all patients included in the study.
Resected specimens were formalin-fixed and preserved in paraffin blocks prior to immunohistochemistry. Patient characteristics were prospectively recorded by the Cancer Board and confirmed in a Clinico-Pathological Conference. The specimens included 57 (41.9%) distal BTCs, 22 (16.2%) intrahepatic BTCs, 49 (36.0%) hilar BTCs, and 8 (5.9%) gallbladder cancers. Of 136 patients, 92 (67.6%) were treated with upfront surgery, 23 (16.9%) were treated with neoadjuvant chemotherapy (NAC), and 21 (15.4%) were treated with neoadjuvant chemo-radiation therapy (NAC-RT).
Immunohistochemistry
Paraffin blocks of resected specimens were cut into 3.5-µm sections, deparaffinized with xylene and ethanol, and bathed in citrate buffer at 95°C for 40 min for antigen retrieval. Endogenous peroxidase activity was inhibited by immersing sections in a 3.0% hydrogen peroxidase solution in methanol for 20 min. Non-specific binding sites were blocked in 1 mol/L PBS with 10% normal rabbit serum from the Avidin/Biotin Blocking Kit (Vector Laboratories Inc., Burlingame, California, USA). Then, sections were incubated at 4°C overnight with 10 µg/ml polyclonal goat anti-SPARC antibody (AF941, R&D systems, Minneapolis, Minnesota, USA). After washing with PBS, sections were loaded with secondary antibody from the Avidin/Biotin Blocking Kit (Vector Laboratories Inc) for 1 h. Sections were stained with avidin-biotin complex reagents (Vector Laboratories Inc) and 3,3’-diaminobenzidine (DAB) and counter-stained with hematoxylin. Finally, sections were dehydrated in graded concentrations of ethanol and xylene and mounted on slides.
SPARC Immunohistochemistry
SPARC immunostaining was evaluated separately in tumor cells and stroma cells. Staining intensities were scored as: absent = 0; weak = 1; moderate = 2; or strong = 3. The area stained was quantified as the ratio of the stained area to the entire section area (e.g., a section with 75% stained cells would be scored as 0.75). The overall staining score was calculated as the intensity score × quantity score. Figure 1a-h demonstrates representative samples of SPARC immunostaining in resected specimens. Data for tumor cells represent the sum of scores in 4 fields (×200) and data for stroma cells represent the sum of scores in 2 fields (×100). Both tumor cells and stroma cells were evaluated at the lesion center and at the invasion front (Fig. 1i-k).
Cell culture
The human gallbladder carcinoma cell line, NOZ (RRID: CVCL_3079), was purchased from the JCRB cell bank (Tokyo, Japan). The NOZ cell line was established from ascites of human gallbladder carcinoma by Homma, et al 17. A human intrahepatic cholangiocarcinoma cell line, CCLP1 (RRID: CVCL_0205) was kindly provided by Dr. Gregory J. Gores (Mayo Clinic, Rochester, Minnesota, USA). A normal human dermal fibroblast cell line (NHDF) was purchased from Ronza, (Tokyo, Japan). All cell lines were grown in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin and streptomycin at 37℃ in a humidified incubator with 5% CO2. Cell lines were sub-cultured every 3 ~ 4 days and used within 10 passages. All experiments were performed with mycoplasma-free cells as tested with MycoAlert (Ronza, Tokyo, Japan)
Cell culture assays were conducted with the recombinant human SPARC protein (rhSPARC; 941-SP, R&D systems, Minneapolis, MN, USA) and the recombinant human CTGF protein (rhCTGF; 9190-CC, R&D systems).
Highly invasive BTC clones established with the in vitro selection method
In vitro selection was carried out to establish HI clones of NOZ and CCLP1 BTC cell lines, as described previously 18. Briefly, highly invasive cells were selected in Corning BioCoat™ Matrigel Invasion chambers (Corning, NY, USA). Each chamber included a companion plate and a cell culture insert that comprised an 8-µm-pore polyethylene terephthalate membrane coated with a thin layer of Matrigel basement-membrane matrix. We seeded 1.0 × 105 cells on each insert, and chambers were incubated at 37°C, 5% CO2 for 24 h. Thereafter, cells that had migrated through the insert pores into the opposite side of the chamber were collected and cultured in a 10-mm dish to 80% confluency. This procedure was repeated six times to establish NOZ-HI and CCLP1-HI clones.
mRNA microarray analysis
mRNA microarray assays were performed with a 3D-Gene Human Oligo chip, 25k (Toray Industries, Tokyo, Japan). We compared gene expression between the NOZ-parent and NOZ-HI cells. We determined that RRM1 and dCK mRNA levels were generally upregulated in the NOZ-HI clone. Normalized data were analyzed to determine whether gene expression was upregulated or downregulated.
Invasion and migration assays
Cell invasion was assayed in Corning BioCoat™ Matrigel Invasion chambers (24-well, 8-µm pores, Corning) according to the manufacturer’s protocol. Briefly, 2.5 × 104 cells were seeded in triplicate on the cell culture inserts. After 24 h, cells that had passed to the opposite side of the membrane were fixed with 100% methanol and 1% toluidine blue. Eight microscopic fields were randomly selected for cell counting.
Cell migration was assayed with TC-inserts (SARSTEDT, Numbrecht, Germany) according to the manufacturer’s protocol. Briefly, 1.5 × 104 cells were seeded in triplicate on the cell culture inserts. After 24 h, cells that had passed to the opposite side of the membrane were fixed with 100% methanol and 1% toluidine blue. Eight microscopic fields were randomly selected for cell counting.
Cell proliferation assay
Cell proliferation was assayed with the Cell Counting kit-8 (Dojindo laboratories, Kumamoto, Japan), according to the manufacturer’s protocol. Briefly, NOZ-parent and NOZ-HI cells were plated at a density of 1.0 × 103 cells/well in a 96-well plate and treated with DMEM containing 0.5% FBS overnight. Cells were then treated with FBS-free DMEM without or with rhSPARC (1 µg/ml or 2 µg/ml). Absorbance at 450 nm was measured with a microplate reader at 24, 48, and 72 h after changing the medium.
Immunocytochemistry
NHDF cells were seeded, in triplicate, at a density of 1.0 × 104 cells in 24-well plates. The next day, the cells were co-cultured with BTC cells for 48 h. Then, NHDF cells were fixed with 4% paraformaldehyde for 15 min. Next, NHDF cells were permeabilized by adding 0.1% TritonX-100, and non-specific binding sites were blocked with Blocking one Histo (Nacalai Tesque, Japan). After washing with a rinse buffer, NHDF cells were stained with anti-SPARC mouse monoclonal antibody (#MA1-21420, Invitrogen, Waltham, MA, USA) at 4℃ overnight. After washing with a rinse buffer, Alexa Fluor 488 anti-mouse IgG Fab2 (Cell Signaling Technology Danvers, MA, USA) was added. After counterstaining with DAPI, we counted the number of SPARC-containing vesicles per field in a Cell Analyzer 6000 (Cytiva, Tokyo, Japan).
Fibroblast cell exposure to recombinant human CTGF protein
NHDF cells were seeded at a density of 3.0 × 105 cells in a 6-well plate overnight. The next day, the medium was exchanged for FBS-free medium without or with 3 µg/ml rhCTGF. After 24 h or 48 h, supernatants were collected for ELISA, and cells were harvested for qRT-PCR and Western blotting.
BTC cell line exposure to gemcitabine and radiation
NOZ-HI and CCLP1-HI cells were irradiated with 2 Gy, from a 137-Cs source, at room temperature. Cells were also exposed to complete medium containing Gemcitabine (1.5 ng/ml for NOZ-HI, 10 ng/ml for CCLP1-HI) for 48 h. Then, these treated cells were co-cultured with NHDF cells for 24 h. Afterward, NHDF cells were harvested and SPARC, FAP, COL1A1 and ACTA2 mRNA expression level were evaluated with qRT-PCR.
Reverse transcription and qRT-PCR
Total RNA was isolated from cell lines with QIAZOL and the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's recommended protocols. Complementary DNA was synthesized with the Reverse Transcription System (A3500, Promega, Madison, WI, USA). qRT-PCR was performed with the SYBR qPCR Mix (TOYOBO, Tokyo, Japan). Gene-specific oligonucleotide primer sequences are shown in Supplementary Table 1. Amplifications were performed with the ViiA7 Real-Time PCR System (Thermo Fischer Scientific, Waltham, MA, USA). β-actin served as the endogenous control. A melting curve analysis was performed to distinguish specific products from non-specific products and primer-dimers. Relative expression was calculated as the ratio of specific mRNA to endogenous β-actin mRNA in each sample.
Western blotting
Total proteins were extracted from cell lines with RIPA buffer (Thermo Fisher Scientific) containing a protease-inhibitor cocktail (Thermo Fischer Scientific) and a phosphatase-inhibitor cocktail (Thermo Fischer Scientific). The protein concentration was measured with the Bradford method. Aliquots of total protein (10 µg) were electrophoresed on sodium dodecyl sulphate polyacrylamide gels containing 10% Tris–HCl (Bio-Rad Laboratories, Hercules, CA, USA). The separated proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). The membranes were blocked with Blocking One (Nacalai Tesque, Kyoto, Japan), then incubated overnight at 4℃ with the following primary antibodies: anti-SPARC (Invitrogen), anti-CTGF (Cell Signaling Technology, Danvers, MA, USA), anti-αSMA (Abcam, Cambridge, UK), and anti-βactin (Sigma Aldrich, Saint Louis, MO, USA). Next, membranes were washed with TBS, then incubated with HRP-linked anti-rabbit IgG and anti-mouse IgG (GE Healthcare Biosciences, Piscataway, NJ, USA) at room temperature for 1 h. Antigen-antibody complexes were detected with ECL Prime Western Blotting Detection Reagent (Cytiva, Tokyo, Japan).
ELISA
The concentration of SPARC protein secreted into NHDF cell culture supernatants was measured with a Human SPARC Quantikine ELISA Kit (R&D systems), according to the manufacturer’s recommended protocol.
Gene silencing with small interfering RNA
We silenced CTGF expression by transfecting cells with CTGF-specific small interfering RNA (siRNA, Invitrogen). We also transfected unrelated siRNAs (Invitrogen) as a negative control. siRNA transfections were performed with lipofectamine RNA iMAX (Invitrogen), according to the manufacturer’s protocols. Transfection efficiencies were confirmed with quantitative real-time PCR (qRT-PCR) and Western blotting.
Paracrine signaling in trans-well co-cultures
We employed trans-well co-cultures to test paracrine signaling between invading tumor cells and stromal fibroblasts. Briefly, 2.0 × 105 NHDF cells were seeded into 6-well plates that contained complete medium. Then, in separate plates, 1.2 × 105 BTC cells (parental NOZ cells and NOZ-HI cells) were seeded onto trans-well inserts with 1-µm pores (BD Biosciences, San Jose, CA, USA). For a control, we prepared trans-well monocultures, with 1.2 × 105 NHDF cells seeded onto trans-well inserts. The next day, the complete medium was removed, and the NHDF cells in plates and the BTC-HI cells on the inserts were washed three times with PBS. Then, the inserts were placed into the NHDF plates, and FBS-free medium was added to the plates (2 mL) and inserts (1 mL). After 24 h of co-culturing, the NHDF cells were harvested and SPARC mRNA was analyzed with qRT-PCR.
Statistical analysis
All data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. Statistical analyses were performed with the student’s t test or Fisher’s exact test for categorical data. Overall survival (OS) and recurrence-free survival (RFS) were estimated with the Kaplan–Meier method and compared with the log-rank test. P-values < 0.05 were considered statistically significant. All statistical analyses were performed with JMP Statistical Software, version 14.2.0 (SAS Institute Inc., Cary, NC, USA).