Xenograft model of cholangiocarcinoma
Human Mz-ChA-1 cholangiocarcinoma cells were resuspended in extracellular matrix (Sigma Aldrich, MO) and injected into male BALB/c nu/nu mice (2 million cells per tumor, subcutaneous in the flank). Once palpable tumors were present (~7 days), the mice were treated with vehicle (10% dimethyl sulfoxide (DMSO) in saline; ip, n = 6), fluoxetine (10 mg/kg in 10% DMSO; ip, n = 6) or sertraline (20 mg/kg in 10% DMSO; ip, n = 6) 3 times per week.
The tumors were measured using electronic calipers and tumor volume was approximated by using the following equation:
Tumor volume (mm3) = 0.5 × [length (mm) × width (mm) × height (mm)].
After 24 days, the mice were humanely euthanized and tumors and serum were collected. All animal experiments were performed with approval from our Institutional Animal Care and Use Committee.
Excised tumors were put into 10% neutral-buffered formalin for 3 days and subsequently processed and embedded in paraffin. Sections were stained using standard immunohistochemistry procedures, using primary antibodies for the cholangiocyte marker CK-19 (Abcam AB52625, Cambridge, MA, 1:400) or the marker of proliferating cells, PCNA (Abcam AB29, 1:100). Sections were counterstained with Hematoxylin QS (Vector Laboratories, Burlingame, CA), and photomicrograph images were taken on a Leica SCN400 slide scanner (Buffalo Grove, IL).
Total RNA was extracted from tumor tissue using an RNeasy minikit (Qiagen, Germantown, MD) following the manufacturer's instructions. Synthesis of cDNA from total RNA was achieved using the iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA). Real time PCR was performed with commercially available and validated PCR primers (Qiagen, Germantown, MD) against human PCNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using an MX3005P real-time PCR machine (Agilent, Santa Clara, CA). A delta delta CT analysis was performed using the untreated cells or the H69 cholangiocyte cell line as the control samples, where appropriate. Data are expressed as relative mRNA levels ± SEM (n = 4).
MTS cell viability assay
Cholangiocarcinoma growth was evaluated in vitro by 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) proliferation assay in the Mz-ChA-1 cell line. After trypsinization, the cells were seeded into 96 well plates (10,000 cells per well) and allowed to adhere overnight. Cells were then stimulated for 48 h with various concentrations of fluoxetine or sertraline (0.1 nM to 1 μM). Cell proliferation was assessed using a colorimetric cell proliferation assay (CellTiter 96AQueous, Promega Corp., Madison, WI), and absorbance was measured at 490 nm by a microplate spectrophotometer (VersaMax, Molecular Devices, Sunnyvale, CA). Data are expressed as the relative proliferation index, which is the average fold change in absorbance compared to vehicle-treated cells (n = 7 per group).
Analysis of cell cycle progression
Mz-ChA-1 cells were stimulated for 24 h with various concentrations of fluoxetine or sertraline (100 nM to 1 mM). The cells were collected by detaching with TrypLE (Invitrogen, Carlsbad, CA) and resuspending in culture medium. Cells were fixed and stained according to the Muse Cell Cycle Kit manufacturer's instructions (EMD Millipore, Billerica, MA) followed by analysis on the Muse Cell Analyzer (EMD Millipore).
Serum was collected from BALB/c nu/nu mice containing xenograft cholangiocarcinoma tumors and treated with sertraline or fluoxetine as described above. Serotonin levels were assessed using a commercially available competitive ELISA kit following the manufacturer's instructions (Enzo Life Sciences Inc, Farmingdale, NY). Data are expressed as average serotonin levels (ng/mL) ± SEM (n = 4).
All data are expressed as avg ± SEM. Statistical significance was analyzed by ANOVA followed by an appropriate post hoc test. For in vivo tumor volume measurements, a two-way ANOVA was used to determine significance. In each case, a p value of <0.05 was used to indicate statistical significance.