Tissue specimen acquisition
Fresh tissue specimens were obtained from 57 treatment naïve gallbladder carcinoma patients who underwent surgery at the Department of Biliary-Pancreatic Surgery of Renji Hospital, School of Medicine, Shanghai Jiao Tong University between September 2014 and September 2016. Gallbladder carcinoma was staged according to the TNM classification (AJCC 7th edition).
Follow-up
Follow-ups were conducted once every month during the first year post surgery and once every 3 months thereafter. Phone calls were made to patients and their relatives according to the follow-up guidelines of the National Comprehensive Cancer Network of China. OS was calculated from the date of surgery until the date of the final follow-up visit or death and DFS was calculated from the date of surgery until the final follow-up visit or tumor recurrence. The final follow-up visit was September 2017.
Mini-PDX models and drug sensitivity assays
Mini-PDX models were established using freshly removed gallbladder carcinoma tissues from 12 patients. Drug sensitivity was examined using the OncoVee™-Mini PDX assay (LIDE Biotech, Shanghai, China). Briefly, gallbladder carcinoma samples were washed with Hank’s balanced salt solution (HBSS) to remove non-tumor tissues and necrotic tumor tissues. After morselization, the tumor tissues were digested with collagenase at 37 °C for 1–2 h. Cells were collected followed by removal of blood cells and fibroblasts. Then, gallbladder carcinoma cell suspension was transferred to the HBSS washed capsules.
Four-weeks-old BALB/c nude mice (SLARC Inc., Shanghai, China), weighing 15–20 g each, were used for subcutaneous implantation. A small skin incision was made and the capsule was embedded in the subcutaneous tissue. Generally, each mouse received 3 capsules. Drugs (gemcitabine, 60 mg/kg, IP, Q4D × 2; oxaliplatin, 5 mg/kg, IP, Q4D × 2; 5-fluorouracil, 25 mg/kg, IP, QD × 5; nab-paclitaxel, 20 mg/kg, IV, QD × 5; irinotecan, 50 mg/kg, IP, Q4D × 2) were administered for 7 days respectively. Normal saline was used as the control. Anti-tumor activity was evaluated based on the relative fluorescence units (RFU) using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI, USA). Proliferation rate was calculated using the equation:
$$ {\text{Proliferation rate}} = {{\left( {{\text{RFU}}^{{{\text{D}}7}} - {\text{RFU}}^{{{\text{D}}0}} } \right)_{{{\text{drug}}}} } \mathord{\left/ {\vphantom {{\left( {{\text{RFU}}^{{{\text{D}}7}} - {\text{RFU}}^{{{\text{D}}0}} } \right)_{{{\text{drug}}}} } {\left( {{\text{RFU}}^{{{\text{D}}7}} - {\text{RFU}}^{{{\text{D}}0}} } \right)_{{{\text{placebo}}}} }}} \right. \kern-\nulldelimiterspace} {\left( {{\text{RFU}}^{{{\text{D}}7}} - {\text{RFU}}^{{{\text{D}}0}} } \right)_{{{\text{placebo}}}} }} $$
The study flowchart is shown in Fig. 1a. All procedures were performed under specific pathogen free conditions and carried out in accordance with the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health.
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissues were immunohistochemically stained as described previously [18]. Primary antibodies against the following proteins were used: p53 (1:200, ab1101, Abcam, Cambridge, UK), Ki-67 (1:600, ab15580, Abcam), P-gp (1:800, 13978, CST, MA, USA), MRP1 (1:200, 72202, CST), Bcl2 (1:400, 15071, CST), TS (1:100, ab58287, Abcam), GST-π (1:200, ab58287, Abcam), and Bcl-2 (1:400, 12286, CST). Immunohistochemical staining was semi-quantitatively scored by rating staining intensity of a protein of interest (I: negative, 0; weak, 1; moderate, 2; intense, 3) and the percentage of positively stained cells (P: 0%–5%, scored 0; 6%–35%, scored 1; 36%–70%, scored 2; and > 70%, scored 3) to obtain a final score (Q), which was defined as the product of I × P. Two senior pathologists evaluated the tissues independently in a blinded manner.
Statistical analysis
Data are presented as mean ± standard deviation (SD). Normally distributed continuous variables were analyzed using unpaired Student’s t-test. For multiple comparisons, the Tukey–Kramer honestly significant difference test was applied following ANOVA. Kaplan–Meier method and log-rank test were used to analyze OS and DFS. Data were censored for patients who were lost to follow-up. Pearson χ2 test was used to analyze the correlation between clinicopathological variables and drug sensitivity. SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. For all analysis, P < 0.05 was considered statistically significant.
Ethical approval
The present study was approved by the Ethical Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University. Written informed consents were provided by all participants before enrollment. All procedures were performed in accordance with the Ethical Standards of Institutional/National Research Committees and the 1964 Helsinki Declaration, its later amendments, or similar ethical standards.
