Patients and specimens
This study was approved by the Ethics Committee of the First Affiliated Hospital, Sun Yat-sen University ([2019] 365) and complied with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Fresh samples (Fig S1) of HCC tumor mass (T) with paired para-carcinoma tissues (P1: ≤ 1 cm and P2: 1-5 cm, distance to tumor, if available) and normal liver tissues (> 5 cm distance to tumor, if available) were obtained from 32 HCC patients who did not received anticancer treatment before surgery and underwent hepatectomy between January 2018 and December 2018 at the First Affiliated Hospital, Sun Yat-sen University. A total of 32 tumoral, 58 para-carcinoma (32 P1 and 26 P2), and 9 normal liver tissue samples were obtained for this study. A series of clinicopathological parameters including age, gender, microvascular invasion (MVI), vascular invasion, etc., were documented (Table 1). Preoperative liver function was assessed by the Child-Pugh scoring system. Tumor differentiation was graded by the Edmondson- Steiner (ES) grading system11. The tumor stage was determined according to the sixth edition of the tumor-node-metastasis (TNM) classification of the International Union Against Cancer (UICC) and the Barcelona Clinic Liver Cancer (BCLC) strategy12.13. Tumor size was defined as the maximum diameter of the lesion and the largest one was selected when there were multiple lesions. The follow-up assessment was implemented using hepatic ultrasonography, computed tomography, and Magnetic Resonance Imaging, every month during the first 6 months after resection, and every 3 months thereafter. New lesions that arose in the residual liver in the imaging study were considered to be tumor recurrence.
Table 1 Clinicopathological features of the HCC patients
Clinicopathological features
|
Results
|
Median age (range, years)
|
55 (29-71)
|
Gender (male/female)
|
27/5
|
Hepatitis (positive/negative)
|
26/6
|
Liver cirrhosis (present/absent)
|
16/16
|
Preoperative AFP(≤20>/20, ng/mL)
|
10/22
|
Tumor size(cm) (≤5/>5, cm)
|
12/20
|
Tumor nodule number (single/multiple)
|
26/6
|
Child-Pugh classification (A /B)
|
26/6
|
BCLC stage (A/B/C)
|
20/0/12
|
ES grade (Ⅰ/Ⅱ/Ⅱ-Ⅲ/Ⅲ)
|
0/17/7/8
|
MVI (present/absent)
|
17/15
|
Vascular invasion (present/absent)
|
9/23
|
Extrahepatic metastasis (present/Absent)
|
4/28
|
TNM stage (Ⅰ/Ⅱ/Ⅲ/Ⅳ)
|
22/0/5/5
|
Reagents
Rabbit polyclonal antibody against CCS (Cat. no. 22802-1-AP, 1:1000 dilution), mouse monoclonal antibody against human beta-actin (Cat. no. 66009-1-Ig, 1:3000 dilution), horseradish peroxidase–conjugated goat anti-mouse IgG, and horseradish peroxidase–conjugated goat anti-rabbit IgG were purchased from Proteintech Group (Chicago, IL, USA). Rabbit polyclonal antibody against Ki-67 (Cat. no. ZM-0165, 1/200 dilution), rabbit polyclonal antibody against E-cadherin (Cat. no. ZM-0092, 1/400 dilution), rabbit polyclonal antibody against vimentin (Cat. no. ZM-0260, 1/500 dilution), rabbit polyclonal antibody against GPC3 (Cat. no. ZM-0146, 1/300 dilution), and rabbit polyclonal antibody against CD34 (Cat. no. ZA-0550, 1/400 dilution) were purchased from Beijing Zhongshan Golden Bridge Biotechnology (Beijing, China) for immunohistochemistry (IHC) assay.
Western blot analysis
Western blotting was performed on samples of HCC tissues, para-carcinoma tissues, and normal liver tissues. The tissues were homogenized on ice in lysis buffer containing PBS, 1.5 mol/L NaCl, 1.0 mol/L HEPES (pH 7.0), 1% NP-40, 0.1mol/L Na4P2O7, 0.1 mol/L sodium fluoride, 0.1 mol/L sodium orthovanadate, protease inhibitors (2 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 10 μg/mL aprotinin) for 30 min. The homogenates were clarified by centrifugation at 12,000 rpm for 15 min at 4°C and the protein concentration was measured by the method of Bradford. Equal amounts of protein (25 ug) were separated on 12% SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) using a mini trans-blot (Tanon, Shanghai, China). After skim-milk blocking, the membranes were incubated with rabbit polyclonal antibody against CCS at 1:1000 dilution and mouse monoclonal antibody against human beta-actin at 1:3000 dilution respectively. Membranes were then washed thrice with PBST and incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG at 1:1000 and horseradish peroxidase–conjugated goat anti-mouse IgG at 1:3000 dilution for 1 h respectively. Signals were detected using Luminol substrate solution. The amount of CCS protein expression was measured semi-quantitatively using the Quantity One software (version 4.6.6, Bio-Rad laboratories, USA) by analyzing the peak area of the CCS protein band relative to the beta-actin within the same sample on the same membrane. The ratio of the gray value of the target protein band to that of the corresponding β-actin was is calculated as the expression level of the target band. The CCS expression levels of the tumor were divided into high and low groups according to their relative expressions as compared to those in the counterpart non-tumor tissues.
Immunohistochemistry examinations
The expression levels of Ki-67, E-cadherin, vimentin, glypican-3 (GPC3), and microvessel density (MVD, as represented by CD34) in HCC and non-tumoral tissues were assessed by IHC. IHC images were evaluated by two independent observers who were blinded to clinical data and were scored semiquantitatively based on staining intensity and distribution according to the immunoreactive score (IRS) scale which took the percentage of positive cells (scale 0 to 4) and the intensity of the color reaction (scale 0 to 3) into account14. The values of positive percentage of counted cells were scored as follows: 0-point, no positive cells; 1-point, < 10%; 2-points,10-50%; 3-points,51-80%; and 4-points, > 80%, respectively. And the values of intensity of color reaction were scored as follows: 0-point, no reaction; 1-point, weak color reaction; 2-points, moderate color reaction; and 3-points, strong color reaction, respectively. The final score ranged from 0 to 12 points which was calculated by multiplying the value of positive percentage of counted cells to the value of intensity of color reaction. Specifically, the expression level of CD34 was scored semiquantitatively based on the "hot spots" method. Each of the isolated microvascular in the tumor tissue was counted as a "hot spots"15. Ten randomly selected images with 40X magnification were used for scoring for each of the biomarker, and 5 images for CD34, specifically.
Bioinformatics analysis
The gene and protein data of CCS, relevant biomarkers, and selected driver gene mutations of HCC, as well as patient prognosis and clinicopathological features, were retrieved from HPA (https://www.proteinatlas.org/, accessed on 30 Jun 2020) and TCGA (https://www.cancer.gov/, accessed on 30 Jun 2020) database. The software included in GEPIA (http://gepia.cancer-pku.cn/, accessed on 30 Jun 2020) and KM plotter (https://kmplot.com/analysis/, accessed on 30 Jun 2020) was used for data processing and data visualization. The correlations between the CCS expression level (high/low) and the aforementioned parameters including relevant biomarkers, driver gene mutations, patient prognosis, and clinicopathological features, were investigated.
Statistical analysis
All statistical analyses were conducted using SPSS 25.0 statistical software. The expression level of biomarkers between high CCS expression group and low CCS expression group was analyzed using two-sided t test. P < 0.05 was considered as statistically significant difference.