Between January 2018 and June 2020, Surgical tissues were collected at the first affiliated hospital of Shandong first Medical University from 132 patients with hepatocellular carcinoma disease confirmed by fast pathology biopsy during the operation after patients’ signed informed consents were obtained. All fresh tissues were restored immediately on the refrigerator of -80℃ and anonymized before transfer to the laboratory for further processing. All the demographic data, including age, sex, clinical classification, survival time, and relative follow-up visits were gathered. All control subjects were followed up for two and a half years and were free of liver malignancy.
Primary TCs Culture
Fresh samples from liver para-cancer tissues and hepatic hemangioma tissues(as the control group) after patients’ operation were selected and cut into fragments, and incubated with 5 mg/ml collagenase type II (Sigma-Aldrich, St. Louis, MO, USA) for 10min. PBS without calcium and magnesium(pH 7.4， G0002，Servicebio, USA ) washing twice and centrifuged at 1000 r.p.m., 5min, then re suspended in DMEM with 10% foetal calf serum. The BJ-40 capillary glass tube (1.0 mm outer diameter, 0.8 mm inner diameter) was soaked in 1 mol/L hydrochloric acid for 24 h, then rinsed continuously with ultrapure water, dried at 65°C and autoclaved. Under 200x magnification of the microscope and selected cells to 0.2 mL centrifuge tube containing 2μL of lysate according to the morphology of TCs[33,34 ].
Liver TCs isolation and identification
After mature C57BL/6 mice(No.4432, Weitonglihua animal company, Beijing, China) were killed with anaesthetic. The hepatic tissue were isolated under sterile conditions and collected into sterile tubes containing DMEM (Gibco-8120217, NY, USA), Suppl.ed with 100 UI/ml penicillin, 0.1 mg/ml streptomycin (20201013, Kaisu biology Co Ltd., Jiangsu, China), transported to the cell culture laboratory. Dispersed cells were separated by filtration through a 40-m-diameter cell strainer(CLS431751，Falcon, NJ, Germany), collected by centrifugation at 1000 r.p.m., 5min., and resuspended in DMEM, Suppl.ed with 10% foetal calf serum, 100UI/ml penicillin, 0.1 mg/ml streptomycin (Sigma-Aldrich). Cells were distributed in 25cm2 plastic culture flasks, at a density of 1×105 cells/cm2, and maintained at 37℃/5% CO2 atmosphere until becoming semi-confluent. Culture medium was changed every 48hrs. The typical TC were photographed by auto-microscopy per 12hrs. After cell adhesion on the plate, Cells were selected, purified and further amplified to the next experiment. The TCs were identified according to the morphology and immunofluorescent staining assays. The protocol followed by the hereinafter.
Lentivirus production and infection
To build recombinant lentivirus, 293T cells were cotransfected with progresses of package, envelop, and expression. The virus-containing supernatant was harvested and concentrated by ultracentrifugation. The viral stock was Suppl.ed with 8 mg/mL of polybrene for infections .
In order to gain stable experimental cell lines, the primary TCs from hepatic tissues(from mice) transfected with SV40 large and small T antigen to constructed TCSV40. The culture medium of TCSV40 cells was Dulbecco’s modified Eagle’s medium/F12 with 10% fetal calf serum (Gibco-8120330, NY, USA) Suppl.ed. HepG2, SNU182 and SK-HEP-1 cell-Lines were cultured in DMEM(GIBCO，Beijing, China) Suppl.ed with 10% FBS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin and then Cells were placed in a humidified atmosphere containing 5% CO2 at 37°C. When cells reached 60– 80% confluence, positive and stable transfectants were selected for further study.
RNA extraction and qRT-PCR analysis
To verify the mRNA expression level of MMP2, MMP3, MMP9, MMP11 and MMP14 in HCC tissues and para-cancer tissues, qRT-PCR analysis was performed. Total RNA was extracted from tissues using Trizol reagent (CW0581, Kangweishiji company, China) according to the manufacturer’s protocol. Reverse transcription and cDNA amplification were performed using the SYBR Master Mixture (CW0957, Kangweishiji company, China), respectively, according to the manufacturer’s guidelines. The β-actin genes were used as endogenous controls. A HiFiScript Primer Assay (CW2569, Kangweishiji company, China) was used to assay MMPs and β-actin(Suppl. Table 1).
For miR-942-3p test, mature miRNA sequence was found by miRBase database, shRNAs and mimics of indicated miRNAs were obtained by RiboBio Company (Shanghai and Wuhan, China). Transfection with shRNAs and miRNAs was completed using riboFECT™ CP (RiboBio) according to the manufacturer’s instructions.
For 3’ -UTR analysis, cells were cotransfected with psiCHECK-2–based construct and pre-miR-942-3p or a negative control. Luciferase assay was conducted with the Dual-Luciferase Reporter Assay System (Promega). Luciferase miRNA Target expression vector (Promega, Madison, WI, USA) to construct the reporter vectors: MMP-9 wild type (WT) , MMP-9 mutant (MUT) and negative vectors-mimics(NC).
Western blot analysis
Human hepatocellular cancer tissues with para-cancer tissues were fetched out from the -80℃ condition, thawed and resuspended using lysis buffer (20% Glycerol, 4% SDS in 100 mM Tris Buffer, pH 6.8). Cell extracts were boiled for 10 min in loading buffer and then equal amounts of cell extracts were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels. Separated protein bands were transferred onto polyvinylidene fluoride membranes. The primary antibodies against MMP2, MMP3, MMP11, MMP9, MMP14 and GAPDH were diluted according to the instructions for each of the antibodies and incubated overnight at 4°C. Then, horseradish peroxidase-linked secondary antibodies were added and samples were incubated at room temperature for 2 h. The membranes were washed with phosphate-buffered saline (PBS), and the immune-reactive bands were colored using an ECL-PLUS/TM (Amersham company, UK) according to the manufacturer’s instructions. The relative protein levels between cancer tissues and para-cancer tissues were normalized to GAPDH concentrations. Three separate experiments were performed for each clone. Furthermore, the primary antibodies against Bax and Cleaved-caspase-3 were incubated for cell apoptosis test(Suppl. Table 6).
Immunohistochemistry (IHC) staining
Formalin fixed paraffin-embedded primary tumor tissue with para-cancer tissue were utilized for IHC. For heat-induced antigen retrieval, slides were soaked in citric acid buffer and heated keeping 1300w for 2 min. After quenching endogenous peroxidase activity with 3% H2O2. Specimens were incubated with antibody at 4°C overnight. Specific signals were developed with second-antibody using diaminobenzidine as chromogen(Suppl. Table 6). Sections were then counterstained with hematoxylin and observed under light Microscope(XSP-C204, CIC, China). Slides were scanned using laster scanning confocal microscope (Eclipse Ti-E, Nikon, Japan) with 40×magnification. Datum were quantified in immunohistochemistry digital slides with Leica Aperio positive pixel count algorithm using whole slide analysis (PANNORAMIC DESK/MIDI/250/1000, 3DHISTECH, Hungary).
All sections were incubated in 2 changes of xylene at 15 min each. A dehydrator was used to dehydrate the sections in 2 changes of pure ethanol and were immersed in EDTA antigen retrieval buffer (pH 8.0, G1206/G1203, Servicebio, USA). The slides were incubated with primary antibodies to double stain for CD34, CD117, PDGFR-α and MMP9 overnight at 4℃ and then with secondary antibody after washing three times with PBS(Suppl. Table 6). Under microscopy, images were collected using fluorescence microscopy (NIKON ECLIPSE C1, Tokyo, Japan) with an imaging system (NIKON DS-U3, Tokyo, Japan). Images were captured at 1 to 400 magnification (Microscope Camera XSP-C204, Olympus Europa GmbH, Hamburg, Germany).
For invasion assays, Transwell migration chambers and Matrigel coated chambers(Becton Dickinson, Waltham, MA) were used. Briefly, 5 × 104 HCC cells were seeded into the upper chamber in serum-free culture medium. The lower chamber was filled with 5 × 104 TCs completed medium with 10% FBS. After 48 h for the invasion assay, cells that have invaded through the membrane were stained with 1% crystal violet and counted in the microscopy(CKX-51,OLYMPUS company, Japan).
CCK-8 cell counting assay
TCs in logarithmic phase were digested and made into cell suspensions. After uniform spreading, the cells were incubated and then 10 μ L of 5 mg/mL cell counting kit-8(CCK-8)(HY K0301, MCE, Shanghai, China) was added to the wells starting from the second day after spreading and 4 h before the termination of the incubation. The OD value was measured at 450 nm by enzyme marker after 4 hours.
Wound healing assay
Use a marker pen on the back of the 6-well plate uniformly with horizontal lines. Add approximately 2.5 × 105 HCC cells to the wells and overnight to reach 100% fusion rate. Prepare co-culture cells-TCSV40, wash the cells 2-3 times with PBS after digestion, resuspending with serum-free medium and add co-culture chambers. MMP9 inhibitor group was a concentration of 3uM, adding each group of co-culture chambers into the corresponding wells. Every chambers incubated in 37℃ 5% CO2 incubator for 48hr. The area was counted using Image J software.
In vivo models
For building metastatic lung cancer, 1 × 107 HepG2 cells in 100 μl of PBS were injected into the tail veins of BALB/c-nu mice(No.4272, Weitonglihua animal company, Beijing, China). 6 × 104 TCs in 50 μl of 0.9% normal saline were injected into mouse tail vein per 7 days after HepG2 cells injection. The mice were sacrificed on day 42. Lung tissues were resected, photoed and fixed with 4% paraformaldehyde, and then strained with hematoxylin and eosin (HE). For subaxillary transplantation, HepG2 cells were injected into the right axilla of nude mice and 6 × 104 TCs in 50 μl of 0.9% normal saline were injected around local tumors per 3 days and TCs with MMP9 inhibitor group built as the compared group. After 28 days, these mice were sacrificed to gain axilla transplanted tumors which were measured weights and volumes(maximum axis × minimum axis2 × 1/2).
Whole-exome Sequencing Technique
A cohort of 23 patients with HCC who underwent surgical resection between 2019 and 2020. Primary culture TCs were selected from fresh para-cancer tissues of Genomic DNA samples of acceptable quality were randomly interrupted by ultrasonic high performance sample processing system (Covaris) into fragments with a major peak of about 200bp-300bp. Subsequent DNA fragments were then end-repaired by adding an "A" base at the 3' end and a library splice at both ends. An appropriate amount of hybridization library was captured and enriched with the exome chip, and the unenriched fragments were eluted and amplified, and the whole exome was captured. The amplification products were subjected to Agilent 2100 bioanalyzer instrument (Agilent DNA 1000 Reagents) and QPCR quality control. We used the Illumina HiSeq family of platforms to perform high-throughput sequencing of each qualified library and to ensure that the data volume of each sample was up to standard. The raw image data obtained from sequencing was converted into raw reads (raw reads) by Illumina Base Calling software, i.e. double-end reads (paired-end reads). The data were stored in FASTQ file format, called raw data.
Variant Calling and Bioinformatics Analysis
The information analysis started with the sequenced downstream data. The raw data contained adapter sequences, bases of low sequencing quality, and undetected bases (expressed as N). Next, the clean data of each sample was compared to the reference genome using the alignment software BWA(Burrows-Wheeler Aligner) to obtain the initial alignment result file in BAM format. To ensure the accuracy of variant detection, we followed the optimal variant detection analysis procedure recommended by the official website of GATK. Based on the alignment results, the evaluation indexes such as sequencing depth, coverage, and alignment rate of each sample were counted. In the process, we wield HaplotypeCaller of GATK v3.4.0, including SNPs(single nucleotide polymorphism) and InDels, and filtered the raw variant detection results with high confidence(Suppl. table 3,4). Next, the variant results were annotated and impact predicted using the in-house software AnnoDB, as well comparison of different types of sample sets using the genome visualization software IGV(Integrative Genomics Viewer; Suppl. figure 3-F ).
SPSS20.0 was utilized for the statistical analyses. Quantitative value was recorded as the mean ± standard(SD). Two-tailed Student’s t-tests, paired t-tests, chi-square tests, and multivariate analysis were used to assess the differences among groups. Pearson correlation analysis was used to analyze MMP9 expression with TCs number in HCC tissues. Survival curves were drawn by the Kaplan–Meier analysis and statistical significance was considered as P < 0.05.