Ethics statement
The animal experiment facilities were approved by the Guangdong Provincial Department of Science and Technology and complied with the guidelines of the Animal Care Committee, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. According to the international regulations, the mice were killed in time within the legal tumor size. All efforts were made to minimize animal suffering.
Antibodies
Western blotting was conducted using the following primary antibodies: GAPDH (Abcam,ab9385), E-Cadherin (Bioworld,Cat#BS1098), Snail (CST, Cat#3879S), p-PI3K p85 (Tyr458)/p55 (Tyr199) (CST, Cat#4228S), Akt (CST, Cat#9272S), p-Akt (Ser473) (CST, Cat#4060S), GSK-3β (CST, Cat#12456S), p-GSK-3β (Ser9)(CST, Cat#5558S), CCR10 (Abcam, Cat#ab3904)
Sources of animals, cells, and parasites
Six to eight-week old female C57BL/6 mice purchased from Vital river Experiment Animal Limited Company (Beijing, China) were bred in the Animal Center of Guangzhou Institute of Biomedicine and Health(GIBH)following the Guide to the Care and Use of Laboratory Animal Committee of the institute. Mice were housed in specific pathogen-free (SPF) conditions, with a 12-hours light cycle and food and water at ad libitum. All animal experiments were performed with the standard guidelines for the care of animals, which were approved by the Welfare Committee of the Center of Experimental Animals (Guangzhou, China). The Hepa1-6 Luciferase cell line, a murine liver cancer cell line derived from C57BL/6 mice was obtained from our deposit at GIBH and was incubated in a humidified atmosphere of 5% CO2 at 37°C. Plasmodium yeolii nonlethal strain (Py17XNL) was obtained from the Malaria Research and Reference Reagent Resource Center (MR4).
Animal models
To better understand the effect of Plasmodium infection on HCC recurrence and metastasis, two animal models were established as follows. Tumor volumes were calculated as follows: tumor volume = (length*width2)/2.
The non-resection model: To determine the effect of Plasmodium infection on HCC metastasis, the non-resection mouse model was established by intrahepatic injection of 5×105 Hepa1-6 Luciferase cells. Female C57BL/6 mice (n=56) were randomly divided into two groups, with 28 in each group. Control group (Hep) were only seeded with 5×105 Hepa1-6 Luciferase cells. Plasmodium infected group (Hep + Py) were orthotopically implanted with 5×105 Hepa1-6 Luciferase cells and intraperitoneally injected with 5×105 Plasmodium yoelli 17XNL parasitized erythrocytes at the same time. Then 26 mice were randomly selected (13 from each group) for observation of survival, dynamics of parasitemia, and weight changes. Tumors in the remaining 30 mice (15 from each group) were harvested 15 days post-inoculation for further experiments.
The resection model: To investigate the effect of Plasmodium infection on post-operation recurrence, the resection mouse model was established by intrahepatic injection of tumor pieces and undergoing tumor resection on day 21 after tumor implantation. Tumors were derived from subcutaneous tumor-bearing mice, which was established by subcutaneous injected with 5 × 105 Hepa1-6 Luciferase cells. Tumors were harvested and cut into 1mm3 pieces on day 14 post inoculation. The tumor pieces were implanted into the livers of 20 mice and Luciferase in vivo imaging was used to monitor the surviving of transplanted tumors in the following days. These mice underwent reoperation on day 21 post inoculation to completely remove the transplanted tumor mass. Then the mice were randomly divided into 2 group according to the inoculation with or without the parasite on day 3 after surgery. Twenty mice (10 each group) were used for the observation of survival and intraperitoneal metastasis of hepatocellular carcinoma.
Luciferase in vivo imaging
To evaluate tumor survival, we used the luminescence properties of luciferase to track HCC in vivo. In the trial, each mouse was injected intraperitoneally with potassium fluorescein as a substrate according to body weight (10 ul/g fluorescein potassium each mouse). IVIS Spectrum was used to monitor the fluorescence emitted by Heap1-6 Luciferase about 10 to 20 minutes later.
Quantitative real-time PCR
Total RNA of tumor tissues were extracted with TRIzol reagent (Invitrogen,Cat#15596018) and the RNA was reverse transcribed with the use of cDNA synthesis kit (TAKARA,Cat#RR047A). Quantitative real-time PCR reactions were performed using TB Green Premix Ex Taq (TAKARA,Cat#RR820A). The mRNA levels of the genes of interest were normalized to that of Actin. The Primer sequences for RT-qPCR were as follows: Actin: forward 5′-TCTGGCACCACACCTTCTAC-3′ and reverse 5′-TCATCTTTTCACGGTTGGCCT-3′, CCR10: forward 5′-CAAGCCCACAGAGCAGGTCTC-3′ and reverse 5′-GATCGGGTAGTTCGTCTGGC-3′, each sample was tested at least three independent replicates.
Protein extraction and Western blotting
Tissues or cells were lysed in RIPA lysis buffer (KeyGene,Cat#KGP702-100) containing 1% phenylmethanesulfonyl fluoride (Beyotime) and phosphatase inhibitor (Cocktail, Cat#HY-K0021) on ice for 30min, and then centrifuged to collect the supernatants. Protein sample with equal quantity was separated by PAGE electrophoresis (SurePAGE, Cat#M00665), and then transferred to PVDF membranes (Millipore, Cat#ISEQ00010). The membrane was probed with anti-E-cadherin (1:500), anti-Snail1 (1:1000), anti-p-AKTSer473 (1:500), anti-AKT (1:300), anti-p-GSK3βSer9 (1:500), anti-GSK3β(1:500), anti-β-actin (1: 2000). After incubation with an appropriate secondary antibody, bands were detected with ECL reagents (Millipore,Cat# WBULS0500). Densitometry of Band signals were quantified using Quantity One. Results were presented as the ratio of the target protein’s densitometry units to GAPDH’s densitometry units.
Statistical analysis
Statistical analysis was performed using GraphPad Prism. Results are presented as means ± standard deviations. A two independent sample t test was applied to compare means of two groups. One- way ANOVA was applied to compare means of multiple groups. Chi-square test was applied to compare the rate of metastasis of two groups. A P-value lower than 0.05 was considered statistically significant. P-value less than 0.05, 0.01, and 0.001 were indicated by *, **, and *** respectively, in each figure.