Preparation and culture of E.granulosus PSCs
E.granulosus PSCs were obtained from the hydatid cysts of sheep livers killed in the local abattoir. The PSCs were aspired into to a 50 ml centrifugal tube (Beyotime Institute of Biotechnology, Jiangsu, China) under aseptic conditions and then centrifugated at 2000 rpm for 2 min. PSCs pellet was obtained by centrifugation three times at 2000 rpm for 2 min. The viability of E.granulosus PSCs was tested by using trypan blue and PSCs inocula with the viability over 95% were used for the transplantation assay.
Cell Lines and Culture Conditions
Human HCC cell lines HepG2 were purchased from the Cancer cell bank of Chinese Academy of Medical Sciences (Chinese Academy of Sciences, Beijing, China), which were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Thermo Fisher Scientific, Waltham, MA) medium, supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco) at 37 °C in 5% CO2 cell incubator.
Animal transplantation assay
All animal experiments were performed in accordance with the approved guidelines and protocols from the Animal Experimental Ethics Committee of Xinjiang Medical University. For the transplantation assay, thirty male nude mice with 8-10 weeks age (15 ± 3 g) were randomly divided into control group and co-transplantation group. Control group mice were subcutaneously transplanted with 5×106 HepG2 cells, while co-transplantation group mice were subcutaneously transplanted with 5×106 HepG2 cells and 2000 E.granulosus PSCs that was defined as the high dosage infection according to the recent findings . Subcutaneous lesion volume was measured every four days from the 25th day after transplantation. Two mice in the control group and three mice in the experimental group were died during the animal studies. Lesion samples from the survived mice were obtained for under anesthetic conditions at the 37th day of transplantation. After fixation in 4% neutral buffered formalin and paraffin-embedding, samples from subcutaneous lesions were sectioned into 4 μm slices, which were then used for the following studies.
Hematoxylin and eosin (H&E) staining
The slices were heated at 60 °C for approximately 1 h. After conventional deparaffinization and hydration with xylene and gradient ethanol, the slices were then totally immersed in the hematoxylin solution for 1.5-2 min, which were washed with water, followed by staining with hydrochloric alcohol for 3-5 s and the eosin solution for 1-1.5 min. Multiple pictures of several areas were taken using a Laser scanning confocal microscope (Leica, Heidelberg, Germany).
Periodic acid–schiff (PAS) staining
After deparaffinization and hydration, the slices were immersed in periodic acid (Solarbio, Beijing, China) for 8 min. Then, the slices were washed with deionized distilled water and subsequently treated with Schiff’s reagent (Solarbio) for 15 min, followed by staining with Mayer’s Hematoxylin (Solarbio) for 1.5-2 min. Images were captured by a Laser scanning confocal microscope (Leica) after conventional dehydration and transparency. Areas of PAS positive cells were calculated through ImageJ (Rawak Software Inc, Stuttgart, Germany).
Picric acid-sirius red staining
After conventional deparaffinization, the slices were added picric acid red solutions (Solarbio) at 37 °C for 30 min, which were then immersed into anhydrous ethanol for 2-3 s. After conventional dehydration and transparency, representative pictures were acquired by the confocal microscope (Leica), which was repeated in triplicate. Positive areas were calculated through ImageJ (Rawak Software Inc).
Paraffin-embed slices were heated at 60 °C for approximately 1 h, which were then deparaffinized in xylene and rehydrated in graded alcohol. After retrieving damaged antigens with citrate buffers (Beyotime), 3% hydrogen peroxide (Beyotime) was used for quenching the endogenous peroxidase activity. The samples were then incubated with the following primary antibodies at 4 °C overnight: rabbit monoclonal anti-mouse antibody α-SMA (1:500 Abcam, Cambridge, MA), CD3 (1:400, Cell Signaling Technology, Danvers, MA), CD4 (1:500, Cell Signaling Technology), CD8 (1:200, Cell Signaling Technology), PD-1 (1:50, Cell Signaling Technology), PD-L1 (1;100, Cell Signaling Technology); rabbit polyclonal antibody Ki67 (1:200; Abcam), Bcl-2 (1:200; Cell Signaling Technology), Caspase3 (1:100; Abcam), CD16 (1:400; Abcam), CD56 (1:50; Abcam). At the next day, after rewarming the slices for 40 min at room temperature, the slices were incubated with HRP‐conjugated secondary antibodies (1:2000; Abcam) for 1.5 h at room temperature. Then, a DAB staining kit (Zhong Shan Jinqiao Biotechnology, Beijing, China) was applied to detect the antibodies. Multiple pictures of several positive areas were captured by the confocal microscope (Leica) and positive areas were calculated through ImageJ (Rawak Software Inc).
Statistical analysis was performed using the Statistical Package for Social Science (SPSS) version 21.0 (SPSS Inc, Chicago, IL, USA) and GraphPad Prism 8.0 (GraphPad Software, San Diego, CA). All data were presented as the Mean (X̅) ± Standard Deviation (SD) from three independent experiments. Statistical comparisons of parameters between groups were made by Student’s t test. Statistical significance was set at the 5% level and P<0.05 was considered as statistically significant.