Cell culture and treatment
Human-derived hepatocellular carcinoma cell line (SK-HEP1) were purchased from Wuhan Servicebio Technology Co., Ltd (Hubei, China),and have been authenticated by STR profiling.
Cell lines were cultured in SK-HEP1 cell-specific culture medium (Servicebio, China) in humidiffed cell incubator at 37 ◦C with 5% CO2. IDO1 inhibitor epacadostat (EPA) was purchased from MedChemExpress LLC (Shanghai, China). According to the reagent instructions, EPA solutions with concentrations of 2.5μM, 5 μM, 10 μM, and 20 μM were prepared using the recommended solvent.Cells were planted in 6-well plates or 96-well plates, and were randomly divided into control group (0μM EPA) and EPA treatment groups with EPA at final concentrations of 2.5, 5,10 and 20 μM in the light of the test data proposed in reference[31-33].According to different experimental purposes, cells were collected for fixation or extraction of RNA and proteins for subsequent assays after incubation for an appropriate period of time.
Tumor models and treatments
All animal experiments were conducted in accordance with the protocols approved by the Laboratory Animal Ethics Committee of Shenzhen Zhongxun Precision Medicine Research Institute and compliance with the relevant ethical regulations for animal research (Approval number:ZXJZ202306100001). Male BALB/c nude mice, 4 - 6 weeks old, were purchased from
Guangdong Pharmalab Biotechnology Co., Ltd (Guangdong, China) and kept under specific pathogen-free conditions. For in vivo xenograft model established and the IDO1 inhibitor treatment was performed as reported in the literature[34, 35]. SK-HEP1 (4 x106 cells/100 μL) in logarithmic growth phase was injected into the right dorsal side of nude mice.Tumor size was measured every 3 days until the end of the experiment, and the tumor volume was calculated according to the formula V = (a × b²)/2, where a is the largest superficial diameter and b is the smallest diameter.When the tumor volume reached approximately 90 mm3, mice were randomly distributed to control and experimental group (five mice in each group), and treatment was initiated. The experimental and control group received intraperitoneal injection of EPA (100 mg/kg) and DMSO, respectively,every 3 days for a total of 3 injections. At the end of the experiment, mice were sacriffced, the blood and fresh tumor tissues were collected and processed for further detection.
Key Fluor 488 Click-iT Edu assay
Effects of IDO1 inhibitor EPA on the proliferation of SK-HEP1 was detected using the Key Fluor 488 Click-iT Edu kit (RiboBio, China) according to the product instructions.
CCK-8 assay
CCK-8 kit (Bioswamp,China) was used to detect the effect of EPA on the viability of SK-HEP1 cells.After co-culturing with medium containing different concentrations of EPA for 24 hours, 10 μL of CCK-8 solution was added to the cells, and the cells were incubated for 4 h at 37 ℃. The absorbance of each group of cells was detected at 450 nm using a multifunctional microplate detector (TECAN Spark, Switzerland). The cell viability was calculated in each group according to the instructions.
Wound healing assay
Total 7×10⁵ SK-HEP1 cells were seeded in a 6-well plate. When the cells reached 85% density, a 10 μL sterile pipette tip was used to draw a straight line vertically on the cell layer. The cells were washed twice with PBS to remove the scratched cell debris. Then fresh complete medium containing different concentrations of EPA was added for culture. The 6-well plates were observed under a microscope and photographed at 0, 24 h and 48 h, respectively.
Transwell assay
SK-HEP-1 cell suspension was prepared by using FBS-free medium and the cell density was adjusted to 1 x106 cells/mL. 200 μL of cell suspension was taken from each group and placed on the upper surface of Transwell, 600 uL of complete medium containing 10% FBS was put into the lower surface of each group. After being cultured in a constant temperature incubator for 24 hours, the medium was aspirated from the lower surface of the chamber, and the cells were washed three times with PBS, and the lower surface of each group was fixed with 4% paraformaldehyde for 30 min, 0.1% crystal violet (Beyotime Biotechnology,China ) staining for 30 min. The cells on the upper chamber surface were wiped off. Under the microscope, six fields of view were randomly selected from each group to observe and count the cells.
RNA extraction, cDNA synthesis, and qPCR
Total RNA was extracted from the cells according to the instructions of the RNA Extraction Kit (Fastagen, China), and then cDNA was synthesized by reverse transcription of total RNA in accordance with the instructions of the HiScript III RT SuperMix for qPCR (Vazyme, China ). qPCR was performed with RealStar Green Fast Mixture (GenStar, China) in Applied Biosystems QuantStudio Dx system. GAPDH was used as endogenous control. Sequence-specific primers for the genes detected are listed as following:
GAPDH (human)-F, 5’-AGAAGGCTGGGGCTCATTTG-3’,
GAPDH (human)-R, 5’-GCAGGAGGCATTGCTGATGAT-3’;
E-cadherin (human)-F, 5’-ATTTTTCCCTCGACACCCGAT-3’,
F-E-cadherin (human)-R, 5’-TCCCAGGCGTAGACCAAGA-3’;
Vimentin (human)-F, 5’-AGTCCACTGAGTACCGGAGAC-3’,
Vimentin (human)-R, 5’-AGTCCACTGAGTACCGGAGAC-3’;
IDO1 (human)-F, 5’- GCCTGATCTCATAGAGTCTGGC-3’,
IDO1 (human)-R, 5’-TGCATCCCAGAACTAGACGTGC -3’;
PCNA (human)-F, 5’- AGAGGAGGAAGCTGTTACCATAGAG-3’,
PCNA (human)-R, 5’-CATACTGAGTGTCACCGTTGAAGAG -3’.
Western blot analysis
Total cellular protein was extracted and quantified according to the instructions of Total Protein Extraction Lysate(Beyotime Biotechnology, China) and BCA protein assay kit (EpiZyme, China) . Then total proteins were separated by 10% SDS-PAGE (EpiZyme, China) and then transferred onto a PVDF membrane (Millipore, USA). After membrane transfer, the membrane was blocked for 10 min with NcmBlot blocking buffer (NCM Biotech, China) . After washing with TBST, incubate the primary antibody overnight at 4 °C, including IDO1 (rabbit, CST), E-cadherin (rabbit, CST), Vimentin (rabbit, CST), GAPDH (rabbit, proteintech) and α-tubulin (rabbit, proteintech) , followed by incubation with HRP-conjugated secondary antibodies (rabbit, proteintech). After imaging by chemiluminescence, Quantity One was used to analyze the gray value of the image and calculate the relative expression level of protein.
Multiplex immunohistochemistry analysis
We performed multiple immunohistochemical staining according to the instructions for the Opal 7-color Manual IHC Kit (PerkinElmer ,USA).The tissue microarray (HLivH180Su31) employed in this study, including total 96 human hepatocellular carcinoma samples and 82 samples of matched adjacent normal tissues, was purchased from Shanghai Outdo Biotech Co. Ltd (Shanghai, China). Tissue microarrays were routinely deparaffinized and antigenically repaired. After blocking, primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were used for staining sequentially, and stained with one of the seven Opal reagents, followed by microwave intervention to remove the primary and secondary antibodies before another round of staining. Anti-IDO1(rabbit, CST), anti-CD11C (rabbit, Proteintech), anti-CD80 (rabbit, Abcam), anti-CD40 (rabbit, Abcam), anti-MHCII (rabbit, HUABIO) and anti-keratin (rabbit, Abcarta) antibody were used as primary antibodies. The dyes Opal520, Opal650, Opal480, Opal690 , Opal570, Opal620 and DAPI were used for staining. Samples were visualized and analyzed using the Tissue Gnostics (TissueFAXS SpectraS, Peking, China).
Histopathology, immunofluorescence and immunohistochemical staining
For histopathological staining, tumor tissues were fixed with 4% paraformaldehyde for 24 h , routinely dehydrated in gradient, embedded in paraffin sections, cut into 4 μm-thick sections. HE staining were conducted according to the reagent instructions (Servicebio, China).
For immunohistochemical staining, we perform routine baking, dewaxing to water for paraffin sections of tumor tissues, antigen repair, blocking endogenous peroxidase. Then the slices were incubated with PCNA antibody(rabbit, proteintech) at 4°C overnight in a wet box.The subsequent steps were carried out according to the instructions of the immunochromatography kit (Servicebio, China).The slides were scaned under the Digital Pathology Slice Scanner (KFBIO, China).
For immunofluorescence staining, before staining, performed routine dewaxing and rehydration, microwave antigen heat repair, and blocking. Next, primary antibodies were added and samples were incubated overnight at 4 °C. After washing with PBS three times, added the corresponding secondary antibodies, incubated at room temperature in the dark for 50 min, followed by staining the nuclei with DAPI (Solarbio Life Science, China) then observed under the fluorescence microscope (Nikon Eclipse Ti-SR, Japan).The following primary antibodies were used: anti-IDO1(rabbit, HUABIO), anti-CD11C (rabbit, Servicebio), anti-CD80 (mouse, Proteintech), anti-CD40 (rabbit, HUABIO), anti-MHCII(rabbit, HUABIO) , anti-CD4 (rabbit, Servicebio) and anti-CD8 (mouse, Servicebio).
Statistical analyses
All data are presented as means ± SEM. Statistical analyses were performed through SPSS22 (IBM Corp, USA) and GraphPad Prism 9 (GraphPad Software, USA). Data were compared using independent-samples t-test for two groups or one-way ANOVA for multi-group variable comparisons. Statistically significant differences were considered at *p < 0.05, **P < 0.01 and ***P < 0.001.