Human HCC cell line HCCLM3 was obtained from Live Cancer Institute of Zhongshan Hospital (Shanghai, China). Mouse HCC cell line Hepa1-6 was purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Science (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin (all from Life Technologies, USA). All cells were kept at 37 °C in a humidified atmosphere of 5% CO2 incubator.
CD8+NKG2D+ cells were generated using our group’s protocol . In brief, human peripheral blood lymphocytes were isolated from healthy volunteers. Cells were cultured in GT-T551 (SK551S, TaKaRa, Japan) medium supplemented with 2% autologous serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2000 IU/ml recombinant human IFN-γ (Chemo Wanbang Biopharma, Shanghai, China) for 24 h. To promote the expression of NKG2D, OK432 (Chugai Pharmaceutical, Chome, Japan), an immunotherapeutic agent prepared from inactivated Streptococcus pyogenes, was added into the culture medium at the concentration of 10 mg/ml . On 2nd day, cells were transferred to a flask pre-coated with anti-CD3 antibody (T&L Biological Technology, Beijing, China). GT-T551 medium supplemented with 700 IU/ml recombinant human IL-2 (BD Biosciences, USA) was used for further culture. After 2 weeks, cells were subjected to flow cytometry to control the phenotype and quality.
Frankincense (#71202000) and myrrh (#71202500) were purchased from Jiangsu Province Hospital of Traditional Chinese Medicine (Nanjing, China). Recombinant human TNF-α (HNAE-10620), mouse TNF-α (MNAE-50349), human IL-6 (HNAE-10395), and mouse IL-6 (MNAE-50136) were all obtained from Sino Biotechnology (Beijing, China).
Water-decocting extract of frankincense and myrrh
The extract of frankincense and myrrh was prepared as previously described . Briefly, powered resin of frankincense (1.0 kg) or myrrh (1.0 kg) was extracted with water (2 × 10 L) under reflux for 1 h. The extraction was repeated twice. Then the extracted solution was boiled in a reflux condensation device for 2 h, cooled to room temperature (RT), and centrifuged at 3000 rpm for 15 min to remove the residue. Finally, the supernatant was evaporated in a rotary evaporator for 12 h to obtain the powder of frankincense or myrrh.
To prepare the solution, 100 mg of frankincense powder and 100 mg of myrrh powder were dissolved in 5 ml cell culture medium (for in vitro experiments) or PBS (for in vivo therapy) in water bath at 90 °C overnight. Then the supernatant was obtained after centrifugation at 12,000 rpm for 30 min followed by filtration through a 0.22 μm filter.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
MTT (M5655, Sigma, USA) (5 mg/ml, 20 μl) was added into each well in 96-well plate and incubated for 3 h at 37 °C. Then, the supernatant was discarded. 100 μl of isopropyl alcohol (12090611516, Nanjing Chemical Reagent Co., China) was then added and agitated for 20 min to dissolve the crystallization. The absorbance was measured using a Multimode Reader (SMP500-13497-JWYK, Molecular Devices, USA) at 570 nm. Cell viability was calculated as the ratio of the absorbance of treated cells to that of controls.
Trypan blue exclusion test
Harvested cells were stained with 0.2% trypan blue (C3601-2, Beyotime, China). Cell numbers and viability were determined with a Countstar Automated cell counter (Inno-Alliance Biotech, USA).
To test the oncolytic effect of CD8+NKG2D+ cells, HCCLM3 cells stably expressing luciferase (HCCLM3-luciferase) were used as target cell. The viability of HCCLM3-luciferase cells was determined by luciferase assay according to manufacturer’s instructions (E1500, Promega, USA). After co-culture in a black 96-well plate, cell lysis buffer was added into each well for 15 min. Luciferase assay substrate was added and then the plate was subjected to GloMax 96 microplate luminometer (Promega, USA).
Western blot analysis
Cells were lysed in radio-immunoprecipitation assay buffer containing a protease inhibitor cocktail (11873580001, Roche, Switzerland). The protein concentration was determined using BCA Kit (P10010, Beyotime, China). Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes (03010040001, Roche, Switzerland). The membranes were blocked, incubated with the specific primary antibodies, washed, and followed with incubation with appropriate horseradish peroxidase-conjugated secondary antibodies. Signals were detected using an enhanced chemiluminescence reagent (WBKLS0500, Millipore, USA) and subjected to the Alpha Innotech Fluor Chem-FC2 imaging system (ProteinSimple, USA). The gray value was analyzed with Software Image J (freely available at https://imagej.nih.gov/ij/download.html). Antibodies used were as follows: anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (MB001, Bioworld, USA; 1:5000 diluted), anti-IκBα (4814S, Cell Signaling Technology, USA, 1:1000 diluted), anti-phospho-IκBα (2859S, Cell Signaling Technology, USA; 1:1000 diluted), anti-STAT3 (9139S, Cell Signaling Technology, USA; 1:1000 diluted), and anti-phospho-STAT3 (9145S, Cell Signaling Technology, USA; 1:1000 diluted).
Quantitative RT-PCR analysis of mRNA expression
Total cellular RNA was extracted with TRIzol (15596-026, Thermo Scientific, USA), and was reverse-transcribed using the synthesis system (RR036A, TaKaRa, Japan). Then SYBR green PCR master mix (04913914001, Roche, Switzerland) reagent was used according to the manufacturer’s protocol and the PCR was performed using the Real-Time PCR system (Viia7, Thermo Scientific, USA). Gene expression was calculated with the comparative threshold cycle (C
) method and normalized to the endogenous levels of GAPDH. Primer sequences used were as follows:
human GAPDH forward 5′- CCATGTTCGTCATGGGTGTGAACCA-3′ and reverse 5′-GCCAGTAGAGGCAGGGTGATGTTC-3′;
human TNF-α forward 5′- CCCAGGGACCTCTCTCTCTAATC-3′ and reverse 5′- ATGGGCTACAGGCTTGTCACT-3′;
human IL-6 forward 5′- GGTACATCCTCGACGGCATCT-3′ and reverse 5′- GTGCCTCTTTGCTGCTTTCAC-3′;
mouse gapdh forward 5′-TCTCCTGCGACTTCAACA-3′ and reverse 5′- TGTAGCCGTATTCATTGTCA -3′;
mouse tnf-α forward 5′- ACGCTCTTCTGTCTACTGA -3′ and reverse 5′- GCCATAGAACTGATGAGAGG-3′;
mouse il-6 forward 5′- CCACCAAGAACGATAGTCA -3′ and reverse 5′- TTGTCACCAGCATCAGTC -3′.
Mice, tumor engraftment, and in vivo experiments
4–6-week-old male C57BL/6 mice and Balb/c nude mice were obtained from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). Mice were kept in the conditional microenvironment (temperature 22 ± 2 °C, humidity 60 ± 10%, 12/12 h light/dark cycle).
To establish HCC xenograft models, each mouse was subcutaneously injected with 5 × 106 Hepa1-6 cells or 1 × 107 HCCLM3 cells suspended in 200 μl PBS containing 10% Matrigel Matrix (354234, Corning, USA). Therapy started on day 7 post tumor cell inoculation, when mice showed visible tumor nodules (4–6 mm in diameter). FM was given daily by oral gavage at 60 mg/kg until the therapy stopped at the indicated time point.
For the CD8 and NK cells depletion experiment, 500 μg of anti-CD8α mAb (clone YTS 169.4, BE0017, BioXCell, USA) or anti-NK mAb (clone PK136, BE0036, BioXCell, USA) was injected intraperitoneally every 2 weeks from 1 day before therapy. Depletion of CD8 or NK cells was confirmed by fluorescence-activated cell sorting (FACS) analysis of peripheral blood.
For all the in vivo experiments, tumor volume was monitored every 2–3 days by caliper measurement and calculated by length × width2/2. Mice were euthanized when tumor volume reached 2 cm3, or when mice appeared moribund.
For CD8+NKG2D+ cells analysis, cells were harvested and stained with anti-CD3-FITC (E10472-1633, eBioscience, USA), anti-CD8-PE (561946, BD, USA), anti-NKG2D-APC (130-099-216, Miltenyi Biotec, Germany), anti-CD27-PE (560985, BD, USA), and anti-CD57-APC (560845, BD, USA) antibodies. To detect the infiltration of CD8 or NK cells in the tumor mass, tumors were removed and crushed into single cell suspension. Then cells were stained with mouse anti-CD8-PerCp (46-0083-80, eBioscience, USA) or anti-NK1.1-FITC (553164, BD, USA) antibodies. To confirm the depletion of CD8 or NK cells, cells were isolated from peripheral blood and stained with anti-CD8-PerCp (46-0083-80) or anti-NK1.1-PerCp antibodies (45-5941-82) (all from eBioscience, USA). The staining was performed on ice for 30 min. Samples were acquired on the FACS Calibur (BD, USA) and data was analyzed using FlowJo software (Version 8.5.3, Tree Star Inc., USA).
IFN-γ enzyme-linked immunosorbent spot (ELISpot) assay
Immunity activity in the TME was measured with mouse IFN-γ ELISpot Kit (3321-2A, Mabtech, Sweden) according to the guidelines. In brief, the plate was coated with mouse INF-γ capture antibodies and kept overnight at 4 °C in the dark. Cells isolated from tumor masses were seeded at a density of 1.5 × 105 cells/well. After 24 h incubation at 37 °C in a humidified atmosphere of 5% CO2 incubator, cells were removed and the wells were rinsed with PBS. The biotinylated detection antibodies against IFN-γ were then added and the plate was incubated for 2 h at RT. Then the wells were rinsed with PBS followed by incubation with Streptoavidin-ALP for 1 h. The substrate BCIP/NBT-plus was then added. The reaction was stopped by washing wells with tap water when spots emerged. The plate was subjected to ELISpot reader system (Autoimmun Diagnostika GmbH, Germany) to detect spots number and activity. The activity of spot was characterized as the average of spot size and intensity for each well.
The results are presented as mean ± SD. Statistical analyses were performed using Student’s t test. Animal survival is presented using Kaplan–Meier survival curves and was statistically analyzed using log rank test (GraphPad Prism version 6). p value < 0.05 was considered to be statistically significant.