Details of animal studies can be found in our previous reports13,15. Briefly, Hepa 1-6 cells (about 2 × 106 cells in 100μl phosphate-buffered saline) were transplanted subcutaneously into 8-week-old female C57BL6 mice (Charles River Laboratories, Wilmington, MA). Body weight, tumor size, and mortality of the mice were monitored daily. After 10-14 days, mice with tumor sizes of 180 mm3 were selected. Tumor volume was examined by using the formula length × width2 × π/6. Each group consisted of seven mice. YIV-906 was administered orally for 7 days (500 mg/kg po, twice per day), while anti-PD1 was administered intraperitoneal on day zero (200ug/mice). In the control groups, mice were administered water orally. On Day 0, YIV-906 was administered 30 minutes prior to anti-PD1 administration. All animal experiments were carried out in accordance with the relevant guidelines and regulations approved Yale University Institutional Animal Care and Use Committee (IACUC) protocol. Animal experimental protocols were approved by Yale University Institutional Animal Care and Use Committee (IACUC). Animal studies were carried out in compliance with the ARRIVE guidelines.
After 4-day treatments, mice were terminated by cervical dislocation two days or four days after initiation of drug treatment (see above). Details of immunohistochemistry protocol can be found in our previous reports13,15. Intestinal and colon tissues were removed, fixed in formalin, embedded in paraffin, and sectioned into 10mm. The sections were mounted on Superfrost slides, dewaxed with xylene, and gradually hydrated. Antigen retrieval was achieved by 10mM Sodium citrate pH6.0 with 0.02% Tween-20 under steaming for 30 minutes. The source and dilution of primary antibody are listed in supplementary methods. The primary antibodies were diluted using Tris-HCl buffer containing 1% BSA and 0.5% Tween-20 and were incubated at room temperature for one hour. As a negative control, a set of slides was processed without primary antibody. Super-picture immunohistochemistry detection kit (Invitrogen, Inc.) was used for detection. The slides were counterstained with hematoxylin and mounted. The antibodies used were: Cleaved Caspase-3 (#9664, Cell Signaling Technology, Inc.), Cleaved Caspase-8 (#9496, Cell Signaling Technology, Inc. Danvers, MA), Cleaved Caspase-9 (#ab52298, Abcam, Cambridge, England), F4/80 (#ab16911, Abcam).
Flow cytometry analysis
Tumor tissues (200mg) were cut into small pieces in 0.5ml RPM1 640 culture medium. Liberase was added to dissociate the connected tumor cells at room temperature for 15 minutes. Dissociated cells were passed through a cell strainer (70um). After spinning down the cells at 1000g centrifugation for 10min, red blood cells were lysed with 1ml BD pharm lyse on ice. Cells were collected at 1000g centrifugation for 10min. 2x106 cells were used for each staining sample. Cells were resuspended in RPM1 640 with 3% FBS. Anti-mouse CD16/CD32 clone 2.4G2 (BD Pharmingen, #553142) was used to block Fc receptors on cells. Total T cells were stained by Anti-CD3-PE (BD pharmingen, clone 145-2c11, #553064) for 30 minutes on ice. Fixation/Permeabilized (eBioscience) was used to fix and permeabilize cells. Then activated cytotoxic T cells was further stained with Anti-Granzyme B-pacific blue (BioLegend, clone GB11, #515408) and T regulatory cells were stained with Anti-FOX3P-APC (eBioscience, clone FJK16s, #17-5773-83). The stained cells were washed and analyzed by flow cytometry LSR II (BD Canto II, New Jersey, USA). Details of flow cytometry analysis can be found in our previous report38.
BMDM or RAW 264.7 cells (American Type Culture Collection) were cultured RPMI supplemented with 5% FBS in 37oC incubator with 5%CO2. 2x106 cells were seeded in 12-well plate. After drug treatment, cells were lysed in 0.3ml protein loading buffer (for 20ml buffer, 10% SDS 4ml, Tris-HCL pH6.8 0.75ml, 10% glycerol 5ml, β-mercaptoethanol 0.5ml, and bromophenol blue) for each well, and sonicated for 30s to break DNA. Then cell extracts were electrophoresed through Mini PROTEAN® TGXTM Precast gels (12%, 15well comb, 15ul/well Cat. #456-1046) in a running buffer (10×, Tris 30g, Glycine 144g, SDS 10g, with DD H2O) and transferred to the nitrocellulose membrane (Bio-Rad Laboratories, Inc) in a transfer buffer (Tris 30g, Glysine 144g, SDS 0.5g). The membrane was blocked and probed in TBS-T buffer (TBST +1% Tween, AB14330-01000, American Bioanalytical) containing non-fat milk 1:5000 (Blotting-Grade Blocker, Cat. #170-0604 Nonfat dry milk). Primary antibodies (PD-1 (D7D5W) XP® Rabbit mAb #84651S Mouse Specific lot:1 Ref: 08/2017) at 1:1000 in TBS-T buffer (TBST +1% Tween, AB14330-01000, American Bioanalytical) were incubated with the membrane for shaking overnight at 4°C. Histone, H3 was used as an internal control for normalization and detected with a monoclonal actin antibody diluted at 1:1000 (H3(D1H2) XP® Rabbit mAb #4499S Ref: 06/2017). After washing with TBS-T three times, each time for 5min, the membranes were then further incubated with goat anti-rabbit IgG-HRP SC-2004, lot #B1711 HRP conjugated 1:5000, and incubated in room temperature for 1 hour. Then the membrane was washed with TBS-T three times again. Stable Peroxide Solution 1ml (SuperSignalTM West Pico PLUS, Prod#1863097) and Luminol/Enhancer Solution 1ml (SuperSignalTM West Pico PLUS, Prod#1863096) were used for visualizing, and scan with densitometer. Antibody list: PD-1 (D7D5W) XP® Rabbit mAb #84651S Mouse Specific lot:1 Ref: 08/2017 (Cell signaling), Anti-PD-L1 antibody [EPR20529]ab213480, Arginase-1 (D4E3MTM) XP® Rabbit mAb#93668 (Cell signaling), iNOS Antibody (Mouse Specific) #2982 (Cell signaling), Jak1(6G4)Rabbit mAb#3344 (Cell signaling), P-Jak1 (Y1034/1035)(D7N4Z) Rabbit mAb#74129 (Cell signaling), Jak2(D2E12) XP® Rabbit mAb #3230(Cell signaling), P-Jak2(Y1008)(D4A8) Rabbit mAb #8082 (Cell signaling), Stat1 Antibody#9172(Cell signaling), Phospho-Stat1(Tyr701)(D4A7)Rabbit mAb#7649(Cell signaling), Stat2(D9J7L)Rabbit mAb#72604(Cell signaling), Phospho-Stat2(Tyr690)-R sc-21689 #K1609(SantaCruz), Stat6(D3H4)Rabbit mAb#5397(Cell signaling), Phospho-Stat6(Tyr641)(D8S9Y)Rabbit mAb#56554(Cell signaling), IRF-1(D5E4) XP® Rabbit mAb #8478(Cell signaling), IRF-4(D9P5H)Rabbit mAb#15106(Cell signaling).
Quantitative real time RT-PCR
Total RNA was extracted with TRIzol reagent (Invitrogen, California, USA). The aqueous phase was collected and then one volume of ethanol was added, following the manufacturer’s instructions. Before centrifuging, this slurry was added to a column (miRNeasy, Qiagen, Venlo, Limburg) for further extraction and simultaneous DNA digestion (RNase-Free DNAse set, Qiagen). cDNA was synthesized using random primers and reverse transcriptase MMLV (New England Biolabs, Ipswich, MA). qPCR assays were performed using iTaq™ SYBR® Green Supermix and the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). Primer sets were listed in the supplementary method. The mouse primer pair sequences are attached in supplementary methods and β-actin serves as internal control (please see supplementary methods for primer sequences). Relative expression of target genes against β-actin was expressed as 2-ΔCt and fold differences was calculated as expressed mRNA of YIV-906 and or antiPD1-treated samples against untreated samples. Primers sequences are listed (Table S1) and the rest of primers that could be found in our previous reports 13,15.
Cytokine analysis by cytometric bead array
Animal plasma and tumor tissue of YIV-906 and or antiPD-1-treated mice and control mice were collected after 96 hours following the treatments. Culture medium of untreated and YIV-906-treated BMDMs were collected after 24 hours exposure. Determination of cytokine expression (IL-6, MIP-1a, IL-5, IL-17A, IL-12p70, TNFa, IL-1B, IL-10, MIG, IFN-r, MCP-1, G-CSF) was performed using cytometric bead array flex set kit by flow cytometry (BD Canto II, New Jersey, USA) according to the manufacturer’s instructions (BD biosciences, UK)15.
Isolation of Bone Marrow Derived Monocytes (BMDMs) and macrophage differentiation
Bone marrow cells were collected from tibias and femurs of 10-week-old C57Bl/6 mice were cultured with complete RPMI-1640 medium (supplemented with 5% Fetal Bovine Serum and 1% Penn/Strep) in the presence of murine M-CSF (10 ng/ml) for 7 days to allow differentiation of monocytes into macrophages39. Macrophage were cultured in 5% FBS RPMI-1640 medium with IFNγ (10 ng/ml) to induce polarization to M1-like macrophage while M2-like macrophage were induced by IL4 (20 ng/ml).
IDO Activity Assay
2x106 HEK293 cells were transfected with mouse IDO (2ug/10cm plate) (OriGene Technologies, Inc., Rockville, MD, Ido1 (NM_008324)) or without IDO DNA as negative control using lipofectamine 3000 for 48h. For one plate, 1ml PBS was used to collect cells into a 2ml tube. Cells were spin down at 3500 rpm 1min. Cells were then sonicated in ice cold 1ml PB buffer pH6.5. Cell lysis was clarified by centrifuging at 12000rpm for 5min at 4oC. 25ul cell lysis will be mixed with 25ul herbal extract at desired concentration and 50ul reaction buffer: (PB buffer 100mM pH 6.5) every 10ml add 70mg Vitamin C, 10ul methylene blue (2.5%), 100ul catalase (20mg/ml), 250ul of 500mM L-tryptophan. Mixture was incubated for 1.5h at 37oC in water bath. Trichloroacetic acid 30% 25ul will be added and incubated at 50oC for 1hr. Finally, Enrlich 0.8% (80mg/10ml in acetic acid) 100ul was added. Absorbance at 540nm will be measure40. Optical density at 540nm (Yellow) has positive correlation to the amount of Kynurenine40.
Each tumor sample were homogenized in 200 μL acetronitrile/methanol/water(2/2,1, v/v/v) and 1 mm glass beads (BioSpec Products, Bartlesville, OK) for 30 s at 3500 rpm twice. The homogenate was then centrifuged at 12000 rpm for 15 min at 4 oC. The supernatant was dried down in a Speedvac. The residue of each tumor sample was re-dissolved in 100 µL of acetonitrile, and vortexed at 3000 rpm for 3 min. The solution was then centrifuged at 12,000 rpm at 4 ℃ for 15 min, and 2 μL supernatant was injected into the UPLC-QTOF system for analysis. All sample analyses were performed on an ACQUITY ultra-performance liquid chromatography (UPLC) system coupled with a quadrupole-time of flight (Q-TOF) MS instrument (UPLC Xevo G2-XS QTOF MS, Waters Corp., Milford, MA, USA) with an electrospray ionization (ESI) source. Separation was carried out on a Waters ACQUITY BEH C18 column (2.1 X100 mm id, 1.7 μ m) with a guard column (Waters ACQUITY BEH C18 column (2.1 X5 mm id, 1.7 μ m)). The mobile phase consisted of acetonitrile (A) and water containing 0.1% formic acid (B) using a gradient elution of 5% A at 0–2 min, 5–10% A at 2–3 min, 10–17% A at 3–10 min, 17–30% A at 10–15 min, 30–40% A at 15–20 min, 40–80% A at 20–25 min, 80% A at 25–30 min, 80–5% A at 30–31 min, and 5% A at 31–35 min. The flow rate was 0.3 mL/min. Mass spectrometry was performed on a Water Xevo G2-XS QTOF. The scan range was from 50 to 1000 Da. For negative electrospray mode, the capillary voltage and cone voltage were set at 2.5 kV and 60 V, respectively. The desolation gas was set to 800 L/h at a temperature of 500 ℃; The cone gas was set to 50 L/h at a temperature of 120 ℃; Data acquisition was achieved using MSE, and the collision energy was 15-60 V.
Data were analyzed by one- or two-way analysis of variance (ANOVA) (GraphPad Prism 7), correlation analysis (GraphPad Prism 7) and Student’s t test (Microsoft Office Excel). The difference was statistically significant when P < 0.05.