Compounds purified from various TCMs, including CT, were obtained from the National Institutes for Food and Drug Control (Beijing, China). CT was dissolved at a stock concentration of 15 ~ 20 mg/ml in DMSO and diluted into physiologic solutions or medium for experiments. LPS (E. coli O55:B5) was from Sigma. Anti-PD-L1 (clone 10F.9G2) and control rat IgG2b (clone LTF-2) were purchased from BioXCell (West Labanon, NH).
A549 or LLC cells were seeded into a 96-well flat-bottomed tissue culture plate at 4 × 103/well in appropriate medium and cultured in a CO2 incubator (37 °C humidified air-containing 5% CO2) overnight. After adding CT, the cells were incubated for 48 h and pulsed with tritiated thymidine (3H-TdR, New England Nuclear, North Billerica, MA) at 0.5 µCi/well for the last 4 h. At the end of culture, the cells were collected onto a membrane with a 96-well harvester (INOTECHAG IH-280, Dottikon, Switzerland) to measure 3H-TdR incorporation (CPM) using an automatic MicroBeta counter (Wallac). The change in the percentage (%) of cell proliferation was calculated as: % Proliferation = (CPM with compound − CPM blank)/(CPM without compound − CPM blank) × 100. The concentration at which 50% of the proliferation was inhibited (IC50) was calculated using GraphPad Prism.
DC generation and treatment
Mouse dendritic cells (DCs) were generated by culturing mouse hematopoietic progenitors isolated from C57BL/6, TLR4−/−, or MyD88−/− mice as previously reported . Mouse DCs at 5 × 105/ml in mGM-CSF-containing RPMI 1640 medium were cultured in a CO2 incubator with CT at indicated concentrations for specified time periods before harvesting culture supernatants and DCs for cytokine measurement and cytometric/signaling analysis, respectively.
Detection of apoptosis
LLC cells in a 12-well plate (3 × 105/ml/well) were cultured for 24 h with CT or NaZ3 (for positive control) at indicated concentrations. The cells were harvested by treating with 0.25% trypsin-2.21 mM EDTA, washed three times, and stained with an apoptosis detection kit (BMS500FI/300, eBioscience) consisting of FITC-conjugated annexin V and propidium iodide (PI) following the vendor’s recommendation. The stained samples were assayed using an LSR II (BD) flow cytometer and analyzed using FlowJo.
LLC cells with 70 ~ 80% confluency were washed and serum-starved in a CO2 incubator in DMEM medium containing 0.2% FBS for 48 h for synchronization. The synchronized LLC cells were plated into a 6-well plate at 5 × 105/well in DMEM medium (10% FBS) containing various concentrations of CT and cultured in a CO2 incubator for 24 h. The resultant cells were washed twice with PBS and fixed in 70% ethanol for 30 min at 4 °C. After fixation, the cells were washed twice with PBS and treated with 50 µl/tube of 100 µg/ml of ribonuclease for 30 min at room temperature. Finally, 200 µl of PI at 50 µg/ml was added into each tube and the cells were analyzed using an LSR II flow cytometer.
Treatment of LLC for signaling analysis
LLC cells were serum-starved in a CO2 incubator in DMEM medium containing 0.1% FBS overnight before they were treated with various concentrations of CT for 24 h. The treated cells were solubilized in 1 × SDS–PAGE sample buffer at 107/ml, boiled for 5 min, and stored at − 20 °C until use.
SDS–PAGE and western blot
Samples and pre-stained standard separated on a 4–12% NuPAGE™ (Invitrogen) were transferred onto a piece of Immobilon™ membrane (Millipore, Bedford, MA). The membranes were rinsed with TBS-T (TBS containing 0.05% Tween 20), blocked with 5% nonfat dry milk at room temperature for 1 h, and incubated with appropriately diluted (1:500 ~ 2000) 1st antibodies overnight at 4 °C. The first antibodies were rabbit IgG purchased from Cell Signaling (Beverly, MA), including anti-I-κBα (#9242), anti-GAPDH (#2118), anti-phospho-p44/42 (#9101, Thr202/Tyr204), anti-p44/42 (#9102), anti-phosphorylated p38 (#9211, Thr180/Tyr182), anti-p38 (#9212), anti-phosphorylated JNK (#9251, Thr183/Tyr185), anti-JNK (#9252), anti-phosphorylated p53 (#9284, Ser15), anti-Cdc2 (#77,055), and anti-cyclin B1 (#4138). After washing three times with TBS-T, the membranes were reacted with 1:2000 diluted HRP-conjugated goat anti-rabbit IgG (Cell Signaling, #70,741) for 1 h at room temperature, washed, and developed in SuperSignal® West Dura Extended Duration Substrate (Thermo Fisher Scientific Inc., Hanover Park, IL). The images were collected using the G BOX Chemi systems (Syngene, Frederick, MD).
TNFα, IL-1β, IL-10, and IL-12p70 in the culture supernatants were quantitated by human and mouse Customary Cytokine Arrays following the manufacturer’s protocol (MesoScale Diagnostics, Rockville, MD).
LLC mouse model, treatment, and tumor tissue processing
C57BL/6 mice (female, 8 ~ 10-week-old, n = 5) were implanted subcutaneously with 0.2-ml sterile PBS containing 5 × 106/mouse of LLC into the right flank. The appearance and growth of tumors were monitored twice a week. The greatest longitudinal diameter (length) and the greatest transverse diameter (width) of a palpable tumor were measured to the nearest 0.1 mm using a caliper. Tumor volume (mm3) was calculated by the formula Tumor volume = (length × width2)/2. LLC-bearing mice were treated every other day, starting on day 7, with intratumoral (i.t.) injection of CT alone or in combination with anti-PD-L1 at various doses for 2 weeks. In experiments determining the contribution of lymphocytes to the development of anti-LLC immune defense, LLC-bearing mice were also simultaneously treated intraperitoneal injection of 0.2 ml PBS containing 200 µg of either control rat IgG (clone 2A3, BioXcell, West Lebanon, NH), anti-mouse CD4 (clone GK1.5, BioXcell), anti-mouse CD8α (clone 53-6.72), or anti-mouse NK1.1 (clone PK136, BioXcell). In accordance with the institutional guideline, mice with big tumors (volume > 2000 mm3) undergoing necrosis were considered morbid and euthanized.
Tumors resected from LLC-bearing mice treated with CT alone or in combination with anti-PD-L1 were used for RNA extraction using TRIZol solution (Invitrogen). Alternatively, the tumors were dissociated into single-cell suspensions using an enzymatic cocktail consisting of collagenase I, II, and VI, deoxyribonuclease I, and elastase as previously reported .
Immunostaining and flow cytometry
DCs suspended in FACS buffer (PBS containing 0.5% BSA and 0.05% NaN3) were blocked with 2% mouse serum on ice for 20 min and stained with various combinations of fluorophore-conjugated antibodies against human or mouse DC surface markers on ice for 30 min in the dark. Mouse DCs were stained with FITC-anti-mouse CD86 (clone GL1, TONBO Biosciences, San Diego, CA), PE-anti-mouse CD80 (clone 16-10A1, TONBO), Pacific Blue-anti-mouse CD83 (clone Michel-19, BD), and APC-anti-mouse I-A/E (clone M5/114.15.2, eBioscience). Single LLC tumor cell suspensions were stained with FITC-anti-mouse CD4 (clone GK1.5, Tonbo), PerCP-Cy5-anti-mouse-B220 (clone RA3-6B2, Tonbo), APC-anti-mouse-CD11c (clone HL3, BD), eFluor450-anti-mouse CD45 (clone 30-F11, eBioscience), and APC-Cy7-anti-mouse-CD8 (clone 53 − 6.7, Tonbo). Data of the stained samples were acquired using an LSR II flow cytometer (BD) and analyzed using the software FlowJo.
Total RNA isolation and cDNA synthesis
RNA samples from LLC tumors were isolated by a combination use of TRIzol (Invitrogen, Cat: 1,559,026) and an RNeasy Micro Kit RNA (Qiagen, Hilden, Germany, Cat: 74,004). The purity and concentration of the extracted RNA was assessed and measured using absorption at the 260 nm wavelength with a Nanodrop ND-1000 spectrometer (Nanodrop Technologies, Wilmington, DE). Next, the cDNA was made from the isolated RNA using the RT2 First-Strand Kit (Qiagen, Cat: 330,401).
Quantitative real-time polymerase chain reaction (qPCR)
The expression of target mouse genes was determined by qPCR using a LightCycler 480 II (Roche Life Sciences, Branford, CT, USA), RT2 SYBR Green/ROX qPCR Master Mix (Qiagen, Cat: 330,523), and the specific primer pairs for CXCL9 [Qiagen, Cat: PPM029723-200], CXCL11 [Qiagen, Cat: PPM03192C-200], Granzyme B [Qiagen, Cat: PPM05303F-200], Perforin [Qiagen, Cat: PPM34456B-200], IFNγ [Qiagen. Cat: PPM03121A-200], IL-10 [Qiagen, PPM03017C-200], and GAPDH [IDT, Cat:135,048,676]. The cycling conditions for the qPCR were: hot start for 10 min at 95 °C; amplification for 40 cycles at 95 °C for 15 s, 55 °C for 35 s, and 72 °C for 30 s; and cool down for 2 min at 37 °C. Finally, the expression levels were normalized to those of GAPDH and the data were analyzed using the ΔΔCt method through Qiagen’s GeneGlobe Data Analysis Center.
All experiments were performed at least three times and the results of one representative experiment or the mean of multiple experiments are shown. The difference between groups in terms of cytokine production was determined by Student’s t test. Differences in the in vivo tumor growth were determined by Repeated Measures of ANOVA.