Mice and cell lines
C57BL/6, TLR7−/−, MyD88−/−, and immunodeficient nude mice (8–12 weeks old, female) were kept under specific pathogen-free conditions with water and food given ad libitum.
Hepa1-6 hepatoma cell line (CRL-1830) and EG7 thymoma cell line (CRL-2113) used in the present study were maintained in DMEM (Meditech) supplemented with 10% FBS (Hyclone) and 2 mM l-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-mercaptoethanol at 37 °C in a humidified incubator with 5% CO2.
Cell proliferation assay
Hepa1-6 cells (5 × 103/well) were seeded in triplicate in round-bottomed 96-well plates in complete DMEM (0.2 ml/well) and incubated in the presence or absence of the indicated concentration of CT at 37 °C in a CO2 incubator for 48 h. Tritiated thymidine (3H-TdR, New England Nuclear, North Billerica, MA) was added at 0.5 μCi/well for the last 4 h of culture. The cultures were harvested on a membrane using a 96-well automatic harvester (INOTECHAG IH-280, Dottikon, Switzerland). The filter mat and scintillation fluid were placed into a bag, which was sealed and assessed for 3H-TdR incorporation (CPM) using an automatic MicroBeta counter (Wallac).
A CCK8 (Sigma-Aldrich, St. Louis, MO, USA) assay was performed to assess cell viability in Hepa1-6 cells treated with different concentrations of cucurbitacin I, according to the manufacturer’s instructions.
Cell cycle and apoptotic assays by flow cytometry (FACS)
The cycle distribution was analyzed by FACS analysis after staining with propidium iodide (PI) solution. Briefly, Hepa1-6 cells were treated with CT for 48 h and fixed with 75% ethanol. Next, the cells were incubated with 500 µL of a solution containing 50 µg/mL PI and 0.1% Triton X-100 in the dark and analyzed by FACS. To further analyze the apoptosis induction effects of CT, the cell apoptosis was detected as described previously . After treatment with CT for 48 h as described above, both attached cells and floating cells were harvested, stained with PI and Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s instructions, and analyzed by FACS.
Generation and treatment of bone marrow-derived macrophages (BMM)
Mouse BMM were generated as described previously . To measure surface marker of mouse BMM, adherent cells were grown to confluence in 24-well plates (at about 5 × 105/well) in a CO2 incubator in the presence or absence of various reagents at concentrations specified for 48 h before immunostaining.
Immunostaining and FACS
Cultured BMMs were detached as described by Han et al. . The BMMs were resuspended at 1.0 × 106 cells/1 ml in PBS and then incubated with FITC-anti-mouse CD86 (clone GL1, TONBO Biosciences, San Diego, CA), PE-anti-mouse CD80 (clone 16-10A1, TONBO), Alexa Fluor® 647-anti-mouse CD206 (clone MR5D3, BD Pharmingen). For immunoprofiling of tumor-bearing mice, single cell suspensions of Hepa1-6 tumors or the draining lymph nodes (dLN) at 1 × 106 cells/sample were immunostained with a combination of some of the following antibodies, such as FITC-anti-mouse CD4 (clone GK1.5, Tonbo), PE-anti-mouse CD11b (clone M1/70, BD), PerCP-Cy5-anti-mouse-B220 (clone RA3-6B2, Tonbo), APC-anti-mouse-CD11c (clone HL3, BD), eFluor450-anti-mouse CD45 (clone 30-F11, eBioscience), APC-Cy7-anti-mouse-CD8 (clone 53-6.7, Tonbo), eFluor450-anti-mouse CD44 (clone IM7, eBioscience), APC-anti-mouse CD62L (clone MEL-14, eBioscience), eFluor450-anti-mouse CD8 (clone 53-6.7, eBioscience), and eFluor660-anti-mouse CD107a (clone 1D4B, eBioscience). Data of the stained samples were acquired using an LSR II flow cytometer (BD) and analyzed using the software FlowJo (Tree Star Inc., Ashland, OR).
Total RNA isolation and cDNA synthesis
Total RNA from mouse BMM was isolated according to the protocol of RNeasy Micro Kit (Qiagen, Hilden, Germany, Cat: 74,004). Total RNA from Hepat1-6 tumors was extracted using TRIzol (Invitrogen, Cat: 1,559,026) and purified using the RNeasy Micro Kit RNA. The purity and concentration of isolated RNA samples were determined by measuring absorption at 260 nm wavelength using an NanoDrop ND-1000 spectrometer (Nanodrop Technologies, Wilmington, DE). cDNA was then synthesized from the RNA using the RT2 First Strand Kit (Qiagen, Cat: 330,401).
Quantitative real-time polymerase chain reaction (qPCR)
qPCR was performed using a LightCycler 480 II (Roche Life Sciences, Branford, CT, USA), the RT2 SYBR Green/ROX qPCR Master Mix (Qiagen, Cat: 330,523), and the specific primer pairs (sTable 1). The cycling conditions for the qPCR amplification 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. The transcript levels were then normalized to that of a house-keeping gene (i.e., β actin or GAPDH) and then the data were analyzed using the ΔΔCT method through Qiagen’s GeneGlobe Data Analysis Center.
Western blot analysis
Western blotting analysis was performed as described previously . Briefly, cells treated with CT for 48 h were lysed in RIPA buffer (Beyotime, Beijing, China). The protein concentrations were quantitated with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Frederick, MD, USA). Total protein 30 µg/lane were loaded onto SDS–polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ). The membranes were blocked and incubated with (1:1000) rabbit anti-p-STAT3 (Cell Signaling, Cat: 9145L, Tyr 705), anti-STAT3 (Cell Signaling, Cat: 4904S), anti-p-JAK2 (Cell Signaling, Cat: 8082), anti-JAK2 (Cell Signaling, Cat: 3230), anti-I-kBα (Cell Signaling, Cat: 9242), anti-GAPDH (Cell Signaling, Cat: 2118) overnight at 4 °C and (1:2000) horseradish peroxidase-conjugated secondary antibody (Cell Signaling, Cat: 70,741) for 1 h at room temperature. The protein bands were visualized using the G-BOX Chemi system (Syngene, Frederick, MD).
TNFα and IL-12p70 in the culture supernatants were quantitated by human and mouse Customary Cytokine Arrays following the manufacturer’s protocol (MesoScale Diagonostics, Rockville, MD).
Mouse tumor model and treatment
Female mice (C57BL/6, n = 5–10, 8–12-weeks old) were injected subcutaneously with 0.1 ml PBS containing Hepa1-6 (2 × 107/ml) or EG7 (2 × 105/ml) or into left or right flank regions as previously reported [16, 17]. The appearance and size of tumors, as well as the mouse body weight, were monitored twice weekly. The length (L) and width (W) of tumors were measured with a caliper. Tumor size was calculated by the formula: (L × W2)/2. Hepa1-6-bearing mice were treated with intratumoral (i.t.) injection of CT alone or in combination with anti-PD-L1 as indicated. In some experiments, tumor-bearing mice were simultaneously treated with intraperitoneal (i.p.) administration of 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). For the analysis of leukocyte infiltration in Hepa1-6 tumor tissue, the tumors were removed and dissociated into single cell suspensions using an enzymatic cocktail consisting of collagenase I, II, and VI, deoxyribonuclease I, and elastase as previously reported .
Student’s t tests were performed for parametric comparisons between two groups. A two-way analysis of variance (ANOVA) was used to analyze tumor volume difference between groups. Differences in survival curves were considered statistically significant by the log-rank survival analysis. All experiments were performed at least three times, and the results of one representative experiment or the mean of multiple experiments are shown. All statistical analyses were conducted using GraphPad Prism software (version 7, GraphPad Software, San Diego, CA).