Cell were cultured in a humidified incubator with an atmosphere of 5% CO2 at 37°C under normal oxygen conditions. Human umbilical vein endothelial cells (HUVECs, #8000, Sciencell, USA) were grown in endothelial cell medium (ECM, # 1001, Sciencell, USA) and only early passages (< p6) were used. CT26 cells were obtained from the Cell Bank of the Chinese Academy of Science s and were cultured in RPMI-1640 containing 10% FBS and 1% penicillin/streptomycin. The STAT3 overexpression plasmid were purchased from GeneChem (Shanghai, CN).
Conditioned medium (CM) preparation
CT26 cells and CAF were confirmed by morphological observation (Fig. S1a) and WB (Fig. S1b). The tumor cell supernatant polarizes the M0 macrophages, which was confirmed by morphological observation and flow cytometry (Fig. S1c-d). After tumour-associated macrophages, cancer-associated fibroblasts, CT26 cells have grown to 80% of the bottom area of the flask, replace with FBS-free medium, at 48h post-treatment, cell suspensions were collected as conditioned medium. The CM was collected after high speed centrifugation and then pass 0.22 μm microporous mebrance, stored at -20˚C. In the same way, the cells were treated with 10 nM of bufalin for 24 hours, and then treated as above to obtain the relevant conditioned medium after drug action.
Tube formation assay.
HUVEC cells were incubated with conditioned medium for 24 hours before tube formation. Matrigel (BD, #356234, USA) was thawed overnight at 4˚C a day in advance, 50 μl of Matrigel was spread in a 96-well plate and polymerized at 37˚C for 30 minutes. Next, 3 × 104 HUVECs in 50 μl ECM were seeded to each well and incubated at 37°C in 5% CO2 for 4 hours, photographed under the microscope.
Cell migration assay
HUVEC cells were incubated with conditioned medium for 24 hours before migration assay. 3×104 HUVECs in 300 μl of serum-free ECM mentioned earlier in the upper chamber and were seeded into a transwell insert (8.0 μm pore size, #353097, FALCON, USA), and allowed to migrate towards 700 μl of complete ECM. The non-migrated cells were removed from the upper part of the transwell with a cotton swab after 6 hours of incubation, and the insert was fixed with 4% paraformaldehyde for 10 minutes at room temperature. Transwell inserts were stained in 500 μl of 0.03% crystal violet solution for 30 minutes at 37℃.Then the insert was immersed and washed three times with PBS solution for 5 minutes each time, dried and photographed under the microscope.
After HUVEC cells were overgrown in a 24-well plate, they were treated with conditioned medium for 24 hours. Then 2×104 HCT116-GFP cells in 300 μl of serum-free ECM were inoculated on the treated HUVEC cells, and then incubated in a 37℃ incubator for 2 hours, washed with PBS three times, and then photographed under a fluorescence microscope.
Total RNA was extracted from HUVEC using TRIzol (Invitrogen). The concentration of total RNA was quantified by measuring the absorbance at 260 nm. For SYBR Green-based quantitative PCR amplification, the reaction were carried out in a 20 μl reaction volume (Applied Biosystems). Using 2−ΔΔCt method to determine the relative expression level of each cell line in each group.
The primer sequences were as follows: VEGF, 5’-TTGCTGCTCTACCTCCAC-3’ and 5’-AATGCTTTCTCCGCTCTG-3’; PDGFA, 5’-AGGCGTCCAGGCAGGTGATC-3’ and 5’-GCTTCTTCCTCGGTGCGTTCC-3’; E-selectin, 5’-ATGTTCAAGCCTGGCAGTTCCG-3’ and 5’-GCAGAGCCATTGAGCGTCCATC-3’; P-selectin, 5’-CGCTCTGGACCAACCCTGTTTC-3’ and 5’-CTCCTGGCTTCTGTGGCTTGTG-3’.
Western Blot (WB)
The proteins separated by SDS-PAGE and subjected to immunoblotting to analyze antibodies (Abcam, USA). Blocked and incubated overnight at 4°C with primary antibodies. The membrane was further probed with horseradish peroxidase-conjugated anti-rabbit/mouse IgG antibody (Cell Signaling Technology, 1:5,000). Using ImageJ software to quantify protein bands.
In Vivo Xenograft Model
To determine the in vivo antiangiogenic activity of bufalin treatment, CT26-LUC cells (2× 106) were injected into the flanks or spleen of male Balb/c mice (6 weeks old). One weeks after injection, bufalin (1 mg/kg) was administered by intraperitoneal (i.p.) injection once every other day for 21 days (flank) or 14 days (spleen). Subcutaneous tumour size was measured every three days and live imaging once a week after the treatment. The estimated tumor volumes (Vs) were calculated by the formula V=W2 ×L×0.5, where W represents the largest tumor diameter in centimeters and L represents the next largest tumor diameter. Tumor-bearing mice were sacrificed after 21 or 14 days of treatment, and tumour tissues, spleen and liver were harvested, weighed, and then immediately fixed in formalin for follow-up experiment.
All experiments conformed to the ethical principles of animal experimentation stipulated by institutional animal care and use committee of Putuo Hospital, Shanghai University of Traditional Chinese Medicine, China.
The tissues were harvested after animals were sacrificed. The procedure of histopathological assay was accomplished by conventional hematoxylin-eosin (H&E) staining in accordance with standard techniques.
2×104 HUVECs were seeded and cultured overnight on microscope coverslips (Thermo Fisher Scientific, Waltham, MA, USA). After treated with CM with or without bufalin for 24 hours as described previously, the cells were washed with PBS twice and fixed in methanol for 15 minutes, then permeabilized with 0.2% Triton X-100 (Beyotime, Shanghai, China) /PBS for 5 minutes and blocked by 3% BSA for 1 hours at room temperature. The coverslips were incubated with primary antibodies at 4°C overnight, and then incubated with secondary antibodies for 2 hours at 37°C (protected from light). Nuclear localization was assessed with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China).
Tissue sections were permeabilized with cold methanol for 5 minutes and incubated with 5% BSA in PBS for 1h. Primary antibodies were applied in blocking buffer and incubated overnight at 4°C. Dye-conjugated secondary antibodies were added in blocking buffer and incubated for 2 hours. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China). Images were taken using Zeiss LSM880 confocal microscopy at the same voltage level and analyzed using ZEN Software.
The tissue was fixed in 10% formalin, embedded in paraffin, and then sectioned (5 mm thick). The IHC of CD31 was performed as follows. The slides were dewaxed and incubated with 3% H2O2 aqueous solution for 10 minutes to quench the endogenous peroxidase activity. The heat-induced antigen recovery method was used to detect the antigen. Incubate the tissue with 5% BSA for 30 minutes at room temperature, and then incubate with the primary antibody in PBS at 4°C overnight. Using the appropriate secondary antibody to apply the indirect avidin-biotin-peroxidase method at room temperature for 30 minutes. EnVision (K4007, Dako) signal enhancement system was used to develop bound antibodies. The sections were stained with Harris hematoxylin, dehydrated and fixed. For quantification, 30 random images (400×) were captured with a microscope (Leica, Wetzlar, Germany) under each microscope.
VEGF in HUVEC cells culture supernatants was evaluated by Human VEGF Quantikine ELISA Kit (R&D, Minnesota, USA) according to the manufacturer’s protocol. VEGF in serum was evaluated by Mouse VEGF Quantikine ELISA Kit (R&D, Minnesota, USA) according to the manufacturer’s protocol.
Each experimental value is expressed as the mean ± SD. Statistical analysis was performed using t-test to evaluate the significance of the difference between the different groups, and the significance was accepted at *p<0.05, **p<0.01 and ***p<0.001. All data points represent the average of three repeated measurements. Spearman rank statistical test and Mann-Whitney test were used for statistical analysis of tissue samples to assess the significance of differences between groups.