Helicobacter strain and infection experiments
H. pylori strain SS1 was cultured in H. pylori fluid nutrient medium (HB8647, Hopebio, QingDao, China) under microaerobic conditions (N2: 85%; O2: 5%; CO2: 10%) at 37°C. H. pylori strain SS1 was collected in phosphate buffered saline PBS (Gibco, ThermoFisher, USA) and male BALB/ c mice (4-6 weeks of age) were fasted for 6H and inoculated by oral gavage of the bacterial suspension (1 ~ 2 x109CFU/mL, 100μl per mice) every other day for 3 days. Non-infected control groups were given PBS alone.
Total bone marrow transplantations (BMT)
Balb/c mice were lethally irradiated using an x-ray machine. 24 h post-irradiation, mice were injected i.v. with unfractionated bone marrow cells harvested aseptically from age-matched lsltdtomato- female mice. Following transplantation, mice received antibiotics for 4 wk in water (Enrofloxacin; 0.2 mg/ml). After three months of chronic H. pylori infection, engraftment of lsltdtomato marrow-derived cells was tracked with lsltdtomato staining.
BM-MSCs extraction and transplantation
After 3 months of chronic H. pylori infection, GFP fluorescent-labeled BM-MSCs transplantation model was established. BM-MSCs were obtained by femoral lavage of 4-week-old male GFP transgenic BALB/c mice. Cells were cultured and identified by flow cytometry for cell surface antigens including CD45, CD73, CD105, Stem cell antigen-1 (Sca1), CD11b (antibodies all from BD-Pharmingen, PaloAlto, CA) (Fig.S1). 2x 106 collected BM-MSCs from GFP mice was resuspended and washed in PBS then injected into the serosal layer of mice gastric antrum using a 50μl Hamilton syringe. After 3 months, the level of engraftment and migration was assessed by confocal microscope observation of GFP fluorescence in gastric tissues.
HUVEC extraction and identification
Human umbilical vein endothelial cells (HUVEC) were obtained from human umbilical vein using Type I collagenase Hank’s solution. HUVEC were cultured in Endothelial Cell Medium (ECM) (cat. No.1001, ScienceCell, San Diego, California, USA) containing 25 ml of fetal bovine serum (FBS, Cat. No. 0025), 5 ml of endothelial cell growth supplement (ECGS, Cat. No. 1052) and 5 ml of antibiotic solution (P/S, Cat. No. 0503). Cells were identified by immunofluorescence staining of anti- von Willebrand factor (VWF) (Bs-0586R, Bioss) and anti-CD31(ab9498, Abcam) for cell surface antigens (Fig.S2).
Cell culture, animals and patient samples
The GC cell lines SGC7901 used in our study were purchased from American Type Culture Collection (ATCC). BALB/c mice and BALB/c nude mice were purchased from the Beijing Huafukang Biological Co., Ltd and Beijing Viton Lihua Biological Co., Ltd., and housed under standard laboratory conditions in Experimental Animal Center of Tongji Medical College, Huazhong University of Science & Technology. The human sample comes from the Endoscopy Center of Union Hospital. All samples were obtained with the patients’ informed consent, and the samples were processed histologically.
SGC7901 xenograft tumor model
Four-week-old male BALB/c nude mice housed under standard laboratory conditions. Human gastric cancer SGC7901 were harvested and resuspended in PBS.2 x106 Cells were injected subcutaneously into the right forelimb armpit of BALB/c nude mice. The tumor size was measured every two days. After 21 days, mice were sacrificed and the tumor growth was evaluated by weight.
Chick embryo chorioallantoic membrane (CAM) assay
The CAM assay was performed to evaluate in vivo angiogenic activity as previously described(18). Briefly, 100 fertilized eggs were incubated at 38 °C and 36.5-38.5% relative humidity in a forced draught incubator. A small window was punctured on each egg of 8-day-old fertilized eggs. 30μl of cell suspension was dropped onto a gelatin sponge and then added onto the CAM. The eggs were sealed with transparent tape and incubated at 37.8 °C and 60% humidity for 4 days. On day 12, the eggs were opened and the chorioallantoic membrane vasculatures were imaged using a stereo microscope equipped with a digital camera. Angiogenesis was quantified by counting the blood vessel density (percentage of blood vessel area over the whole area under a microscopic field) using the image analysis program IPP 6.0 (Image-Pro Plus, version 6.0, Media Cybernetics).
Tube formation assay
HUVECs were resuspended to a final concentration of 2 x105cells/ml. μ-Slide Angiogenesis (ibidi, Martin Reid, Germany) were pre-coated with 10μl Matrigel (Corning, BD) and then incubated at 37 °C and 5% CO2 for 30 min. After Matrigel was solidified, resuspended HUVECs (approximately 1x 104 cells) were seeded into the μ-Slide Angiogenesis. After a 7-h incubation, the tube network was photographed using a phase contrast microscope and confocal microscope. The NIH Image J with the Angiogenesis plugin software were used on the tube network to detect total nodes/tubes /mesh in combination.
Angiogenesis fluorescence imaging in vivo
The AngioSense 750EX Fluorescent Imaging Agent (Perkin Elmer) was injected into the mouse model of gastric cancer cell xenograft by tail vein (2 nmol/100μl per mouse, respectively). After 4H, the probe was accumulated in tumors and enabled imaging of vascularity, perfusion, and vascular permeability at a wavelength of 750nm.The angiogenesis fluorescence imaging in vivo was detected by Bruker In-Vivo Xtreme (Germany) and quantified by measuring the tumor uptake of the probe AngioSense750 EX.
RNA-SEQ
Total RNA was obtained using Tripure Reagent (Roche Applied Science, Indianapolis, IN, USA), following manufacturer’s instructions. Image analysis, per-cycle base-calling and quality score assignment was made using Illumina Real Time Analysis software (Illumina, San Diego, CA). Firstly, the magnetic beads with Poly(T) probes were hybridized with total RNA to purify mRNA. Then the mRNA with Poly(A) was eluted from the magnetic beads and broken into fragments with a magnesium ion solution. The mRNA fragments were reversely transcribed to cDNA and synthesized to double-stranded cDNA. The amplified cDNA was repaired and purified. Directional RNA-SEQ libraries resulting from these analyses were sequenced in paired-end format in two different rounds (Illumina HiSeq2000).
RNA extraction and gene expression analysis
Total RNA was isolated from triplicate wells in each condition with RNAiso Plus reagent (Code No.: 9109, TAKARA, Japan) and reversely transcribed into cDNA using a Reverse Transcription system (Code No.: RR036A, TAKARA, Japan) according to the manufacturer’s instructions. The quality and quantity of RNA were measured with a Nanodrop (Thermo Scientific). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the TB Green Premix Ex Taq (Tli RNaseH Plus) (Code No.: RR420A, TAKARA, Japan). The fold changes in gene expression were obtained after normalized to GAPDH and the 2−(ΔΔCt) was used to calculate the relative abundance of mRNA. Primers used in this study are shown in Supplemental Table1.
SDS–PAGE and Western blotting analysis
Proteins were extracted in RIPA buffer containing phosphatase and protease inhibitor mixes (Beyotime Biotechnology, China) at 4℃ for 10-20 min. The quantity of proteins was measured using BCA protein concentration determination kit (Beyotime Biotechnology, China). After removing DNA and impurities, proteins were separated by SDS–PAGE (WB2102, Biotides, BeiJing, China) and transferred to nitrocellulose membranes. The membrane was blocked using 10% skimmed milk before detecting with specific antibodies and HRP-conjugated secondary antibodies in combination with enhanced chemiluminescence. The Signal was acquired with Millipore immobilon western chemilum HRP substrate and grey value was quantified with Image J. The primary antibodies used were sheep anti-mouse THBS4(AF7860-SP, R&D Systems), rabbit anti-AKT (#4691T, CST), and rabbit anti-Phospho-AKT(p-AKT) (Ser473) (#4060T, CST).
Histology, immunohistochemistry (IHC) and Immunofluorescence (IF) staining
The tissues were embedded and sectioned. After dewaxed and hydrated, the sections were incubated overnight with antibodies against human THBS4(YT4646, immunoWay), human PECAM-1(ab9498, Abcam), mouse CD31(AF3628-SP, R&D Systems), mouse NG2(sc-5389, Santa Cruz), mouse THBS4(AF7860-SP R&D Systems), mouse Ki67(ab1667, Abcam), and identified with Alexa 488, Alexa 594 or cy3 secondary antibodies or HRP-conjugated secondary antibodies the next day. The sections were detected by phase contrast microscope and confocal microscope and raw image data quantity were processed with Image-Pro Plus.
Cell migration assay
The twenty-four-well plate Transwell was performed to determine the ability of HUVEC migration. 1000 serum-starved HUVEC were plated on the upper chambers of 3.0-μm pore size Transwell inserts and culture ECM medium containing 20% FBS was introduced to the lower chambers. After incubating the cells for 24h at 37° incubator, unmigrated cells on the top membrane was removed with cotton swabs. The remaining cells were fixed in 4% paraformaldehyde for 15min and stained with 1% crystal violet for 10 min at room temperature and then washed in PBS for 3 times. The image of migratory cells was acquired with inverted light microscope and the cells were counted to quantify cell migration.
Lentivirus construction and transfection
Lentivirus for THBS4 short hairpin RNA (shRNA) and negative control (NC) sequences were designed and constructed by Obio Technology Co. Ltd., Shanghai, China. The shRNA Lentivirus were transfected into BM-MSCs using HitransG A (Genechem, Shanghai, China) according to the manufacturer's protocol. Selection of stably transfected cells was performed with puromycin(8μg/ml). The targeted THBS4 sequences are as follows: GGATGAGTGCAAATACCAT.
Statistical analysis
All experiments were repeated at least three times, and the results were expressed as means ± SD. All statistical analyses were performed using GraphPad Prism software. Student’s t-test or the one-way analysis of variance (ANOVA) were used to analyze the data, and chi-square test was used to analyze differences in other variables, as appropriate. P value of ≤0.05 was considered statistically significant for all datasets. (*p < 0.05, **p < 0.01, ***p < 0.001).