THBS4/Integrin α2 axis mediates BM-MSCs promote angiogenesis of gastric cancer associated with chronic Helicobacter pylori infection LingNan He Wuhan Union Hospital https://orcid.org/0000-0002-0760-9789 WeiJun Wang Wuhan Union Hospital HuiYing Shi Wuhan Union Hospital Chen Jiang Wuhan Union Hospital HaiLing Yao Wuhan Union Hospital YuRui Zhang Wuhan Union Hospital Wei Qian Wuhan Union Hospital Rong Lin ( [email protected] ) Wuhan Union Hospital Research Keywords: Bone marrow-derived mesenchymal stem cells (BM-MSCs), Helicobacter Pylori, Gastric cancer (GC), Tumor angiogenesis, THBS4, Integrin α2, PI3K/AKT pathway Posted Date: April 3rd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-366308/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/27 Abstract Background BM-MSCs contribute to Helicobacter pylori (H. pylori)-induced gastric cancer, but its mechanism is still unclear. The aim of our study is to investigate the specic role and mechanism of BM-MSCs in H. pylori- induced gastric cancer. Main methods Mice were received total bone marrow transplantations and then infected with H. pylori. BM-MSCs were extracted and transplanted to gastric serosal layer of mice infected with H. pylori chronically. Hematoxylin and eosin staining, immunohistochemistry staining and immunouorescence were performed to detect the tumor growth and angiogenesis in mice stomach tissues. Chicken chorioallantoic membrane assay, xenograft tumor model, and Human umbilical vein endothelial cells tube formation assay were used for in vivo and in vitro angiogenesis study. THBS4 was screened out from RNA-SEQ Analysis with gastric tissues of BM-MSCs transplantation into H. pylori infected mice. Results BM-MSCs can migrate to the site of chronic mucosal injury and promote tumor angiogenesis associated with chronic H. pylori infection. Migration of BM-MSCs to the site of chronic mucosal injury induced the up-regulation of THBS4, which was evident also in human gastric cancer, and correlated with more blood vessel formation and worse outcome. THBS4/Integrin α2 axis exhibited promoting angiogenesis by facilitating PI3K/AKT pathway in endothelial cells. Conclusions Our results discover that BM-MSCs have a new pro-angiogenic effect in chronic H. pylori infection microenvironment, primarily through THBS4/Integrin α2 axis, which activated PI3K/AKT pathway in endothelial cells and eventually induced formation of new tumor vessels. Introduction Gastric cancer is the fth most common cancer and the third most common cause of cancer death globally(1). Many factors are implicated in the carcinogenesis and progression of gastric cancer, including prevalence of Helicobacter pylori (H. pylori) infection, the main cause of gastric cancer(2). Chronic H. pylori infection of the gastric mucosa leads to stepwise progression from atrophic gastritis and intestinal metaplasia, and eventually adenocarcinoma (3) However, the mechanism by which H. pylori infection mediates gastric cancer progression is incompletely dened. Recent studies on gastric cancer demonstrated that cancer stem cells (CSCs) exist in gastric cancer. These cells could originate from bone marrow–derived cells (BMDCs) (4–7). BM-derived mesenchymal Page 2/27 stem cells(BM-MSCs) are among the BMDCs that have been shown to be recruited to gastric cancers and to promote tumor growth(8, 9). Houghton et al demonstrated chronic infection of C57BL/6 mice with Helicobacter, induces an environment conducive to BMDCs recruitment, with the BM-MSCs the most likely candidate(7). Varon demonstrates that infection by H. pylori leads to BM stem cell and particularly BM- MSCs recruitment and homing in the gastric mucosa and that BM-MSCs reconstituted gastric glands can evolve toward metaplasia and dysplasia(10). Quante et al demonstrated that a signicant percentage of carcinoma-associated broblasts (CAFs)in Helicobacter felis infected inammation-associated gastric cancer originates from BM-MSCs, which promote cancer growth and progression(11). Studies using bone marrow transplantation in mice have shown that MSCs display a remarkable feature called tumor- specic tropism, in which they actively migrate to tumor sites(12).However, the specic mechanism of BM-MSCs involved in the occurrence of H. pylori-related gastric cancer remained uninvestigated. Several studies have pointed to BM-MSCs promote tumor growth by enhancing angiogenesis(13–15). Nevertheless, whether BM-MSCs participate in the occurrence of gastric cancer associated with chronic H. pylori infection by promoting tumor angiogenesis is unknown. Since the in vivo distinct functional role of facilitating blood vessel formation of BM-MSCs in spontaneous primary gastric tumors associated with chronic H. pylori infection remain unexplored, the aim of this research was to investigate the role of BM- MSCs and its specic mechanism in angiogenesis associated with chronic H. pylori infection. In this study, we demonstrate that BM-MSCs can migrate to the site of chronic mucosal injury with chronic H. pylori infection and promote tumor angiogenesis in vitro and in vivo. BM-MSCs in mouse models of chronic infection of BALB/c mice with H. pylori induced the up-regulation of Thrombospondin 4(THBS4), which was previously shown to affect the functions of microvascular endothelial cells and promote angiogenesis(16, 17). BM-MSCs derived THBS4 exhibits promoting angiogenesis by facilitating PI3K/AKT pathway in endothelial cells. Thus, our ndings that BM-MSCs and its derived THBS4 promote angiogenesis may provide a therapeutic target to inhibit angiogenesis in H. pylori infection related gastric cancer. Materials And Methods Helicobacter strain and infection experiments H. pylori strain SS1 was cultured in H. pylori uid 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. Page 3/27 Following transplantation, mice received antibiotics for 4 wk in water (Enrooxacin; 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 uorescent-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 identied by ow 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 uorescence in gastric tissues. HUVEC extraction and identication 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 identied by immunouorescence 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 sacriced and the tumor growth was evaluated by weight. Chick embryo chorioallantoic membrane (CAM) assay Page 4/27 The CAM assay was performed to evaluate in vivo angiogenic activity as previously described(18). Briey, 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 quantied by counting the blood vessel density (percentage of blood vessel area over the whole area under a microscopic eld) using the image analysis program IPP 6.0 (Image-Pro Plus, version 6.0, Media Cybernetics). Tube formation assay HUVECs were resuspended to a nal 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.