Endothelial Differentiation Of Gastric Cancer Stem-like Cells

IntroductionGastric cancer (GC) has been causing devastating damage to people’s health in China,which accounts for23.2%of cancer related death. The main therapy for GC includeresection, chemotherapy and radiotherapy. Anti-angiogenesis therapy has been drawingattention these years because of its high specificity and ideal therapeutic effect incombination with chemotherapy and radiotherapy. However, the drug resistance againstanti-angiogenesis drugs is a critical problem remaining to be solved.Current drugs,mostlyuse VEGF as a target of conventional regulator of angiogenesis, has enabled signifcantadvances in the therapy of cancer and neovascular age-related macular degeneration.However, similar to other therapies, inherent/acquired resistance to anti-angiogenic drugsmay occur in patients, leading to disease recurrence and limited effcacy（20%to37%）.The emerging principle of endothelial cells, with cytogenetic abnormalities in theirchromosomes, derived from putative cancer stem cells, which is an important way amongseveral known methods of blood vessel formation in normal tissues and tumors. Recentreports in nature have documented the ability of glioblastoma cancer stem cells (CSCs) totransdifferentiate into endothelial-like cells and70%of endothelial cells inside the tumorare derived from CSCs. Here in our study of gastric cancer, we discuss could SGC7901-derived CSCs give rise to endothelial cells and how much the potentialcontributions of CSCs give to the angiogenic process, for provide new insight into thebiology of gastric cancer and the definition of cancer stemness, as well as the mechanismsof tumor neo-angiogenesis.Objective：1. Induction and identification of gastric cancer stem-like cells (Cancer Stem LikeCells, CSLCs);2. Verification of the phenotypic and functional differentiation of gastric cancerstem-like cells to endothelial cells in vitro;3. Validation of the differentiating ability of gastric cancer stem-like cells toendothelial cells in vivo;Methods：1.Induce gastric cancer stem like cells: Use FACS screened with GFP positiveSGC7901cells and were treated with vincristine (1μg/ml) for3days (VCR-inducedcancer cells). The surviving cells were inoculated in the wells (200cells per well) ofultra-low-attachment96-well plates. The cancer stem cell medium was supplemented withDMEM/F12, human recombinant epidermal growth factor(EGF,20ng/ml) and humanrecombinant basic fibroblast growth factor (bFGF,20ng/ml).2. Identification of cancer stem-like cells in Vincristine preconditioned SGC7901gastriccancer cell line：Spheroid Colony Formation Assay、Plate Clone formation Assays、Differentiation Assays、Self-Renewal Assay （PKH-26Staining、BrdU and EDU LablingAssay）、Western Blot Analysis、Immunofuorescence Staining、Real-time PCR (RT-PCR)、In Vitro Drug Sensitivity Assay、In Vivo Experiments.3. Phenotype validation: To determine the potential contribution to the angiogenic processof CSLCs,we cultivated one part of the identification cells under endothelialconditions(in the M200medium containing such as VEGF vascular endothelial growthfactor) and evaluate endothelial markers CD31, CD34expression level of the fourgroups (SGC7901group, gastric cancer stem-like cells group, endothelial environmentcultivated group and the human umbilical vein endothelial cell group) through Western blot and Immunofluorescence methods.4. Functional test In vitro: we used Tube-forming assay to detect the endothelial functionalfeatures of the four groups.5. Subcutaneous xenograft transplantation:15mice/group were injected ofSGC7901(5X106cells) and another part of the identification CSLCs(5X104cells). After4weeks,do statistics of size and weight.6. Immunohistochemistry xenografts: Immunohistochemistry of SGC7901,CSLCsxenografts and human gastric cancer using anti-human CD31and anti-human CD34, toinvestigate the ability of CSLCs to form endothelial vessels in vivo.Results:1．Identification of gastric cancer stem-like cells in spherical clone culture andverification of tumorigenicitySignificant spheroid formation of VCR-preconditioned cells was observed with theexpansion of time.Tumor sphere bodies formed in significant numbers in cells withtransient VCR treatment, while few SGC7901cells formed sphere bodies without VCRtreatment. VCR-preconditioned SGC7901significantly increased rate of tumor sphereformation (P<0.05).2. In vitro validation of endothelial phenotypes and functions for gastric cancerstem-like cellsThe differentiation ability of VCR-induced CSLCs was explored in a2D Matrigeldifferentiation assay. The self-renewal property in CSLCs, we employed a lipophilicfluorescent dye, PKH-26.Gastrointestinal genes CDX2and SOX2were significantlyup-regulated, explored by real-time PCR.Obvious asymmetric ability of CSLCs wasobserved when comparing with its parent cell line SGC7901(25%vs.8-11‰, P<0.05,paired student t-test). Tumorigenicity in vivo was assessed by inoculating CSLCs ortheir parental cells subcutaneously into nude mice.Western blot and Immunofluorescence evaluation of human CD31and human CD34in the four group cells. Whereas human CD31and human CD34enriched in gastriccancer stem-like cells cultivated under endothelial conditions and human umbilical vein endothelial cell (HUVEC), neither SGC7901cells nor the CSLCS were found.Such gastric cancer stem-like cells cultivated under endothelial conditions andHUVEC showed considerable tube-forming ability, which were completely absent in theCSLCS and in the SGC7901cell line.5．Validation of endothelial differentiation from gastric cancer stem-like cells in vivoExplanted subcutaneous xenograft obtained by injection of CSLCs and SGC7901.Immunohistochemistry of subcutaneous xenografts showed that CSLCS-derived tumourscontained human vessels labelled by human-specific anti-CD31, whereas xenograftsgenerated with SGC7901cell lines did not.Conclusions：The way of VCR-preconditioned cells to form spheroids (considered to be onecharacteristic of CSCs) was a confirmed approach for obtaining CSLCs from SGC7901cell lines. Gastric CSLCs exhibited differentiation ability and self-renewal ability in vitroand remarkable tumorigenicity in vivo. CSLCs under endothelial differentiation conditionsdeveloped phenotypical and functional features of endothelial cells. Subcutaneousxenografts showed that CSLCs-derived tumors contained human vessels. The ability ofcancer stem-like cells to directly contribute to the tumour vasculature by endothelial celldifferentiation represents a new mechanism of angiogenesis.