Isolation And Characterization Of Gastric Cancer Stem Cells And Their Roles In Invasion And Metastasis Of Gastric Cancer

Gastric cancer is the second leading cause of cancer related mortality, especially in EastAsia, East European and South America. Despite of the development of surgery andchemotherapy, the five year survival rate remains low. Recently, emerging of cancer stem cell(CSC) hypothesis provides us a new way insight into the mechanism underlining gastriccancer initiation and progression. To demonstrate the existence of gastric cancer stem cells(GCSCs) and explore their malignant behavior, we established a method of sphere formingculture to gain the tumor spheres from gastric cancer cell lines. Tumor sphere cells possessedself-renew, multi-potent differentiation ability and highly tumorigenicity, indicating thattumor spheres enriched GCSCs. Moreover, GCSCs exhibited properties of high invasion,metastasis and multi-drug resistance. A series of genes and miRNAs differential expressionbetween GCSCs and Monolayer cells were screened by gene microarray and miRNA arrayanalysis. Among the differential expression molecular, ANTXR2was particularlyover-expressed in GCSCs. Then, roles of ANTXR2in gastric cancer stemness, invasion andmetastasis were explored.Main methods, results and conclusion as follow:1. Tumor spheres formed from gastric cancer cells possess stem cell properties.1)Gastric cancer cells of SGC7901，MGC803and BGC823could grow as tumor sphere inserum free conditioned medium.2) Compared with monolayer cells (MN), sphere cells(SC)highly expressed stemness genes Sox2，Oct4and Bmi1;3) SGC7901-SC possessed highercolony formation ability than SGC7901-MN cells (156±5vs35±2, p<0.05);4) CulturedSGC7901-SC in medium supplemented with10%FBS, the cells could produce more maturecells expressing CK18and H-K-ATPase;5) Xenograft assay showed that SGC7901-SC werehighly tumorigenic, and the xenograft formed by SGC7901-SC with lower proportion ofCK18and H-K-ATPase positive cells compared to xenograft formed by SGC7901-MN（13%±4%vs94%±2%，p<0.05and20%±4%vs42%±3%, p<0.05respectively）, indicating that xenografte formed by SGC7901-SC are immature. Those data suggesting that sphere cellspossess stem cell properties.2. GCSCs possess highly invasive and metastatic ability in association with epithelialmesenchymal transition.1) We found xenograft tumors formed by SGC7901-SC cells invadedinto the capsule and metastasized to lymph nodes. In contrast, there were compact capsulessurrounding SGC7901-MN-xenografts without any invasion or metastasis；2）Invasive abilityof SGC7901-SC were measured by Transwell invasion assay, and we found that SGC7901-SCwith higher invasive ability in vitro compared to SGC7901-MN（153±16vs51±12，p<0.05）;3) Expression of invasive related genes in SGC7901-SC were detected by Real time PCR andWestern blot, we found that SGC7901-SC with increased expression of Vimentin, MMP2anddecreased expression of E-cadherin.3. GCSCs show the property of multi-drug resistance.1) SGC7901-SC were moreresistant to chemo-drugs5-Fu，DDP and ADR compared to SGC7901-MN;2) SGC7901-SCshowed higher expression of ABCC4, lower expression of MDR1compared to SGC7901-MN.There was no significant difference of MRP1and ABCG2expression between SGC7901-SCand SGC7901-MN.4. We found a series of genes and miRNAs differentially expressed betweenSGC7901-SC and SGC7901-MN by gene array and miRNA array analysis. Signalingpathways of Wnt, Notch, TGFβ, VEGF, p450and anti-apoptosis were activated in GCSCs,indicating these signaling pathways may be involved in the self-renewal, invasion, metastasis,multi-drug resistance and angiogenesis in GCSCs.5. ANTXR2plays important roles in gastric cancer stemness maintenance, invasion andmetastasis.1) ANTXR2was found highly expressed in GCSCs by gene array analysis andconfirmed by real time PCR and FACS;2) ANTXR2was co-localized with stem cell markerCD44in gastric cancer samples;3) Silenced ANTXR2expression in gastric cancer cells byshRNA, the ability of sphere formation, proliferation, invasion and metastasis was impaired;4)Over-expressing ANTXR2, the colony formation ability of gastric cancer cells was increased.6. The possible molecular mechanisms of ANTXR2in stemness maintenance, invasionand metastasis of gastric cancer cells were explored.1) Silenced ANTXR2expression ingastric cancer cells by shRNA, the expression of stemness and EMT related genes Sox2，Bmi1，CD44, β-catenin and Vimentin was down-regulated, meanwhile, the expression of E-cadherin was up-regulated;2) Gene array analysis showed that silencing ANTXR2expression mainly resulted in change of MAPK signaling pathway. Using the inhibitors ofMAPK signaling pathway, the result was confirmed. ERK inhibitor PD98059and JNKinhibitor SP600125could significantly inhibited the colony formation of gastric cancer cellswith over-expression of ANTXR2, and the ERK inhibitor PD98059is more effective;3)Silencing ANTXR2expression resulted in decreased phosphorylation of ERK, Src-Tyr416and increased phosphorylation of Src-Tyr527in gastric cancer cells, indicating that thefunction of ANTXT2may be dependent on the Src/ERK signaling pathway.7. ANTXR2expression has important clinical significance in patients with gastric cancer.Immunohistochemistry(IHC) was used to detect the expression of ANTXR2in181specimensof gastric cancer, the result showed that the expression of ANTXR2was correlated withinvasive depth (p=0.03）, TNM stage (p=0.024) and shorter survival time (p=0.006),indicating that ANTXR2is an indicator of poor prognosis in gastric cancer.In summary, sphere formation culture is an effective method for isolation/enrichment ofGCSCs. GCSCs possesses highly invasive, metastasis and multi-drug resistant properties.Multi-signaling pathways, such as Wnt, Notch, TGF-beta, p450metabolism enzyme familyand anti-apoptosis signaling pathway, may be involved in regulation of the GCSCs behaviors;Our results also demonstrated that ANTXR2play important roles in stemness maintenance,invasion and metastasis of GCSCs. The function of ANTXR2may be dependent on Src/ERKsignaling pathway. Expression of ANTXR2is positively correlated with clinic pathologicparameters and negatively correlated with survival time, indicating that it can be used as anindicator of poor prognosis of patients with gastric cancer.