LAUSR

Introduction Gastric cancer (GC) is the fifth most common cancer worldwide and the third leading cause of cancer-related deaths. To date, gastrectomy and chemotherapy are the only therapeutic options, but drug resistance is the main cause for treatment failure. Vasculogenic mimicry (VM) is a new model of neovascularization in aggressive tumours and has been correlated with poor prognosis in GC patients. Our group has developed chemotherapy-resistant GC cells using the Caucasian adenocarcinoma cell line AGS and three drugs among the most used in clinic (5-fluorouracil, cisplatin and paclitaxel) henceforward denominated 5FUr, CISr, TAXr. Our study has highlighted phenotypical differences among chemo-sensitive and chemo-resistant cell lines such as acquisition of stem-like phenotype and increased capacity to form vessels. Material and methods Establishment of AGS resistant cell lines exposing cells to increasing dilution of drugs for over 9 months up to dilutions higher than IC50 values initially verified on AGS cells through MTT analysis. Quantitative RT-PCR, flow cytometry and western blot analysis for stemness and VM markers. Vasculogenic mimicryassay Results and discussions AGS cells acquired chemoresistance as indicated by the increase of IC50 values in drug-treated cells with respect to AGS. Furthermore, MTT assay highlighted that there is not cross-resistance among 5FUr, CISr and TAXr. Supportive data is that cells are MDR1 negative. Resistant cells showed an upregulation of Yamanaka factors either in qPCR and flow cytometer analysis, and particularly interesting is ALDH overexpression in 5FUr. TWIST upregulation suggested the investigation of VM which resulted particularly enhanced in 5FUr cells which demonstrated their ability to form and sustain vessels up to 96 hours in the tube formation assay. Markers of VM such Laminin γ2 and Ephrin A2 showed an increase in resistant cells and especially in 5FUr. Conclusion One of the most interesting result is that 5FUr cells acquire stemness properties and are positive to the tube formation assay suggesting that VM might be one mechanisms adopted by cells to avoid drugs exposure. These findings suggest that acquisition of chemoresistance could cause a relapse of disease in which tumour cells take advantage of their capability to perform VM in order to self-sustain their growth and that may be cause of poor outcomes.