Establishment Of 5-FU-resistant Canine Mammary Tumor Cell Line And Evaluation Of Its Biological Features

Introduction:Mammary tumor is one of the most common tumors in canine,as most of the neoplasms are malignant,surgery can't always solve the problem.In order to improve the quality and quantity of life,chemotherapy is recommended to these patients.Unfortunately,with the progress of chemotherapy,cancer cells would be resistant to chemo-drugs.In order to disclose the mechanism of drug resistance,we established a 5-FU-resistant canine mammary tumor cell line and investigate the growth features of this cell line,the relationship between drug resistance and cancer stem cells,the function of Notch 1 pathway in maintaining drug resistance.Method:1.Establishment of 5-FU-resistant canine mammary tumor cell line through stepwise increasing concentration of drug in vitro.Morphology study,cross resistance,growth manner,expression level of drug resistant proteins and animal experiment were preformed to analyze the different biological features between resistant cells and parental sensitive cells.2 Primary culture the cells that derived from the experiment animal which accepted 5-FU treatment.Differential adhesion and limiting dilution methods were used to purify the cells.Then assess the drug resistant property of the purified cells.3.In order to verify the relationship between drug resistance and cancer stem cells,we studied the stem cell property and metastasis property of drug resistant cells.4.Investigate the activation status of Notchl pathway in drug resistant cells,whether Notchl inhibitor DAPT can reverse drug resistance,and how did DAPT works.Results:1.A 5-FU-resistant cell line CMT7364/5-FU was established through 8months culture in vitro to investigate the acquired resistance mechanism.Compare with parental sensitive cells,resistant cells tended to grow scattered and polygonal,the PDT of resistant cells were much longer,and they were cross-resistant to four other chemo-drugs,ABCBland ABCG2 were up-regulated,CMT7364/5-FU was much more resistant to 5-FU in vivo.2.The primary tumor cells were purified through differential adhesion and limiting dilution methods.After identified by karyotype analysis,we verified the purified cells were canine mammary cells.The PDT of these cells were similarly to resistant cells,and these cells were much more resistant to 5-FU than CMT7364/5-FU.3.Flow cytometry analysis revealed that the proportion of CD24-/CD44+ cells were much bigger in CMT7364/5-FU.The number of spheres under suspension culture and clones under adhesion culture in CMT7364/5-FU cells were much more than sensitive cells.CSCs related proteins were up-regulated in CMT7364/5-FU than CMT7364.104 resistant cells can initiate tumor in mice.Wound healing assay and transwell assay indicated that resistant cells possess much more power of immigration and invasion.Western blotting assay showed that MMP-2 and vimentin were over-expressed in resistant cells.Resistant cells were much easier transfer to lung than sensitive cells in vivo.4.Notchl pathway were over-activated both in gene and protein level in resistant cells,DAPT as the inhibitor of Notchl can reverse drug resistance,DAPT plays its role through inhibiting the migration and self-renew ability of resistant cells.Conclusion:All the study above indicated that,stepwise increasing concentration of drugs is a good way to obtain drug resistant cell line.5-FU-resistant canine mammary tumor cell showed different biological features,and showed CSCs properties.Notchl pathway were over-activated in 5-FU-resistant cells,Notchl pathway inhibitor can reverse drug resistance in vitro,so Notchl maybe a new target for treating canine mammary tumors.