The Isolation,Identification,Drug Resistance,and The Migration Mediated By The Chemokine CXCL12 Of Human Hepatic Cancer Stem Cells

Object Hepatocellular carcinoma(HCC)is the third cause of death related to tumor in china.The post operation recurrence is the major cause of death.The hepatic cancer stem cells has been valued to be one of the sources of recurrence.Here we show the isolation,identification,drug resistance,and the migration mediated by the chemokine CXCL12 on human hepatic cancer stem cells(HCSCs).Methods Based on the characteristic of the cancer stem cells in which the different efflux of the DNA-binding dye Hoechst33342 could be observed by the flow cytomety,the cell line HepG2 and the fresh tumor tissue of 21 HCC patients were sorted to separate the side population(SP).Properties of the SP cells were established by cell proliferation assay,capacity to form spheroids in the serum-free medium,invasion and migration assays,tumorigenicity in NOD/SCID mice and mRNA microarray analysis.The concentration gradients of trypan blue and Sorafenib were established in the microfluidic chips to investigate the different effects to SP and non-SP cells.The concentration gradient of CXCL12,a chemokine,was also established in the microfuidic chip to study the different migration rates of SP and non-SP cells sorted from HepG2.AMD3100,the inhibitor of CXCL12 receptor CXCR4,was used to study the effect of CXCL12/CXCR4 axis in the migration of HCSCs.Results SP cells were observed in HCC cell line HepG2 and 18 of the 21 fresh tissues,the rates were 3.24±0.012%and 3.43±0.025%.SP cells showed higher abilities of invasion,migration,proliferation and forming spheroids,Tumours could generate from SP but not non-SP cells in low doses of subcutaneous injection to the NOD/SCID mice(5×10~2cells/mouse),the tumorigenicities of SP cells sorted from 2 patients’fresh tissue were equal to that from HepG2.There was no difference in the morphology of the tumors generated from SP and non-SP cells,which was shown by the HE staining.The mRNA microarray analysis in HepG2 SP cells revealed that 42405 genes were investigated and 3079 of them were up-regulated.The survive rates of SP cells were significantly higher than that of non-SP cells in the trypan blue or the sorafenib concentration gradients.In the microfluidic chips,SP cells could migrate to the areas with higher concentration of CXCL12,and could be inhibited by AMD3100.On the contrary,the non-SP cells did not show significant migration.Conclusions The SP sorting is comparatively reliable to isolate the HCSCs.The HCSCs has higher abilities of invasion,migration,proliferation,tumorigenicity and drug resistance.Some of the HCSCs can migrate under the mediation of the chemokine CXCL12,and can be inhibited by AMD3100.