Inhibitory effect of anti-EGFRv Ⅲ CAR-T lymphocytes combined with DAPT on self-renewal ability of human glioma initiating cells
Objective To observe the inhibitory effect of anti-epidermal growth factor receptor vⅢ chimeric antigen receptor gene-modified T lymphocytes(anti-EGFRvⅢ CAR-T lymphocytes)combined with Notch1 pathway specific inhib-itor DAPT on the self-renewal ability of human glioma initiating cells(GICs).Methods The primary glioblastoma(GBM)cells were divided into two parts,including DT007 and DT008.After culturing stem cells into spheroids,they were sorted using immunomagnetic bead method,were identified as GICs by Western blotting and immunofluorescence,and were named PA37S and PA38S,respectively.These two types of cells were randomly divided into the control group(added with cell culture medium),DAPT group(added with 10 μ mol/L DAPT),CAR-T group(added with anti-EGFRvⅢ CAR-T lymphocytes obtained by lentivirus transfection that inhibit EGFRvⅢ expression),and CAR-T+DAPT group(added with anti-EGFRvⅢ CAR-T lymphocytes and 10 μ mol/L DAPT),respectively;all cells from the above groups were cultured for 24 h.Western blotting was used to detect the relative expression levels of EGFRvⅢ and Hes1 proteins in cells.The diameter and number of cell spheres were measured by spheroid formation experiment,and the number of cell spheres was measured by extreme dilution assay at cell densities of 5,10,20,50,100,200,and 500/well.Results In PA37S and PA38S cells,the relative expression levels of EGFRvⅢ protein in the CAR-T group and CAR-T+DAPT group were lower than those in the control group and DAPT group;the relative expression levels of Hes1 protein in the DAPT group and CAR-T+DAPT group were lower than those in the control group and CAR-T group(all P<0.05).The results of the spheroid formation experiment showed that in PA37S and PA38S cells,the number and diameter of cell spheroids in the DAPT group,CAR-T group,and CAR-T+DAPT group were lower than those in the control group,with the CAR-T+DAPT group showing a more significant decrease(all P<0.05).The results of the extreme dilution experiment showed that there was no statistically significant difference in the number of cell spheres between the 5/well and 10/well cell densities in PA37S and PA38S cells(P>0.05).At a cell density of 20/well,the number of cell spheres in the CAR-T+DAPT group was smaller than that in the control group.At the cell densities of 50/well,100/well,and 200/well,the number of cell spheres in the CAR-T+DAPT group was smaller than that in the control group,DAPT group,and CAR-T group.At a cell density of 500/well,the number of cell spheres in the DAPT group,CAR-T group,and CAR-T+DAPT group was smaller than that in the control group,and the CAR-T+DAPT group showed a more significant decrease(all P<0.05).Conclusion The combination of anti-EGFRvⅢ CAR-T lymphocytes and DAPT can inhibit the self-renewal ability of GICs.
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