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. 2021 May 27;14(8):101138. doi: 10.1016/j.tranon.2021.101138

Fig. 3.

Fig. 3

CAR-T cells kill cancer stem cells. Single cell suspension of adherently cultured ESCC tumor cells (adherent) and sphere-derived CSCs (sphere) was separately prepared. (A-C) Surface expressions of CD133 (A), CD44 (B), and CD276 (C) were detected via FACS in tumor cells (adherent) and CSCs (sphere), respectively. Mean fluorescence intensity (MFI) of the target molecules was compared with t-test. (D) Tumor cells (adherent) and CSCs (sphere) were suspended in DMEM/F12 media and added into ultra-low attachment plate, respectively. Then CAR-T cells were added at E/T = 1 without exogenous cytokines. At 6 h and 24 h, apoptosis of tumor cells (EpCAM+CD3−) was determined by Annexin V staining. (E) FFluc+ tumor cells were incubated with indicated CAR-T cells at E/T = 1 without exogenous cytokines for 24 h. Then cell viabilities were calculated relatively to CAR19.28ζ group according to BLI intensity. (F and G) ELISA of effector cytokines from CAR-T cells incubated with adherent or sphere tumor cells. t-test was performed in D-G. Data represent 3 independent repeats. **, P < 0.01; ***, P < 0.005.