Fig. 5
Intercellular transfer of FMR1-AS1 by exosomes disseminates ESCC stem-like phenotypes. a Size distributions and statistics graph of exosomes isolated from CM of ECA-109 and KYSE-150 cells (Upper) and serum of female ESCC patients (Lower). b FACS analysis of exosomes isolated from the CM of ECA-109 and KYSE-150 cells (Upper) and serum of female ESCC patients using CD63 and CD81(Lower). c qRT-PCR analysis of FMR1-AS1 in exosomes isolated from the CM of FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells (n = 3). d qRT-PCR analysis of FMR1-AS1 in ESCC cells after 48 h incubation with indicated exosomes (PBS as control, n = 3). e qRT-PCR analysis of FMR1-AS1 in serum exosomes from female ESCC patients (n = 48, ***P < 0.001), compared with that from healthy control. f qRT-PCR analysis of FMR1-AS1 in serum exosomes from female ESCC patients (first visit patients group versus recurrent patients group, n = 66, ***P < 0.001). g The consistency between FMR1-AS1 tumor expression and serum expression within individual ESCC patients (R2 = 0.7529, P < 0.001). h Kaplan-Meier plots for the disease-free survival rate of female ESCC patients in groups of serum FMR1-AS1 high or low expression levels in the Suzhou cohort (n = 146, discovery set), Guangzhou cohort (n = 107, validation set), and the overall populations (n = 253, pooled analysis). i, j Flow cytometry assay using antibodies against CD44 cell surface markers to obtain two subtypes of tumor cells (CD44 high and CD44 low) from 12 female ESCC patients. The histograms showing expression levels of FMR1-AS1, c-Myc and TSIX in these two cell subtypes (*P < 0.05). k, l CD44 immunostaining in female ESCC samples and FMR1-AS1, c-Myc expression levels in CD44 strong, medium and weak samples (*P < 0.05). m Western blotting of the c-Myc protein level and TSIX expression level on ESCC cells treated with or without c-Myc inhibitor (*P < 0.05). n NFκB activities of ESCC cells after 48 h incubation with indicated exosomes, examined by EMSA. o Western blotting showing c-Myc levels in ESCC cells after 48 h incubation with indicated exosomes. p, q Subcutaneous xenograft assay of FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells in nude mice with intratumoral injection of indicated exosomes (n = 5 per group)