Fig. 4

CD73 is an effector not a regulator of EGF-EMT. A Schematic representation of possible roles of CD73 in EGFR-EMT. CD73 may act as a regulator of proliferation/metabolism or EMT, or, alternatively, may represent an effector molecule downstream of EGFR-mediated EMT. B FaDu cell spheroids in Matrigel were treated mitomycin C (0.02 µg/mL) and with the indicated compounds. EGF: 50 ng/mL; IgG: immunoglobulin control 5 µg/mL; 22E6: antagonizing anti-CD73 monoclonal antibody 5 µg/mL. Yellow contours and lines served for quantification of invasive area. All treatment were performed for 72 h in the absence of fetal bovine serum. C Quantitative analysis of invasive area from n = 3 independent experiments with multiple spheroids per treatment. Singular data points are depicted. ns not significant, **** p < 0.0001. D FaDu cells were maintained under control condition (serum-free), treated with an EMT-inducing concentration of EGF (50 ng/mL; 72 h), or with a combination of EGF and immunoglobulin IgG or 22E6 (5 µg/mL; 72 h). Shown are representative bright light microscopy images of each treatment. E FaDu cells treated as in (C) were subjected to quantitative RT-PCR with primers specific for E-cadherin, N-cadherin, Twist1, and vimentin. Shown are mean relative mRNA expression levels normalized to control treatments from n = 3 independent experiments. ns: not significant, * p < 0.05, ** p < 0.01. F FaDu cell spheroids in Matrigel were treated with the indicated compounds. CD73 inhibitor APCP was used at 50 µM concentration. Quantitative analysis of invasive area from n = 3 independent experiments with multiple spheroids per treatment are shown in the lower right panel. Singular data points are depicted. ns not significant, **** p < 0.0001. G CD73 knock-down (KD), over-expression (OE), and CD73 re-expression in knock-down cells (KD-OE) in FaDu cells were analyzed for CD73 expression by flow cytometry with specific antibodies (CD73) or isotype control (iso.). Shown are representative histograms for each cell lines with cognate control cells. H Standardized mean fluorescence intensities of CD73 cell surface expression are shown as means with SD from n = 3 independent experiments. * p < 0.05, ** p < 0.01, **** p < 0.0001. I, L, N FaDu control (Ctrl.), CD73 knock-down (CD73-KD) (I), CD73 over-expression (CD73-OE) (L), and CD73 re-expression in knock-down cells (CD73-KD-OE) (N) cell spheroids in Matrigel were treated with the indicated compounds. EGF: 50 ng/mL; IgG: immunoglobulin control 5 µg/mL; 22E6: antagonizing anti-CD73 monoclonal antibody 5 µg/mL. Yellow contours and lines served for quantification of invasive area. All treatment were performed for 72 h in the absence of fetal bovine serum. J, M, O Quantitative analysis of invasive area from n = 3 independent experiments with multiple spheroids per treatment from spheroids shown in I, L, N, respectively. Singular data points are depicted. ns not significant, **** p < 0.0001. K Controls and CD73 knock-down (KD) FaDu cells were analyzed for CD73 expression by flow cytometry with specific antibodies (CD73) or isotype control (iso.). Cells were either kept under serum-free condition (Ctrl.-SF, KD-SF) or treated with EGF (50 ng/mL) (Ctrl.-EGF, KD-EGF). Shown are representative histograms for each cell lines with cognate control cells. Standardized mean fluorescence intensities of CD73 cell surface expression are shown as means with SD from n = 3 independent experiments. * p < 0.05, **** p < 0.0001