Fig. 3

CD73 blocking inhibits EGF-induced local invasion in a 3D model. A FaDu cell spheroids in Matrigel were treated with the indicated compounds. EGF: 50 ng/mL; Cet.: cetuximab 10 µg/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 and invasive distance, respectively. All treatment were performed for 72 h in the absence of fetal bovine serum. B Quantitative analysis of invasive area (left) and invasive distance (right) from n = 3 independent experiments with multiple spheroids per treatment. Singular data points are depicted. **** p < 0.0001. C FaDu cell spheroids in Matrigel were treated with the indicated compounds. EGF: 50 ng/mL; Cet.: cetuximab 1 µg/mL; 22E6: antagonizing anti-CD73 monoclonal antibody 2.5 µg/mL, and a combination of Cetuximab and 22E6. All antibody concentrations were equalized upon addition of immunoglobulin control antibody. Yellow contours and lines served for the quantification of invasive area and invasive distance, respectively. All treatment were performed for 72 h. D Quantitative analysis of invasive area (left) and invasive distance (right) from n = 3 independent experiments with multiple spheroids per treatment. Singular data points are depicted. **** p < 0.0001. E Schematic representation of the assessment of functional IC50 values of Cetuximab in the absence and presence of 22E6. F FaDu cell spheroids in Matrigel were treated with the indicated concentrations of Cetuximab in the absence or presence of 22E6 at the individually ineffective concentration o 2.5 µg/mL. Micrographs were taken at 72 h and are representative of n = 3 independent experiments with multiple spheroids. G Dose–response curve of the EGFR-mediated invasive area as a function of Cetuximab concentration without or with additional treatment with 22E6. Functional IC50 values of Cetuximab regarding the inhibition of EGFR-mediated invasion are indicated