Fig. 7: AREG enhances mesothelial clearance ability of CL31.

a Representative differential interference contrast (DIC) and pseudocolored confocal fluorescence images of ability of vehicle- or AREG-treated CL31 cell clusters expressing red fluorescent protein (RFP) to clear a mesothelial monolayer expressing green fluorescent protein (GFP) at the indicated time points. Scale bar, 50 μm. b Quantification of the mesothelial clearance area (black area within the green monolayer) cleared in 24 h by CL31 spheroids treated with vehicle or AREG from three independent experiments. Mesothelial area cleared at the end point was normalized to the initial (1 h) area of CL31 clusters (measured from the DIC images), as previously described³⁹. Relative clearance area of 20–30 clusters of CL31 of each condition per experiment were analyzed and averaged. Data shown as mean ± SEM of three independent experiments, shown in arbitrary units (AU), p value from Welch’s t test of the means. c Model illustrating the transient cooperative interactions involved in metastasis in this model system. CL31, which carries amplified ERBB2 and displays anchorage-independent growth in vitro, forms only malignant ascites, but is unable to form solid peritoneal metastasis. Transient and non-tumorigenic AREG-high clones can act at an early temporal window, and are later dispensable, to induce CL31 solid peritoneal metastasis. AREG is required for metastasis of CL31, but not expansion in ascites or metastatic sites.