Fig 1: 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.
Fig 2: The expression of miR-103 regulates the LATS2/YAP signaling pathway. (A) miR-103 regulates the protein level of LATS2, YAP, p-YAP (p-S127), CYR61, AREG, CTGF and CXCL5 (*P<0.05). (B) RT-qPCR analysis suggested that an increased miR-103 expression regulated the expression level of downstream of YAP target genes (*P<0.05). LATS2, large tumor suppressor kinase 2; YAP, yes-associated protein; CYR61, cysteine-rich angiogenic inducer; AREG, amphiregulin; CTGF, connective tissue growth factor; CXCL5, C-X-C motif chemokine 5.
Fig 3: Overexpression of amphiregulin induces peritoneal metastasis of CL31.a Heatmap of ligands identified by RNA-sequencing. Growth factors and cytokines that were expressed by one or more of the other clones and poorly expressed in CL31 were identified and filtered for those whose corresponding receptor(s) were expressed in CL31 (cpm > 2). The relative mRNA levels of the identified ligands/growth factors are shown. Data are mean centered Log2. FDR-corrected p values were calculated using the exact binomial test in edgeR. b Levels of AREG secreted into the media by the indicated clonal populations over the course of 36 h, as determined by an ELISA assay. Data represent as mean ± SEM from three independent experiments. c Tumor burden in vivo assessed by measurement of luciferase activity in whole blood samples collected at the indicated time points. Data presented as fold change in luciferase activity compared to 24 h post injection. Representative of two independent experiments. Data summarized by mean ± SD, p values were computed using the Student’s t test and FDR corrected. d Fold change in luciferase activity in blood samples of individual mice collected at the end point (10 weeks) relative to the 24 h time point. The data shown as mean ± SD. p Values were computed using the one-way ANOVA test and Dunnett’s multiple comparison test. e Representative H&E images of solid peritoneal metastases on the diaphragm of mice inoculated with CL31 expressing AREG or vector control. T tumor cells, D diaphragm. Scale bar, 2 mm. f Tumor burden score of solid metastases on the diaphragms of mice inoculated with CL31 expressing AREG or vector control, determined as in Fig. 2e. p Value was computed using the chi-square test with Monte Carlo simulation. d–f Data from two independent experiments, total n = 8 mice.
Fig 4: AREG blocking antibody reduces solid peritoneal metastasis.a Tumor growth dynamics of an equal mixture of CL31 and CL17 (CL31:CL17) treated with 200 µg monoclonal AREG blocking antibody or vehicle control, assessed by measurement of luciferase activity in whole blood samples collected at the indicated time points. Data presented as fold change in luciferase activity compared to 24 h post injection. Data represents mean ± SD from two independent experiments, n = 9 mice each group. b Fold change in luciferase activity per mouse injected with CL31:CL17 determined as in (a) at the 10-week end point. p Value was computed using the Mann–Whitney test, two-tailed. c Malignant ascites cell pellet volume at the 10-week end point. p Value was computed using the Mann–Whitney test, two-tailed. d Tumor burden score of solid metastases on the diaphragm at 10-week end point generated by CL31:CL17 in mice treated with AREG blocking antibody or vehicle control. p Value was computed using the chi-square test with Monte Carlo simulation. e Weight of solid tumors on the diaphragms at 10-week end point of same tumors in (d). Data from two independent experiments, n = 9 mice in total for each group, and shown as mean ± SD. p Values from Mann–Whitney test, two-tailed. NS not significant. f Individual diaphragms from mice in one of the two experiments at the 10-week end point.
Fig 5: Transient exposure to CL17 or AREG is sufficient to promote peritoneal metastasis of CL31.a Schematic depiction of experimental design of short-term supplementation with human recombinant AREG in vivo. b Representative H&E images of diaphragms collected from mice at the 10-week end point supplemented with AREG or BSA vehicle control as depicted in (a), representative images from one of two independent experiments. T tumor cells, D diaphragm, B bone. Scale bar, 2 mm. c Tumor burden score of solid metastases on the diaphragm generated by CL31 in mice treated with AREG or BSA vehicle control. Data from two independent experiments, total n = 8 mice. p Value was computed using the chi-square test with Monte Carlo simulation. d Fold change in luciferase activity in blood samples collected from individual mice at the end point (10 weeks) relative to the 24-h time point. Data from two independent experiments (total n = 8 mice) and shown as mean ± SD. p Value was computed using the Mann–Whitney test, two-tailed. NS not significant. e Representative H&E images of the diaphragms of mice inoculated with CL31 alone (3 × 106 cells) or a 1:1 mixture of CL31 and CL17 (3 × 106 total cells). T tumor cells, D diaphragm, L liver, B bone. Scale bar, 2 mm. f Tumor burden score of solid metastases on the diaphragm generated by the indicated cell inocula. Data from two independent experiments (total n = 6–7 mice) and shown as mean ± SD. p Values were computed using the chi-square test with Monte Carlo simulation. NS not significant. g Fold change in luciferase activity in blood samples collected from individual mice at the end point (10 weeks) relative to the 24-h time point. Data from two independent experiments (total n = 6–7 mice) and shown as mean ± SD. p Values were computed using the one-way ANOVA test and Dunnett’s multiple comparison test. NS not significant.
Supplier Page from Abcam for Human Amphiregulin ELISA Kit (AREG)