Figure 5. Suppressing amphiregulin expression in tumor-supportive fibroblasts reduces tumorigenicity and amount of mesenchymal cells.

(A) Reduced amphiregulin protein levels in HFFF2 fibroblasts expressing two distinct shRNAs directed against AREG compared to cells expressing non-targeting shRNA (immunoblotting). β-actin was used as a loading control. shAREG-2 was more potent at suppression both by immunoblotting and quantitative RT-PCR (see also Figure S3). (B) Reduced tumorigenicity of Cal51 cells co-injected with HFFF2 cells expressing either shRNAs targeting AREG as compared to control shRNA. Tumor-take rates for each group are indicated. Asterisks denote significant differences in tumor volumes for shAREG-1 or shAREG-2 compared to control (p<0.05) Data are expressed as the mean ± SEM. (C) As with (B) but for MDA-MB-231 breast cancer cells. p<0.05. (D) Immunohistochemical analysis of the effects of suppressing amphiregulin expression in tumor-supportive fibroblasts HFFF2 on Cal51 tumor cell proliferation and the tumor microenvironment (bottom panel) compared to tumors formed using Cal51 co-injected with control HFFF2 (top panel) as described in the legend to Figure 2. (E) Quantification of Ki-67 positive tumor cells in Cal51 tumors injected with control HFFF2 fibroblasts or shAREG-2 HFFF2 fibroblasts. Five different fields of three different tumors per group were scored. Error bars represent SEM. There was a small decrease in proliferation in the shAREG-2 HFFF2 fibroblast group (not statistical significance, p = 0.06). (F) Similarly performed quantification of monocytes and neutrophils, with a slight decrease observed in the shAREG-2 HFFF2 fibroblast group (not, significant, p = 0.14). (G) Similarly performed quantification of blood vessel endothelial cells, with no significant difference (p = 0.24). (H) Similarly performed quantification of activated mesenchymal cells, asterisk indicates a significant difference (p = 0.002). (I) Pericyte coverage of tumor associated blood vessels in tumors derived from injection of Cal51 cells with HFFF2 fibroblasts expressing shRNA-targeting AREG compared to control. Fifteen vessels from three tumors were examined for each group were analyzed for the ratio of pericytes (α-SMA⁺) to endothelial cells (CD31⁺). Data are expressed as the mean ± SEM. (J) The effect of amphiregulin on the replicative rate of wild-type mouse embryonic fibroblasts (WT-MEFs) measured by quantifying BrdU incorporation. Data representative of three independent experiments are shown. Concentration of amphiregulin (AREG ng/ml) is indicated on the x-axis. Asterisk indicates significant differences (p<0.05) between the control (0 ng/ml AREG) and experimental groups (10, 50, 100 and 200 ng/ml AREG). (K) As in J, but assaying the replicative rate of human fibroblasts, HFFF2.