br Next we investigated the expression levels of integrin
Next, we investigated the expression levels of integrin β3 in normal breast tissue samples, ductal carcinoma in situ (DCIS), and in invasive and metastatic breast tumour tissues by qRT-PCR and IHC staining. We found that integrin β3 was undetectable or under-expressed in normal mammary tissues, but integrin β3 levels gradually increased during progression of tumour tissues from DCIS to metastasis (Fig. 1H and I). Collectively, these data indicated that the expression of integrin β3 is a pro-invasive or pro-metastatic factor toward breast cancer.
3.2. CAFs promote breast cancer cell invasion via integrin β3
To confirm the correlation between integrin β3 expression and the invasive ability of breast cancer, normal mammary epithelial Bay 11-7821BAY 11-7082 MCF10A, breast cancer cells with a low invasive potential (e.g. cell lines SKBr3, T47D, MDA-MB-453, BT474), and breast cancer cells with a high invasive potential (e.g. MDA-MB-468, BT549, and Hs578T) were
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Fig. 1. Integrins are dysregulated during EMT in mammary cells. (A) The expression levels of the integrin family in MCF10A/Twist and MCF10A control cells were identified by cDNA array and proteomics analyses. Data are presented as relative fold changes (> 1.2) of MCF10A/Twist vs MCF10A/Vector. (B) mRNA expression levels of integrins were determined by qRT-PCR analysis in MCF10A/Twist and MCF10A/vector cells. The data are presented as mean ± SD. (C, D) mRNA expression levels of ITGB1, ITGB3, and ITGB4 were assessed by qRT-PCR analysis of normal breast cells MCF10A and breast cancer cell lines (MCF7, BT549, and Hs578T) (C) and in BT549 cells transfected with shRNA against Twist (BT549/shTwist) or control shRNA (BT549/shCtrl) (D). The data represent mean ± SD. (E, F) A Transwell assay was carried out to test cell invasion abilities of MCF10A cells transfected with Twist, ITGB1, ITGB3, and ITGB4 (E); or BT549 cells transfected with shRNA against Twist (BT549/shTwist) or control shRNA (BT549/shCtrl) (F) in the presence of a culture supernatant derived from CAFs or NFs (*P < 0.05, **P < 0.01). (G) Integrin β3 mRNA levels were detected by qRT-PCR in normal breast tissues, ductal carcinoma in situ (DCIS), and samples of breast cancer tissues with invasive and metastatic lymph nodes. Data are shown as relative fold changes of each group compared with the normal group (n = 30 per group). (H) Representative images of IHC staining of integrin β3 in normal breast tissues, DCIS, and in breast cancer tissues with invasion and metastatic lymph nodes (n = 27 per group). Magnification: × 200. (I) Semi-quantitative analysis of integrin β3 expression presented in (H); the MOD represents the mean value of integrin β3 staining at diﬀerent clinical stages (*P < 0.05).
Fig. 2. CAFs promote breast cancer cell invasion via integrin β3 (A, B) Integrin β3 mRNA (A) and protein (B) levels in normal mammary epithelial cells and in diﬀerent breast cancer cell lines. (C) The in-vasion potentials of normal mammary epithelium and of the indicated breast cancer cell lines in the presence of a culture supernatant (CM) derived from CAFs or NFs (**P < 0.01). (D, E) Integrin β3 mRNA
subjected to determination of integrin β3 levels. Consistent with the above findings, the mRNA and protein expression levels of integrin β3 turned out to significantly increase in the malignant breast cancer cells (Fig. 2A and B). Indeed, the invasive ability of breast cancer cells co-cultured with CAFs significantly increased (Fig. 2C), this phenomenon was closely associated with their integrin β3 levels (Fig. 2A). To clarify whether CAFs can promote breast cancer cell invasion via
integrin β3, the invasive potentials of integrin β3 knockdown BT549 and Hs578T cells were evaluated next. The knockdown of integrin β3 (Fig. 2D and E) reduced tumour cell invasion (Fig. 2F). Notably, during co-culture of integrin β3 knockdown tumour cells with CAFs or NFs, a significant attenuation of the invasive ability was detected only in tu-mour cells co-cultured with CAFs (not with NFs; Fig. 2G), suggesting that integrin β3 is essential for CAF-stimulated tumour cell invasion.
Fig. 3. Diﬀerential cytokine levels between CAFs and NFs. (A) The diﬀerentially expressed cytokine genes identified by microarray analysis of CAFs and NFs. Data are shown as relative fold changes (> 1.2) in CAFs vs NFs. (B) Expression of 10 randomly selected cytokine genes was verified by qRT-PCR in CAFs and NFs. (C) Three dysregulated cytokines containing the RGD motif in CAFs were identified by a bioinformatic analysis. (D) mRNA levels of IL32, CD70, and BMP1 were determined by qRT-PCR in CAFs and NFs (**P < 0.01). (E) IL32 protein levels in the culture supernatant of CAFs and NFs were measured by an ELISA (**P < 0.01). (F) IL32 mRNA expression levels were detected in CAFs and their paired NFs derived from breast tumour tissues (**P < 0.01, n = 18). (G) IL32 protein levels in the culture supernatant of primary CAFs and their paired NFs were measured by an ELISA (*P < 0.05). (H) The dose eﬀects of rIL32 (0–40 ng/ml) on the invasiveness of BT549 cells (*P < 0.05, **P < 0.01). The vehicle (PBS) served as a control for rIL32.