Cancer-associated fibroblasts induce epithelial-mesenchymal transition of breast cancer cells through paracrine TGF-β signalling

Abstract

Background: Cancer-associated fibroblasts (CAFs) activated by tumour cells are the predominant type of stromal cells in breast cancer tissue. The reciprocal effect of CAFs on breast cancer cells and the underlying molecular mechanisms are not fully characterised.

Methods: Stromal fibroblasts were isolated from invasive breast cancer tissues and the conditioned medium of cultured CAFs (CAF-CM) was collected to culture the breast cancer cell lines MCF-7, T47D and MDA-MB-231. Neutralising antibody and small-molecule inhibitor were used to block the transforming growth factor-β (TGF-β) signalling derived from CAF-CM, which effect on breast cancer cells.

Results: The stromal fibroblasts isolated from breast cancer tissues showed CAF characteristics with high expression levels of α-smooth muscle actin and SDF1/CXCL12. The CAF-CM transformed breast cancer cell lines into more aggressive phenotypes, including enhanced cell-extracellular matrix adhesion, migration and invasion, and promoted epithelial-mesenchymal transition (EMT). Cancer-associated fibroblasts secreted more TGF-β1 than TGF-β2 and TGF-β3, and activated the TGF-β/Smad signalling pathway in breast cancer cells. The EMT phenotype of breast cancer cells induced by CAF-CM was reversed by blocking TGF-β1 signalling.

Conclusion: Cancer-associated fibroblasts promoted aggressive phenotypes of breast cancer cells through EMT induced by paracrine TGF-β1. This might be a common mechanism for acquiring metastatic potential in breast cancer cells with different biological characteristics.

Figure 1

Stromal fibroblasts isolated from breast cancer tissues exhibit characteristics of CAFs. (A) H&E staining of paraffin-embedded breast cancer tissue sections ( × 400). There were stromal cells surrounding the cancer nests and distributed among the invasive cancer cells in the heterogeneous cancer tissue. (B) Activated fibroblast marker α-SMA expression in paraffin-embedded primary breast cancer tissue sections was detected by immunohistochemistry ( × 400). Stromal fibroblasts surrounding cancer cells highly expressed α-SMA, present characteristics of CAFs. (C) Morphological features of primary cultured stromal fibroblasts isolated from the primary breast cancer tissue ( × 200). (D) Mesenchymal marker vimentin expression in stromal fibroblasts grown on a coverslip was detected by immunohistochemistry ( × 400). The stromal fibroblasts expressed vimentin highly. (E) Epithelial marker E-cadherin expression in the stromal fibroblasts was detected by immunohistochemistry ( × 400). The stromal fibroblasts did not express E-cadherin. (F) The expression of α-SMA and E-cadherin in stromal fibroblasts isolated from different primary breast cancer tissues was detected by immunobotting. All the primary cultured stromal fibroblasts expressed α-SMA highly but did not express E-cadherin, presenting characteristics of CAFs.

Figure 2

CAF-CM enhances the abilities of migration and invasion of breast cancer cell lines with different characteristics. (A) Morphological features of breast cancer cells. Compared with untreated control cells, the MCF-7 and T47D cells cultured in CAF-CM had fewer cell junctions, and scattered cells had elongated pseudopodia; pseudopodia in MDA-MB-231 cells in particular were significantly elongated. (B) Cell adhesion ability was measured using an cell–ECM adhesion assay. Compared with control cells, the adhesion rates of all the three cell lines cultured with CAF-CM were higher. (C) Cell migration ability was measured by a wound-healing assay. Compared with control cells, the migration distances of all the three cell lines cultured with CAF-CM were increased. (D, E) Cell migration ability was measured using a Transwell cell migration assay. The migration ability of all the three cell lines cultured with CAF-CM was significantly greater than that of the corresponding control cells. (F, G) Cell invasion ability was measured using a Transwell cell invasion assay. The invasion ability of all the three cell lines cultured with CAF-CM was significantly greater than that of the corresponding control cells. *P<0.05, **P<0.01.

Figure 3

CAF-CM induces EMT programming and phenotype in different breast cancer cell lines. (A) The expression of epithelial marker E-cadherin and mesenchymal marker vimentin was detected by immunoblotting. Compared with control cells, the cells cultured with CAF-CM had decreased E-cadherin expression in MCF-7 and T47D cells, and increased vimentin expression in MDA-MB-231 cells. (B) The expression of mesenchymal marker VIM and FN1 was detected by reverse transcription (RT)-quantitative PCR (QPCR). Compared with control cells, VIM and FN1 expression levels were upregulated in all the three cell lines cultured with CAF-CM. (C) The expression of epithelial marker E-cadherin and mesenchymal marker vimentin was detected by immunofluorescence staining. Compared with control cells, the cells cultured with CAF-CM had decreased E-cadherin expression in MCF-7 cells and increased vimentin expression in MDA-MB-231 cells cultured with CAF-CM. Nuclei were visualised with DAPI staining (blue). (D) The expression of MMPs was detected by RT-QPCR. MMP2 and MMP9 expression levels were upregulated in all the three cell lines cultured with CAF-CM. (E) The expression of EMT-TFs was detected by RT-QPCR. SNAI1 expression levels were upregulated in all the three cancer cell lines cultured with CAF-CM. SNAI2 expression levels were upregulated in MCF-7 and T47D cells. TWIST1 expression levels were upregulated in MDA-MB-231 cells, and ZEB1 expression levels were upregulated in MCF-7 cells. *P<0.05, **P<0.01.

Figure 4

CAFs activated TGF-β/Smad signalling of breast cancer cells through secreted TGF-β1. (A) TGF-β1, TGF-β2 and TGF-β3 secreted by CAFs in CAF-CM were detected by cytokine antibody array. Cancer-associated fibroblasts secreted significantly more TGF-β1 than TGF-β2 and TGF-β3. (B) TGFB1 expression in CAFs and breast cancer cell lines was detected by reverse transcription (RT)-quantitative PCR (QPCR). Cancer-associated fibroblasts expressed significantly higher TGFB1 expression levels than breast cancer cell lines MCF-7, T47D or MDA-MB-231. (C) The expression of TGF-βRII, total Smad2 and phosphorylated Smad2 in breast cancer cell lines cultured with CAF-CM was detected by immunoblotting. Phosphorylated Smad2 expression levels in MCF-7, T47D and MDA-MB-231 cells cultured with CAF-CM were significantly higher than their control cells, whereas total Smad2 and TGF-βRII were not significantly changed. (D) TGFB1 expression in breast cancer cells cultured with CAF-CM was detected by RT-QPCR. TGFB1 expression levels in MCF-7, T47D and MDA-MB-231 cell lines were not significantly affected by culturing in CAF-CM.

Figure 5

EMT phenotype was reversed by blocking TGF-β1 in CAF-CM cultured breast cancer cells. (A) The expression of epithelial and mesenchymal markers in breast cancer cells was detected by immunoblotting or reverse transcription (RT)-quantitative PCR (QPCR), respectively. After the TGF-β1 signalling pathway was blocked by neutralising antibody, the reduced E-cadherin in MCF-7 cells, increased vimentin in MDA-MB-231 cells and upregulated FN1 expression levels stimulated by CAF-CM in all the three cell lines were reversed. (B) The expression of EMT-TFs was detected by RT-QPCR. After the TGF-β1 signalling pathway was blocked by neutralising antibody, the upregulated SNAI1 expression levels in MCF-7 and MDA-MB-231 cells stimulated by CAF-CM were reversed. (C) The expression of epithelial and mesenchymal markers in breast cancer cells was detected by immunoblotting or RT-QPCR, respectively. After the TGF-β1 signalling pathway was blocked by adding SB-431542 in CAF-CM, the reduced E-cadherin in MCF-7 cells, increased vimentin in MDA-MB-231 cells and upregulated FN1 expression levels in both cancer cells stimulated by CAF-CM were reversed. (D) The expression of EMT-TFs was detected by RT-QPCR. After the TGF-β1 signalling pathway was blocked by adding SB-431542 in CAF-CM, the upregulated SNAI1 expression levels in MCF-7 and MDA-MB-231 cells stimulated by CAF-CM were reversed. (E) Cell migration ability was measured using a Transwell cell migration assay. The migration ability of MCF-7 and MDA-MB-231 cells cultured in CAF-CM were reversed by adding anti-TGF-β1 antibody. (F) Cell invasion ability was measured using a Transwell cell invasion assay. The invasion ability of MCF-7 and MDA-MB-231 cells culturing in CAF-CM were reversed by adding anti-TGF-β1 antibody. *P<0.05, **P<0.01.