Fibroblasts are known to contribute to tumor development. In order to more effectively examine the influence of fibroblasts on breast carcinoma cells, we developed a three dimensional tumor growth assay that allows for quantitation of fluorescent tumor growth over 8-10 days. Using this assay, we demonstrated that ERα positive breast carcinoma cell lines exhibited increased growth in co-culture with bone and breast fibroblasts, while ERα negative breast tumor cells showed no increased growth. This enhanced growth was due mainly to soluble factors.
IL-6 has been implicated in breast cancer progression. Bone and breast fibroblasts produce large amounts of soluble IL-6, as do ERα negative tumor cell lines. In contrast, ERα positive tumor lines produce negligible IL-6. Also, we show that ERα negative cells have constitutively active STAT3, a transcription factor downstream of IL-6. The ERα positive cells exhibited low to undetectable levels of phosphorylated STAT3. When ERα positive breast tumor cells are cultured in the presence of fibroblasts or fibroblast-conditioned media (Fibro-CM), increased phosphorylation of STAT3 is seen. This phosphorylation of STAT3 is due to IL-6, since fibro-CM stripped of IL-6 attenuates the response. We demonstrate that IL-6 is the primary factor responsible for the increased growth response of ERα positive tumor cells in the presence of fibroblasts. In addition, MCF-7 cells engineered to express IL-6 (MCF-7IL-6) were able to form xenograft tumors in athymic mice without any estrogen supplementation. They also grew at a faster rate compared to wild-type MCF-7 cells, which did not form tumors without estrogen supplementation.
We examined the functional consequences of chronic exposure to IL-6 in the ERα positive breast carcinoma cells. MCF-7IL-6 cells demonstrated epithelial to mesenchymal transition (EMT) by increased expression of mesenchymal markers (Vimentin and N-cadherin) and decreased expression of the epithelial marker (E-cadherin). These cells also exhibited increased invasiveness when compared to MCF-7 wild-type cells. These data demonstrate the importance of IL-6 in breast cancer development and provide a novel mechanism that may help explain the ability of fibroblasts to promote breast tumor growth, progression, and metastasis.