Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial-mesenchymal transition in vitro.

Epithelial-mesenchymal transition (EMT) acts as a facilitator of metastatic dissemination in the invasive margin of malignant tumors where active tumor-stromal crosstalks take place. Co-cultures of cancer cells with cancer-associated fibroblasts (CAFs) are often used as in vitro models of EMT. We established a tumor-fibroblast proximity co-culture using HT-29 tumor spheroids (TSs) with CCD-18 co fibroblasts. When co-cultured with TSs, CCD-18 co appeared activated, and proliferative activity as well as cell migration increased. Expression of fibronectin increased whereas laminin and type I collagen decreased in TSs co-cultured with fibroblasts compared to TSs alone, closely resembling the margin of in vivo xenograft tissue. Active TGFβ1 in culture media significantly increased in TS co-cultures but not in 2D co-cultures of cancer cells-fibroblasts, indicating that 3D context-associated factors from TSs may be crucial to crosstalks between cancer cells and fibroblasts. We also observed in TSs co-cultured with fibroblasts increased expression of α-SMA, EGFR and CTGF; reduced expression of membranous β-catenin and E-cadherin, together suggesting an EMT-like changes similar to a marginal region of xenograft tissue in vivo. Overall, our in vitro TS-fibroblast proximity co-culture mimics the EMT-state of the invasive margin of in vivo tumors in early metastasis.