Carcinoma-associated fibroblasts (CAFs) are a key determinant in malignant progression of cancer and represent an important target for cancer therapies. In this work, we present a microfluidic-based 3D co-culture device to reconstruct an in vitro tumor microenvironment and firstly investigate the effect of CAFs on cancer cell invasion in 3D matrix. This device is composed of six co-culture units, which enable parallel co-culture assays to be run in the presence of 3D extracellular matrix. Salivary gland adenoid cystic carcinoma (ACC) cells and CAFs embedded in matrix were co-cultured without direct contact on the device. Communication between ACC cells and CAFs could be established via medium diffused in matrix. It was observed that CAFs promoted ACC cell invasion in 3D matrix in a spheroid fashion, indicating that CAFs play a critical role in cancer invasion. We further demonstrated the effect of MMP inhibitor as an agent against CAF-promoted cancer invasion. This co-culture device reproducibly reflected the in vivo growth and invasion pattern of ACC and recreated the stroma-regulated ACC invasion. Thus, it provides a suitable platform for elucidating the mechanism of CAF-regulated cancer invasion and discovering anti-invasion drugs in a well defined 3D environment.
To investigate the molecular mechanism underlying the differentiation of osteoblasts and chondroblasts, we established a clonal cell lines, RD-C6, from Runx2-deficient mouse embryos. RD-C6 cells expressed almost undetectable levels of phenotypes related to osteoblast and chondroblast differentiation at basal culture condition, whereas treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2) or transduction of BMP-2 by adenovirus effectively induced this cell line to express mRNA related to the differentiation of osteoblasts and chondroblasts including alkaline phosphatase, osteocalcin, and osterix. Transduction of Runx2 also induced the expression of these mRNA in RD-C6 cells. BMP-2 transduction increased expression levels of mRNA for Msx2 and Dlx5, but Runx2 transduction induced no significant increases in expression levels of these mRNA. Microarray analysis using RD-C6 cells with or without rhBMP-2 treatment demonstrated that BMP-2 upregulated 66 genes including 13 transcription-related molecules such as Id1, Id2, Id4, Hey1, Smad6, Smad7, and Msx2. To confirm bone and cartilage formation ability of RD-C6 cells, we transplanted RD-C6 cells into the peritoneal cavity of athymic mice using diffusion chambers with rhBMP-2. RD-C6 cells generated unmineralized cartilage but not bone. These results indicate that BMP-2 induces Runx2-deficient cells to express markers related to osteoblast and chondroblast differentiation using a Runx2-independent pathway, but it failed to induce these cells to differentiate into bone-forming osteoblasts and mature chondrocytes.
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