Cancer-associated fibroblasts contribute to cancer progression that is caused by epithelial-mesenchymal transition (EMT). Recently, mesenchymal stem cells (MSCs) were found to be the major candidate involved in the development of tumor-promoting cancer stroma. Here we report that a-smooth muscle actin-positive myofibroblast-like cells originating from MSCs contribute to inducing EMT in side population cells of pancreatic cancer. More importantly, MSC-derived myofibroblasts function to maintain tumorinitiating stem cell-like characteristics, including augmenting expression levels of various stemness-associated genes, enhancing sphere-forming activity, promoting tumor formation in a mouse xenograft model, and showing resistance to anticancer drugs. Furthermore, both c-secretase inhibitor and siRNA directed against Jagged-1 attenuated MSC-associated E-cadherin suppression and sphere formation in pancreatic cancer side population cells. Thus, our results suggest that MSC-derived myofibroblasts play important roles in regulating EMT and tumor-initiating stem cell-like properties of pancreatic cancer cells through an intermediating Notch signal. (Cancer Sci 2013; 104: 157-164) D uring tumor progression, epithelial-mesenchymal transition (EMT) contributes considerably to the malignant characteristics of tumors such as local invasion and distant metastasis.(1,2) Epithelial-mesenchymal transition has recently been reported as the key phenomenon that tightly regulates the stem cell-like characteristics of both normal and malignant cells. (3,4) Side population (SP) technology has been widely used to isolate the stem cell-enriched fraction in a variety of tissue. Side population cells are detected by their own ability to efflux Hoechst33342 dye through an ATP-binding cassette membrane transporter. We recently found that SP cells from pancreatic cancer cells are highly responsive to transforming growth factor-b (TGF-b)-mediated EMT, invasion, and metastasis.(5) Our results suggest that SP cells are enriched with cells that undergo mesenchymal-epithelial transition (MET) after TGF-b-associated EMT. Thus, our results indicated that an EMT ⁄ MET conversion is tightly linked to malignant potential in pancreatic cancer, such as invasion ⁄ metastasis. However, the mechanisms by which the EMT ⁄ MET status is regulated within a tumor in vivo remains undetermined.The tumor microenvironment consists of various stromal cells, including tumor-associated fibroblasts, endothelial cells, pericytes, adipocytes, and immune cells.(6) Among these cell types, cancer-associated fibroblasts (CAFs) and/or myofibroblasts have been recently implicated in regulating tumor progression, invasion, and metastasis. (7,8) Cancer-associated fibroblasts and myofibroblasts secrete a number of important inflammatory mediators, including MMP-2, -3, and -9, that can alter the stromal ECM and potentiate invasion, cell motility, and metastasis.