Ingenieria Bioquimica

INTERNATIONAL JOURNAL OF ONCOLOGY 34: 1433-1448, 2009

Three-dimensional culture using a radial flow bioreactor induces matrix metalloprotease 7-mediated EMT-like process in tumor cells via TGFß1/Smad pathway
SHUN-ICHI SHIBATA1, HIDEKI MARUSHIMA1, TADASHI ASAKURA1, TOMOKAZU MATSUURA2, HOMARE EDA1, KATSUHIKO AOKI1, HIROSHI MATSUDAIRA2, KAZU UEDA3 and KIYOSHI OHKAWA1 Departments of1Biochemistry, 2Internal Medicine and 3Obstetrics and Gynecology, Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan Received December 11, 2008; Accepted January 29, 2009 DOI: 10.3892/ijo_00000272 Abstract. To confirm the usefulness of the radial flow type bioreactor (RFB) for a three-dimensional (3D) culture system, which provides a tissue architecture andmolecular function mimicking the in vivo environment, molecular expression in the A431 human squamous carcinoma cell line during culture were analyzed under the physically different environments of 3D culture in the RFB, 2D culture in a monolayer as well as in nude mice. Time-dependent accumulation of autocrine transforming growth factor (TGF) ß1 was found in spent culture media obtained only from 3Dcultured A431 cancer cells, which grew well with a stratified-sheet morphology. Cells in the RFB overexpressed matrix metalloproteinase 7 (MMP7) and showed an increased release of soluble 80-kDa fragments of E-cadherin into the media time-dependently, resulting in the reduction of E-cadherin protein at the cell surface without down-regulation of the mRNA. ß-Catenin and its nuclear partner, LEF1,were up-regulated and Wnt protein secretion was also accelerated. Additional up-regulation of the transcriptional factors, HMGA2 and down-stream Slug, was noted. TGFß1-dependent, MMP7-mediated up-regulation of ß-catenin/LEF1 signaling and TGFß1-activated HMGA2 pathways consequently converged with Slug overexpression, due to disassembly and further repression of E-cadherin expression, which wasreproducible in the epithelial mesenchymal transition process without any manipulation. Other transcriptional factors, Notch/HEY1 and NF-κB, were also up-regulated in 3D-cultured cells. These signals recruited molecules related to extracellular matrix-cell remodeling and angiogenesis. Expression of several representative molecules in the 3D cultured cells was parallel with that in xenotransplantedA431 tumor tissues in nude mice. 3D culture of tumor cells in the RFB is a useful tool for cancer experimental biology and evaluation of cancer therapeutic-like systems in nude mice. Introduction In vitro tumor growth in a three-dimensional (3D) architecture has been demonstrated to play an important role in cancer biology research (1-3) and in experiments not only on tumor expansion and metastasis(1,4,5), but also on the resistance of cancers to various anticancer agents or irradiation (6,7). In general, the conventional monolayer (2D) cell culture method can be used more easily for the manipulation of cells, especially genes, to investigate the genetic and biochemical changes during carcinogenesis and cancer therapy, but the induced effects can only be observed in a flat, 2D sheet ofculture cells. To resolve this dilemma, a novel culture system that closely mimics the living environment in which cancer pathogenesis occurs is required (1,2). Animal models yield a more accurate morphological representation of the tumor environment, but there may be unknown biological effects of the specific animal itself on the tumor cells; moreover, animal models are less amenable tolarge-scale studies (2,8). To this end, as mentioned by Friedrich (2), a 3D cell culture system, which combines the benefits of animal models and 2D cell culture, appears to be an in vitro culture system that allows cells to develop into structures similar to those in living organisms, thereby enabling the investigation of some of the biological changes occurring after the manipulation of cancer cells....