Influences of tumor stroma on the malignant phenotype Jørgen Dau Nielsen 1 , Mette Moeslund 1 , Hans H. Wandall 2 , Sally Dabelsteen 3 1 Department of Oral Diagnostics, Dental School, Faculty of Health Sciences, Copenhagen University, Copenhagen; 2 Department of Cellular and Molecular Medicine, Faculty of Health Sciences, Copenhagen University, Copenhagen; 3 Department of Oral Medicine, Clinical Oral Physiology, Oral Pathology & Anatomy, Faculty of Health Sciences, Copenhagen University, Copenhagen, Denmark BACKGROUND: The microenvironment makes a significant contribution to the progression of oral carcinomas. Many different stromal events, such as vas- cularization, fibroblast activation, myofibroblast differ- entiation, and the presence of specific stromal proteins, such as proteolytic enzymes, fibronectin and laminin 5 are all characteristics of the tumor stroma. Less is, however, known of the significance of the biophysical properties of the tumor stroma. The purpose of the present study was to investigate how cellular and mechanical properties of the three-dimensional collagen matrix may influence cell proliferation and invasion of oral carcinoma cell lines. METHOD: Oral cancer cells were cultured in an orga- notypic culture system on different collagen concentra- tions, and invasion was measured. Furthermore, the presence of cancer associated proteins such as glyco- sylated oncofetal fibronectin and laminin 5 was investi- gated. RESULTS: We found that expression of glycosylated oncofetal fibronectin was increased in the invasive phe- notype of oral carcinoma cell lines. Furthermore we demonstrated that certain concentrations of collagen in the connective tissue equivalent, appears to stimulate invasiveness of oral carcinoma cells. J Oral Pathol Med (2008) 37: 412–416 Keywords: invasion; oncofetal fibronectin; oral cancer; tumor stroma Introduction The induction and progression of human cancers have conventionally been attributed to the stepwise accumu- lation of genetic lesions within the target epithelium. Recent years, however, have witnessed a paradigm shift in which the microenvironment is increasingly recog- nized to make a significant contribution to tumor progression (1–5). Tumor growth and invasion involves multiple inter- actions not only between tumor cells, but also between tumor cells and stromal cells and between tumor cells and the extracellular stroma. The importance of vascu- larization of tumor stroma is well established (6). Activation of fibroblasts also seems to be a central event that occurs in carcinoma-associated stroma (5, 7– 11). Myofibroblast differentiation along with increased ability to secrete growth and invasive stimulating factors is a characteristic of tumor-associated fibroblasts (1, 4, 12–14). Furthermore, the presence of specific stromal proteins, such as proteolytic enzymes, fibronectin and laminin 5 may also be a characteristic of the invasive phenotype (5, 8, 13, 15). Less is, however, known of the significance of the biophysical properties of the tumor stroma. It appears that cell behavior in vitro differs when cells are cultured in matrix systems rather than two- dimensional substrates (16, 17). In three-dimensional gels, gene expression and cellular behavior can simply be changed by altering the density of the collagen gel (18– 21). Breast epithelial cells differentiate into tubules if the cells are cultured in three-dimensional collagen matrix floating in medium, but if the matrix is denser, prolif- eration is favored. This observation is notable, because women with dense breast tissue, which is linked to a substantial increase in collagen deposition in the stroma, have a four- to sixfold increased risk of developing breast cancer (22). These observations are of special interest to the development of oral cancer in patients with submucous fibrosis; a chronic disease characterized by progressive fibrosis of subepithelial connective tissue and increased risk of malignant transformation (23). Previous studies of the interaction of oral squamous cell carcinomas and their non-malignant fibroblast neighbors are based mainly on in vitro studies using different invasion assays (5, 12, 24) including organo- typic systems (4, 25) that consist of a connective tissue equivalent where fibroblasts are cultured in an extra- cellular matrix of collagen I (10). Tumor cells are then cultured on top of the connective tissue equivalent. The organotypic system is a good in vivo equivalent in which epithelial mesenchymal interaction can be studied (17, Correspondence: Sally Dabelsteen, Department of Oral Medicine, Clinical Oral Physiology, Oral Pathology & Anatomy, Faculty of Health Sciences, Nørre Alle¢ 20, DK-2200, Copenhagen, Denmark. Tel: + 45 3532 66 52, Fax: + 45 3532 6722, E-mail: sally@odont.ku.dk Accepted for publication December 17, 2007 J Oral Pathol Med (2008) 37: 412–416 ª 2008 The Authors. Journal compilation ª 2008 Blackwell Munksgaard Æ All rights reserved www.blackwellmunksgaard.com/jopm doi: 10.1111/j.1600-0714.2008.00655.x