Author Proof A Journal of Cellular Biochemistry 9999:1–9 (2006) Interactions of B16F10 Melanoma Cells Aggregated on a Cellulose Substrate M. Hindie ´, 1 M. Vayssade, 1 M. Dufresne, 1 S. Que ´ant, 1 R. Warocquier-Cle ´rout, 1 G. Legeay, 2 P. Vigneron, 1 V. Olivier, 1 J-L. Duval, 1 and M-D. Nagel 1 * 1 Domaine Biomate ´riaux-Biocompatibilite ´, UMR CNRS 6600, Universite ´ de Technologie de Compie `gne, BP20529 60205 Compie `gne Cedex, France 2 De ´partement Mate ´riaux, Centre de Transfert de Technologie du Mans, Le Mans, France Abstract There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca 2þ dependent and mediated by N-cadherins. The levels ofN-cadherin and b-catenin transcripts in cells cultured on CELand tPS were similar, but those on CELcontained less b-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and b-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CELand the cellulose coatingcaused mostofthem to arrestin G 1 . We also compared the melanin contents and tyrosinase activity ofcells on CEL and controlsgrown on tPS. Melanogenesiswasinduced in cellsaggregated on CEL. Acellulose substrate thusappearsto be an outstanding tool for studying cell–cell interactions and cell functions in 3D cultures. J. Cell. Biochem. 9999: 1–9, 2006. ß 2006 Wiley-Liss, Inc. Key words: cell aggregation; cellulose substrate; N-cadherin; b catenins; melanogenesis; melanoma cells Thephysicalpropertiesofa substrateandthe density of extracellular matrix (ECM) mole- cules can prevent or encourage cell spreading [Mooney et al., 1992; Hohn and Denker, 1994; Hohn et al.,1996].Hence,cellshape and contact with the matrix direct the growth and differ- entiation of several types of normal and cancer cells [Archer et al., 1982; Mooney et al., 1992; Hohn and Denker, 1994; Hohn et al., 1996; Bae et al., 1999; Dike et al., 1999; El-Sabban et al., 2003]. Cells in 3D cultures resemble more closely the in vivo situation with regard to cell shape and cell environment than do cells in conventional cultures [Mueller-Klieser, 1997]. Swiss 3T3 fibroblasts grown on cellulose sub- strates that poorly adsorb serum adhesive proteins, adopt a rounded shape of cells in aggregatesattachedtothesubstrate[Faucheux et al., 1999, 2004]. They proliferate more slowly and programmed cell death begins 24 and 48 h post-seeding [Ge ´kas et al., 2004]. Highly meta- static B16F10 murine melanoma cells also aggregate on cellulose (CEL), but no foca l contacts or stress fibers are formed, although these cells for m well-defined foca l adhesion complexes and stress fibers when they are spread on tissue culture polystyrene (tPS). This reflects the effective ‘‘outside-inside’’ transmembrane signaling produced by the attachment of integ- rins to substrate-adsorbed proteins [Pankov and Yamada, 2002; Wierzbicka-Patynowski and Schwarzbauer, 2003]. Our previous study [Hindie ´ et al., 2005] focused on cell/matrix relationships when melanoma cells are aggre- gated on CEL for 48 h. We found that integrins interact with the fibronectin secreted at the surface of the aggregates. The present work JCB/05-0313(20833) ß 2006 Wiley-Liss, Inc. Grant sponsor: French Ministry of Research and Technol- ogy; Grant number: ACI 2001; Grant sponsor: French Cancer League (Comite ´ de l’Oise). *Correspondence to: Prof. M-D. Nagel, Universite ´ de Technologie de Compie `gne, Domaine Biomate ´riaux-Biocom- patibilite ´ UMR CNRS 6600, BP 20529, 60205 Compie `gne Cedex, France. E-mail: Marie-Danielle.Nagel@utc.fr Received August, 4th 2005; Accepted December, 21st 2005 DOI 10.1002/jcb.20833 Published online 00 Month 2006 in Wiley InterScience (www.interscience.wiley.com).