Interactions of B16F10 melanoma cells aggregated on a cellulose substrate

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 Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

Generation of a tumor spheroid in a microgravity environment as a 3D model of melanoma

“…This suggests that the cells were functional and that there was signaling between B16.F10 and HaCaT. Natural occurring HaCaT cells react similar in the 3D model regulating B16.F10 growth and survival through cell-cell interaction, promoting the production of extracellular matrix, displaying organized growth patterns and formation of dendrite-like processes (Li and Herlyn 2000; Hindie et al 2006, Hsu et al 2002). HaCaT and B16.F10 cells remained viable in the center of the scaffold because they received even exchange of nutrients and oxygen while rotating in this system uninterrupted.…”

“…On the other hand, fibronectin is found to be secreted at the cell surface of B16.F10 aggregates when interacting with integrins. Fibronectin and integrins are involved in cell attachment and signaling between the inside and outside of the cells (Hindie et al 2006). Keratinocytes are known to regulate melanocyte growth and proliferation through expression of adhesion molecules such as cadherins.…”

Study of cell behaviour on a cellulose anti-adhesive substratum

“…PolyHEMA, which has been widely used for contact lens and intra-ocular implants, prevents cell adhesion and spreading (Bretland et al 2001;DiazMontero and McIntyre 2005;Feder-Mengus et al 2007;Folkman and Moscona 1978;Ghosh et al 2005;Gilmore et al 2000;Krylov and Dovichi 2000). Our previous studies showed that a novel anti-adhesive cellulose substratum, CEL, is suitable for studying the molecular mechanisms underlying cell-substratum and cell-cell interactions (Hindie et al 2005(Hindie et al , 2006. CEL was designed as an original and sterile bi-layered coating capable of securing an anti-adhesive long-term effect: indeed, HPMC was suitable for short-time assays only (\24 h) and CMC used as a direct coating did not cover completely the bottom of the Petri dish.…”

“…According to Hindie et al (2006), the cells were suspended in 250 lL PBS-EDTA, fixed by adding 750 lL ice-cold absolute ethanol and stored at -20°C for 1-2 weeks. These cells were washed with PBS-EDTA and suspended in PBS-EDTA containing 0.1% Triton X-100 (Sigma Aldrich, St. Quentin Fallavier, France), mixed with 40 lg RNase A (Sigma Aldrich, St. Quentin Fallavier, France) and 25 lg propidium iodide (PI, Sigma Aldrich, St. Quentin Fallavier, France) and incubated for 15 min in the dark.…”

“…We have shown that bi-layered hydroxypropylmethylcellulose-carboxymethylcellulose-coated Petri-dishes (CEL) provide an antiadhesive substratum suitable for studying the molecular mechanisms underlying cell-substratum and cell-cell interactions. They also induce the differentiation of B16F10 melanoma cells (Hindie et al 2005(Hindie et al , 2006. This report describes the behaviour of three adherent cell lines: Swiss 3T3 fibroblasts, MC-3T3 pre-osteoblasts and B16F10 highly metastatic melanoma cells, cultured on CEL for 24 and 48 h. Cell morphology, growth, cell cycle progression and anoikis were analysed.…”