Figure 5

HLA class I expression in melanoma cells cultured in 2D or in MCTS. (A) Flow cytometric analysis of HLA-A^*0201 and HLA Class I expression in melanoma cells cultured in 2D or in MCTS at different cell densities. The HBL, D10, and NA8 cells were cultured for 7 days in 2D or 3D at the indicated cell densities. Cells were then harvested, and aggregates were disrupted by vigorous pipetting and trypsinisation. Cells were stained either with control antibodies (thin lines) or with mAbs recognising HLA-A^*0201 or a monomorphic epitope on HLA class I heavy chains (bold lines). Relevant mean fluorescence intensities, reported in individual histograms, were calculated by subtracting values deriving from isotype control staining from experimental values. (B) Expression of c-myc and IRF-1 genes in melanoma cells cultured in 2D or in MCTS at different cell densities. HBL, D10, or NA8 cells were cultured for 3 days in 2D (□) or in 3D (: 5000 cells – ▪: 30 000 cells). Gene expression was analysed by quantitative real-time PCR and results are expressed as ratio to specific gene expression, as observed in the corresponding 2D sample used as reference.