Many strategies have been proposed to circumvent
cancer development or prevent its growth. One of the promising strategies is to direct the immune response toward tumour
antigens. This can be achieved by loading dendritic cells, the most potent antigen presenting cells, with tumour
antigens. Fusion of dendritic cells (DC) with tumour cells is an attractive way to load the DC with all tumour
antigens regardless of their immunogenicity status and the fact that they have, or not, been identified. The aim of our study was to characterise the immunophenotype of fused cells, monitor the evolution of the fusion interface and the distribution of
surface antigens over time and assess for their maturation status and functionality in vitro. We used
polyethylene glycol to fuse DC with Her2/neu positive
breast cancer cell line T-47D. We demonstrate that false positive events accounted in flow cytometry can be identified using confocal microscopy to avoid an overestimation of fusion efficiency and to distinguish clearly hybrid cells from aggregated or phagocytosed cells. We used imaging means to demonstrate the conservation of presentation
molecules (MHC II, CD1a), co-stimulatory molecules (CD40, CD80, CD86), as well as tumour
antigens (Her2/neu, cytokeratins) in optimised conditions. Fused cells were only recognisable for 48 h as assessed by membrane staining and membranous
antigen distribution. Fusion was necessary for their maturation to be accompanied by functional activity such as secretion of
cytokines and
perforin. These results suggest that hybrid cells generated by the fusion of DC and tumour cells can be easily identified and characterised using imaging techniques, and that, regarding functionality and
cytokine secretion, they appear to be good candidates for anti-tumour
therapies namely in
breast cancer.