Live cell division dynamics monitoring in 3D large spheroid tumor models using light sheet microscopy

Here, we report a major breakthrough based on the engineering of multicellular tumor spheroids expressing an histone H2B fluorescent nuclear reporter protein, and specifically designed sample holders to monitor live cell division dynamics in 3D large spheroids using an home-made selective-plane illumination microscope.As illustrated using the antimitotic drug, paclitaxel, this technological advance paves the way for studies of the dynamics of cell divion processes in 3D and more generally for the investigation of tumor cell population biology in integrated system as the spheroid model.Cell proliferation deregulation is a hallmark of tumor cells. The knowledge of the mechanisms of regulation of cell cycle control and cell proliferation is fundamental to a better understanding of the consequences of their misregulation in tumorigenesis, as well as to manipulate them in cancer therapy. The study of these mechanisms already contributed to a large extent to the improvement of targeted therapies. Nevertheless, most of these works suffer a major limitation that might account for the often-observed failure of new therapeutic strategies. Indeed, most, if not all of the studies performed so far relied on in vitro analyses of rapidly growing cancer cell lines in monolayer. These 2D models do not take into account tissue heterogeneity, cellular interactions and tumour microenvironment that have been shown to be of major relevance in tumour development [1,2]. Cell cycle control mechanisms are also dependent on cell-cell and cell-extracellular matrix interactions. For example, the orientation of the future cleavage plane at the mitosis is dependent on interaction of the cytoskeleton with intrinsic cortical factors but also with extrinsic cues including cell shape, cell-cell interactions and cell adhesion with extracellular matrix[3,4]. Therefore, studying mitosis ongoing in a 3D integrated cellular context would be of great interest.Multicellular tumor spheroids (MCTS) generated