Abstract
Multicellular tumour spheroids are used as a culture model to reproduce the 3D architecture, proliferation gradient and cell interactions of a tumour micro-domain. However, their 3D characterization at the cell scale remains challenging due to size and cell density issues. In this study, we developed a methodology based on 3D light sheet fluorescence microscopy (LSFM) image analysis and convex hull calculation that allows characterizing the 3D shape and orientation of cell nuclei relative to the spheroid surface. By using this technique and optically cleared spheroids, we found that in freely growing spheroids, nuclei display an elongated shape and are preferentially oriented parallel to the spheroid surface. This geometry is lost when spheroids are grown in conditions of physical confinement. Live 3D LSFM analysis of cell division revealed that confined growth also altered the preferential cell division axis orientation parallel to the spheroid surface and induced prometaphase delay. These results provide key information and parameters that help understanding the impact of physical confinement on cell proliferation within tumour micro-domains.
Highlights
Multicellular spheroids reproduce the three-dimensional (3D) multicellular architecture, cell-cell interactions as well as oxygen and proliferation gradients observed in tumour micro-domains[1,2]
We previously reported that light-sheet fluorescence microscopy (LSFM) is well suited to perform 3D imaging of large samples, such as spheroids derived from HCT116 cancer cells[11,12,14]
We adapted clearing with benzyl alcohol-benzyl benzoate (BABB) to spheroids stained with propidium iodide after fixation to improve in depth resolution for the analysis of the 3D shape of nuclei (Fig. 1b)
Summary
Multicellular spheroids reproduce the three-dimensional (3D) multicellular architecture, cell-cell interactions as well as oxygen and proliferation gradients observed in tumour micro-domains[1,2]. We explored the effect of a confined environment on spheroid growth, and showed that multicellular aggregates grown in PDMS micro-channels for several days adopt an elongated rod-like shape with the longer axis parallel to the channel axis[10]. Most of the studies on the impact of confinement on spheroid growth and proliferation were based on the analysis of the spheroid global shape, or on the characterization at the cell scale using imaging strategies based on 2D optical sections or cryosections These approaches are not appropriate to characterize the impact of confinement on cell behaviour parameters, such as shape and orientation of nuclei, as well as the orientation of cell division that should be performed directly using 3D data. These results are consistent with the growing body of evidence that the physical microenvironment is an essential actor of tumour growth, and suggest that confinement could impair cell division and might contribute to genomic instability
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