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 from cancer cells are attractive models to study cancer proliferation in 3D. Indeed these 3D complex multicellular systems reproduce cell-cell and cell-matrix interactions as found in solid tumors . Moreover, MCTS can grow to diameters of several hundred micrometers, progressively developing a gradient of proliferating cells similar to that found in non-vascularised micro-regions of a tumor: dividing cells are located in the outer layers and quiescent cells are located more centrally in regions that are hypoxic and receive few nutrients[6, 7].
Despite our extensive knowledge of the molecular mechanisms that control the cell cycle and cell proliferation, and the consequences of their misregulation for tumorigenesis, we have only a rudimentary understanding of the spatio-temporal dynamics of tumor cell proliferation in complex 3D systems such as spheroids. Expression of specific cell markers within spheroids can be detected by immunofluorescence microscopy of paraffin-embedded or frozen sections or by using flow cytometry after enzymatic dissociation of the cells, but these methods do not allow us to consider the 3D organization of spheroids[8, 9]. A few studies have reported 3D imaging of tumor spheres enriched for cancer stem cells, however, these were small spheres (< 150 μm diameter) comprising few cells [10, 11]. The strategies employed cannot be applied to larger spheroids (> 300 μm diameter) that reproduce tumor organization, and they are incompatible with real-time imaging. Indeed, investigation of the dynamics of living cellular processes in 3D inside large spheroids remains technically very challenging.
A light-sheet-based microscopy method known as selective plane illumination microscopy (SPIM) is well adapted to imaging large samples in 3D [12–14]. In SPIM, a sheet of light illuminates the sample perpendicular to the axis of detection at the focal plane of the microscope objective, thus providing optical sectioning of the whole sample. Images are recorded with a CCD camera one plane at a time with high temporal resolution, thus limiting phototoxicity and facilitating imaging of live samples . Furthermore, the combination of several views permits merging of the data obtained at various angles to produce an image in 3D . One study has reported the use of SPIM to image chemically fixed, small (~140 μm diameter) spheroids of BxPC-3 cells - a human pancreatic cancer cell line - stained with the fluorescent DNA dye DRAQ5 , reviewed in .
In this publication we report the first demonstration that SPIM 3D imaging technology is also adapted to live imaging of MCTS that have been engineered to express fluorescent reporter. It is therefore an attracting new approach to explore unsolved issues on the cell division and proliferation dynamics of cancer cells in 3D using the Multicellular Tumor Spheroid model.