Abstract
BackgroundMulticellular tumor spheroids are models of increasing interest for cancer and cell biology studies. They allow considering cellular interactions in exploring cell cycle and cell division mechanisms. However, 3D imaging of cell division in living spheroids is technically challenging and has never been reported.ResultsHere, 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.ConclusionsAs 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.
Highlights
Multicellular tumor spheroids are models of increasing interest for cancer and cell biology studies
To validate the performance of this setup according to the results obtained by Verveer and collaborators [14], we first performed 3D imaging of fixed large spheroids (> 400 μm diameter) of the human pancreatic cancer cell line Capan-2 stained with DRAQ5 (Additional file 2)
We developed sample holders made from a biocompatible material with a refractive index close to that of water, in which spheroids can grow without mechanical stress so avoiding the regional effects on cell proliferation that are seen with the agaroseembedding procedure commonly used to present samples for selective plane illumination microscopy (SPIM) (Additional file 8)[26,27]
Summary
Multicellular tumor spheroids are models of increasing interest for cancer and cell biology studies They allow considering cellular interactions in exploring cell cycle and cell division mechanisms. 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. 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]. 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]
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