Single cell organization and cell cycle characterization of DNA stained multicellular tumor spheroids

Abstract

Multicellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.

Miniaturized and multiplexed high-content screening of drug and immune sensitivity in a multichambered microwell chip

“…To perform drug screening assays with 3D cell cultures, we used USW-assisted formation of tumor spheroids previously developed in our labs ( Christakou et al., 2015 ; Olofsson et al., 2018 , 2021 ; Vanherberghen et al., 2010 ; Wiklund et al., 2014 ). A498 renal carcinoma cells were seeded and exposed to ultrasound for 24 h, followed by 24 h of standard cell culture.…”

“…In contrast, paclitaxel-induced apoptosis was confined to the periphery of OVCAR8 spheroids ( Figure S3 G). Using image-analysis-based DNA content quantification, we have previously shown that cells in an active cell cycle stage are more common in the cell layers close to the spheroid surface ( Olofsson et al., 2021 ). Thus, it is possible that paclitaxel-induced apoptosis is preferentially observed in dividing cells in the periphery of the spheroids.…”

ARTseq-FISH reveals position-dependent fate decisions driven by cell cycle changes

“…6d-f, centre). DAPI intensity differences [57][58][59] indicate temporal changes in the cell cycle at both the edge and centre (Fig. 6g-h, Supplementary figure 20a and SN2 Fig.…”

Computational multifocus fluorescence microscopy for three-dimensional visualization of multicellular tumor spheroids

“… 2 Since most cellular components are colorless, to observe for example the nuclei in MCTS, cells are usually stained with DNA binding fluorescent probes such as 4’,6-diamidino-2-phenylindole (DAPI) and observed through a fluorescence microscope. 3 Fluorescent staining of DNA by DAPI then allows to visualize of cell nuclei within 3D MCTS 4 to provide morphological information about the architecture of the MCTS. 5 …”