Dataset Information

Microstructured soft devices for the growth and analysis of populations of homogenous multicellular tumor spheroids.

ABSTRACT: Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple method to obtain custom-designed self-built microwell arrays made of polydimethylsiloxane or agarose for uniform 3D cell structure generation. Such materials can provide an environment of tunable mechanical flexibility. We developed protocols to culture a variety of cancer and non-cancer cell lines in such devices and to perform molecular and imaging characterizations of the spheroid growth, viability, and response to pharmacological treatments. Hundreds of tumor spheroids grow (in scaffolded or scaffold-free conditions) at homogeneous rates and can be harvested at will. Microscopy imaging can be performed in situ during or at the end of the culture. Fluorescence (confocal) microscopy can be performed after in situ staining while retaining the geographic arrangement of spheroids in the plate wells. This platform can enable statistically robust investigations on cancer biology and screening of drug treatments.

SUBMITTER: Tartagni O

PROVIDER: S-EPMC10020259 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Microstructured soft devices for the growth and analysis of populations of homogenous multicellular tumor spheroids.

Tartagni Ottavia O Borók Alexandra A Mensà Emanuela E Bonyár Attila A Monti Barbara B Hofkens Johan J Porcelli Anna Maria AM Zuccheri Giampaolo G

Cellular and molecular life sciences : CMLS 20230316 4

Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple ...[more]

PMID: 36929461

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Project description:Multicellular tumor spheroids (MCTS) are valuable in vitro tumor models frequently used to evaluate the penetration and efficacy of therapeutics. In this study, we evaluated potential differences in epigenetic markers, i.e., histone post-translational modifications (PTMs), in the layers of the HCT116 colon carcinoma MCTS. Cells were grown in agarose-coated 96 well plates, forming reproducible 1-mm-diameter MCTS. The MCTS were fractionated into three radially concentric portions, generating samples containing cells from the core, the mid and the external layers. Using mass spectrometry (MS)-based proteomics and EpiProfile, we quantified hundreds of histone peptides in different modified forms; by combining the results of all experiments, we quantified the abundance of 258 differently modified peptides, finding significant differences in their relative abundance across layers. Among these differences, we detected higher amounts of the repressive mark H3K27me3 in the external layers, compared to the core. We then evaluated the epigenetic response of MCTS following UNC1999 treatment, a drug targeting the enzymes that catalyze H3K27me3, namely, the polycomb repressive complex 2 (PRC2) subunits enhancer of zeste 1 (EZH1) and enhancer of zeste 2 (EZH2). UNC1999 treatment resulted in significant differences in MCTS diameter under drug treatment of varying duration. Using matrix-assisted laser desorption/ionization (MALDI) imaging, we determined that the drug penetrates the entire MCTS. Proteomic analysis revealed a decrease in abundance of H3K27me3, compared to the untreated sample, as expected. Interestingly, we observed a comparable growth curve for MCTS under constant drug treatment over 13 days with those treated for only 4 days at the beginning of their growth. We thus demonstrate that MS-based proteomics can define significant differences in histone PTM patterns in submillimetric layers of three-dimensional (3D) cultures. Moreover, we show that our model is suitable for monitoring drug localization and regulation of histone PTMs after drug treatment.

Dataset Information

Microstructured soft devices for the growth and analysis of populations of homogenous multicellular tumor spheroids.

Publications

Microstructured soft devices for the growth and analysis of populations of homogenous multicellular tumor spheroids.

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