Dataset Information

Microscale Mass Spectrometry Analysis of Extracellular Metabolites in Live Multicellular Tumor Spheroids.

ABSTRACT: Extracellular compounds in tumors play critical roles in intercellular communication, tumor proliferation, and cancer cell metastasis. However, the lack of appropriate techniques leads to limited studies of extracellular metabolite. Here, we introduced a microscale collection device, the Micro-funnel, fabricated from biocompatible fused silica capillary. With a small probe size (?25 ?m), the Micro-funnel can be implanted into live multicellular tumor spheroids to accumulate the extracellular metabolites produced by cancer cells. Metabolites collected in the Micro-funnel device were then extracted by a microscale sampling and ionization device, the Single-probe, for real-time mass spectrometry (MS) analysis. We successfully detected the abundance change of anticancer drug irinotecan and its metabolites inside spheroids treated under a series of conditions. Moreover, we found that irinotecan treatment dramatically altered the composition of extracellular compounds. Specifically, we observed the increased abundances of a large number of lipids, which are potentially related to the drug resistance of cancer cells. This study provides a novel way to detect the extracellular compounds inside live spheroids, and the successful development of our technique can benefit the research in multiple areas, including the microenvironment inside live tissues, cell-cell communication, biomarker discovery, and drug development.

SUBMITTER: Sun M

PROVIDER: S-EPMC5912160 | biostudies-other | 2017 Sep

REPOSITORIES: biostudies-other

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Microscale Mass Spectrometry Analysis of Extracellular Metabolites in Live Multicellular Tumor Spheroids.

Sun Mei M Tian Xiang X Yang Zhibo Z

Analytical chemistry 20170816 17

Extracellular compounds in tumors play critical roles in intercellular communication, tumor proliferation, and cancer cell metastasis. However, the lack of appropriate techniques leads to limited studies of extracellular metabolite. Here, we introduced a microscale collection device, the Micro-funnel, fabricated from biocompatible fused silica capillary. With a small probe size (∼25 μm), the Micro-funnel can be implanted into live multicellular tumor spheroids to accumulate the extracellular met ...[more]

PMID: 28753268

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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

Microscale Mass Spectrometry Analysis of Extracellular Metabolites in Live Multicellular Tumor Spheroids.

Publications

Microscale Mass Spectrometry Analysis of Extracellular Metabolites in Live Multicellular Tumor Spheroids.

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