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Open AccessArticle

Single Spheroid Metabolomics: Optimizing Sample Preparation of Three-Dimensional Multicellular Tumor Spheroids

1
Institute of Inorganic Chemistry, University of Vienna, Währinger Str. 42, 1090 Vienna, Austria
2
Institute of Analytical Chemistry, University of Vienna, Währinger Str. 38, 1090 Vienna, Austria
3
Vienna Metabolomics Center (VIME), University of Vienna, Althanstraße 14, 1090 Vienna, Austria
4
Research Network Chemistry Meets Microbiology, Althanstraße 14, 1090 Vienna, Austria
*
Author to whom correspondence should be addressed.
Metabolites 2019, 9(12), 304; https://doi.org/10.3390/metabo9120304
Received: 9 November 2019 / Revised: 5 December 2019 / Accepted: 12 December 2019 / Published: 14 December 2019
(This article belongs to the Special Issue Sample Preparation in Metabolomics)
Tumor spheroids are important model systems due to the capability of capturing in vivo tumor complexity. In this work, the experimental design of metabolomics workflows using three-dimensional multicellular tumor spheroid (3D MTS) models is addressed. Non-scaffold based cultures of the HCT116 colon carcinoma cell line delivered highly reproducible MTSs with regard to size and other key parameters (such as protein content and fraction of viable cells) as a prerequisite. Carefully optimizing the multiple steps of sample preparation, the developed procedure enabled us to probe the metabolome of single MTSs (diameter range 790 ± 22 µm) in a highly repeatable manner at a considerable throughput. The final protocol consisted of rapid washing of the spheroids on the cultivation plate, followed by cold methanol extraction. 13C enriched internal standards, added upon extraction, were key to obtaining the excellent analytical figures of merit. Targeted metabolomics provided absolute concentrations with average biological repeatabilities of <20% probing MTSs individually. In a proof of principle study, MTSs were exposed to two metal-based anticancer drugs, oxaliplatin and the investigational anticancer drug KP1339 (sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]), which exhibit distinctly different modes of action. This difference could be recapitulated in individual metabolic shifts observed from replicate single MTSs. Therefore, biological variation among single spheroids can be assessed using the presented analytical strategy, applicable for in-depth anticancer drug metabolite profiling. View Full-Text
Keywords: multicellular tumor spheroids; metabolomics; metallodrugs; oxaliplatin; KP1339; method development; LC-MS; IT-139 multicellular tumor spheroids; metabolomics; metallodrugs; oxaliplatin; KP1339; method development; LC-MS; IT-139
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MDPI and ACS Style

Rusz, M.; Rampler, E.; Keppler, B.K.; Jakupec, M.A.; Koellensperger, G. Single Spheroid Metabolomics: Optimizing Sample Preparation of Three-Dimensional Multicellular Tumor Spheroids. Metabolites 2019, 9, 304.

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