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

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

Mate Rusz et al. Metabolites.

Abstract

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.

Keywords: IT-139; KP1339; LC-MS; metabolomics; metallodrugs; method development; multicellular tumor spheroids; oxaliplatin.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure A1
Figure A1
Relative responses (12C/13C) of alanine and glutamate as selected examples from a single (N = 3), 5 (N = 3), 10 (N = 3), 15 (N = 2) multicellular tumor spheroids. Sample preparation applied boiling ethanol extraction with U13C internal standardization and LC high-resolution mass spectrometry. Investigated metabolites showed high linear correlation.
Figure A2
Figure A2
Scaled relative responses (12C/13C) of alanine and glutamate as selected examples from a single (N = 3), 5 (N = 3), 10 (N = 3), 15 (N = 2) multicellular tumor spheroids: relative responses were scaled to the mean relative response in single multicellular tumor spheroids (N = 3). Sample preparation applied boiling ethanol extraction with U13C internal standardization and LC high-resolution mass spectrometry. Alanine and glutamate are shown from the investigated compounds.
Figure A3
Figure A3
Absolute amounts in nmol of amino acids after acidic hydrolysis of the pellet containing 1 (N = 3), 5 (N = 3), 10 (N = 3) and 15 (N = 2) multicellular tumor spheroids. The pellets are resulting from the high molecular fraction of the boiling ethanol extraction and centrifugation. Amino acid amounts from the proteins show a strong linear correlation with the number of spheroids in the sample.
Figure A4
Figure A4
Swarm and violinplot of total protein content [µg] in single 3D MTS cultures (10 × 103 cells seeded, 8 days cultivation diameter 790 ± 22 µm at time of treatment) after 24 h of incubation with 200 µM KP1339, 20 µM Oxaliplatin, growth medium (CntrlOxPt), growth medium with 0.5% DMSO (CntrlKP1339). Protein content is determined by the Thermo micro BCA kit from the high molecular fraction in the pellet.
Figure A5
Figure A5
PCA scores plot of absolute amounts of metabolites normalized to protein content of samples from single 3D MTS. Spheroids were treated for 24 h with either 200 µM KP1339, 20 µM Oxaliplatin (OxPt), medium (CntrlOxPt) or medium with 0.5% DMSO (CntrlKP1339). Extraction with cold methanol and internal standardization, measurement by HILIC high-resolution Orbitrap MS.
Figure 1
Figure 1
Correlation matrix of number of multicellular tumor spheroids (MTS), relative responses (12C/13C) of 29 metabolites from boiling ethanolic extracts with 13C internal standardization, absolute amounts of 8 amino acids [nmol] measured from hydrolyzed pellet (indicated with the “pellet” suffix) from samples containing 1 (N = 3), 5 (N = 3), 10 (N = 3), and 15 MTS (N = 2) from the same population. Most metabolites show a strong correlation with all the amino acids hydrolyzed from the pellet and the number of MTSs.
Figure 2
Figure 2
The study design for speeding up the sample preparation. Two different washing procedures (“ON”: washing the multicellular tumor spheroid once with PBS on the 96-well plate vs. “OFF” washing three-times in an Eppendorf-tube after transfer from cultivation well) and two alternative extractions (boiling ethanol vs. cold methanol extraction) were tested on large multicellular tumor spheroids (744 ± 15 µm), which in combination resulted in four sample groups.
Figure 3
Figure 3
Barplots of the absolute amounts (pmol) of 26 selected metabolites extracted from single multicellular tumor spheroids with four different sample preparation method as well as two washing procedures (“ON”: washing the multicellular tumor spheroid once with PBS on the 96-well plate vs. “OFF” washing three times in an Eppendorf-tube after transfer from the culture plate) and two alternative extractions (boiling ethanol, “BE” vs. cold methanol extraction “CM”). OFF/BE (N = 6), ON/BE (N = 4), OFF/CM (N = 5), ON/CM (N = 7) Measurement with liquid chromatography (HILIC separation) high-resolution Orbitrap mass spectrometry. All extractions utilized U13C internal standardization. For the abbreviations of compounds, see Appendix A.
Figure 4
Figure 4
Heatmap displaying absolute amounts of metabolites normalized to total protein content (pmol/µg) of samples from single 3D MTS. Spheroids were treated for 24 h with either 200 µM KP1339, 20 µM oxaliplatin (OxPt), medium (CntrlOxPt) or medium with 0.5% DMSO (CntrlKP1339). Numbers in sample names refer to independent biological replicates. Extraction with cold methanol and internal standardization, measurement by HILIC high-resolution Orbitrap MS.
Figure 5
Figure 5
KEGG pathways with MetaboAnalyst affected by oxaliplatin treatment (a) and KP1339 treatment (b) using pathway enrichment and topology analysis with the MetaboAnalyst pathway analysis module.

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