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

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.

Metabolic Drug Response Phenotyping in Colorectal Cancer Organoids by LC-QTOF-MS

“…Thus, protocol C (37 °C PBS washing and "in well" sampling) offers a fast and simple procedure for repeatable metabolic phenotyping of colon cancer organoids with reasonable coverage of metabolites and lipids. In particular, the protocol enables rapid quenching of metabolic reactions in less than 1 min and metabolite extracts from 30 samples are ready for LC-MS analysis within less than 2 h. Such advantages of fast extraction with minimal cell manipulation are in accordance with recent findings from protocol optimization experiments for tumor spheroid metabolomics, where the optimized protocol consisted of rapid on plate washing followed by cold methanol extraction [31]. Extraction protocols evaluated for metabolomic and lipidomic profiling of colorectal cancer (CRC) organoids using LC-QTOF-MS after dual LC separation by HILIC and RPLC.…”

Inhibition of DNA Repair Pathways and Induction of ROS Are Potential Mechanisms of Action of the Small Molecule Inhibitor BOLD-100 in Breast Cancer

“…Monitoring of DSB in human tissues such as blood or skin provides a minimally invasive strategy to monitor efficacy of therapeutics in patients [28], and, therefore, could also be a useful biomarker to evaluate the efficacy of BOLD-100. Furthermore, recent studies have reported changes in cellular metabolite profile [33] or protein associated with specific metabolic pathways [34] including DDR following treatment with BOLD-100. Consistently, our data show an increase in DNA damage in breast cancer cells, as shown by an increase in gamma-H2AX.…”

Thermodynamic genome-scale metabolic modeling of metallodrug resistance in colorectal cancer

“…Accordingly, response profiling with metabolomics analysis can be a powerful tool to investigate drugs and drug candidates [28,27] and dissecting emerging resistance [85]. Currently, only a handful of studies consider metallodrugs applied to cancers with metabolomics [86,87,88], and even fewer investigate acquired metallodrug resistance [52].…”