Preclinical studies on metal based anticancer drugs as enabled by integrated metallomics and metabolomics

Abstract

Resistance development is a major obstacle for platinum-based chemotherapy, with the anticancer drug oxaliplatin being no exception.

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

“…The stronger metabolic change caused by the ruthenium drug (KP1339) treatment compared to oxaliplatin treatment was further confirmed by hierarchical cluster analysis, where KP1339-treated samples were separated from both controls, which was not the case for oxaliplatin-treated samples (Figure 4). Overall, the fact that we see a stronger effect in the metabolome with KP1339 treatment than with oxaliplatin treatment is not surprising, since in a study with monolayer cell cultures of the same cell line, we observed considerably milder effects with oxaliplatin, as well [37].…”

“…The stronger metabolic change caused by the ruthenium drug (KP1339) treatment compared to oxaliplatin treatment was further confirmed by hierarchical cluster analysis, where KP1339-treated samples were separated from both controls, which was not the case for oxaliplatin-treated samples (Figure 4). Overall, the fact that we see a stronger effect in the metabolome with KP1339 treatment than with oxaliplatin treatment is not surprising, since in a study with monolayer cell cultures of the same cell line, we observed considerably milder effects with oxaliplatin, as well [37]. Pathway enrichment analysis using targeted data revealed that oxaliplatin exposure affected purine metabolism (GMP and adenosine being most significantly down-regulated; glutamine, IMP, inosine, guanosine, guanine, adenine, and AMP were also affected) and pyrimidine synthesis (UMP and CMP being most significantly down-regulated; but cytidine, uracil, glutamine and thymine were also affected), which is in agreement with the accepted mechanism of action of DNA targeting [45] Pathway enrichment analysis using targeted data revealed that oxaliplatin exposure affected purine metabolism (GMP and adenosine being most significantly down-regulated; glutamine, IMP, inosine, guanosine, guanine, adenine, and AMP were also affected) and pyrimidine synthesis (UMP and CMP being most significantly down-regulated; but cytidine, uracil, glutamine and thymine were also affected), which is in agreement with the accepted mechanism of action of DNA targeting [45] ( Figure 5a, Table S3, "pathways_pathways_oxaliplatin").…”

“…Four different sample preparation strategies were compared, namely OFF and ON plate washing, both followed by either BE or CM, respectively, all involving U 13 C internal standardization upon extraction. The extracts were measured as described in [37]. In brief, a hydrophilic interaction liquid chromatography (HILIC) separation at pH 4 was used combined with high-resolution MS.…”

“…The choice of incubation time and drug concentration was based on previous studies using monolayer cultures, which showed prominent metabolomic shifts only after 24 h exposure to sub-cytotoxic drug concentrations [37]. Specifically, the applied concentrations were 20 and 200 µM for oxaliplatin and KP1339, respectively.…”

“…Finally, the ON/CM sample preparation procedure optimized for single MTS analysis comprised the addition of yeast 13 C standards. The measurements relied on hydrophilic interaction chromatography-Orbitrap MS and included external calibrants with internal standards for a large panel of metabolites [37]. Implementing the internal standardization approach together with the use of high-resolution Orbitrap MS enabled us to perform targeted and non-targeted metabolomics in a single analytical run.…”