The Anticancer Ruthenium Compound BOLD-100 Targets Glycolysis and Generates a Metabolic Vulnerability towards Glucose Deprivation.

Cellular energy metabolism is reprogrammed in cancer to fuel proliferation. In oncological therapy, treatment resistance remains an obstacle and is frequently linked to metabolic perturbations. Identifying metabolic changes as vulnerabilities opens up novel approaches for the prevention or targeting of acquired therapy resistance. Insights into metabolic alterations underlying ruthenium-based chemotherapy resistance remain widely elusive. In this study, colon cancer HCT116 and pancreatic cancer Capan-1 cells were selected for resistance against the clinically evaluated ruthenium complex sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (BOLD-100). Gene expression profiling identified transcriptional deregulation of carbohydrate metabolism as a response to BOLD-100 and in resistance against the drug. Mechanistically, acquired BOLD-100 resistance is linked to elevated glucose uptake and an increased lysosomal compartment, based on a defect in downstream autophagy execution. Congruently, metabolomics suggested stronger glycolytic activity, in agreement with the distinct hypersensitivity of BOLD-100-resistant cells to 2-deoxy-d-glucose (2-DG). In resistant cells, 2-DG induced stronger metabolic perturbations associated with ER stress induction and cytoplasmic lysosome deregulation. The combination with 2-DG enhanced BOLD-100 activity against HCT116 and Capan-1 cells and reverted acquired BOLD-100 resistance by synergistic cell death induction and autophagy disturbance. This newly identified enhanced glycolytic activity as a metabolic vulnerability in BOLD-100 resistance suggests the targeting of glycolysis as a promising strategy to support BOLD-100 anticancer activity.

Structure-Activity Relationships of Triple-Action Platinum(IV) Prodrugs with Albumin-Binding Properties and Immunomodulating Ligands.

Chemotherapy with platinum complexes is essential for clinical anticancer therapy. However, due to side effects and drug resistance, further drug improvement is urgently needed. Herein, we report on triple-action platinum(IV) prodrugs, which, in addition to tumor targeting via maleimide-mediated albumin binding, release the immunomodulatory ligand 1-methyl-d-tryptophan (1-MDT). Unexpectedly, structure-activity relationship analysis showed that the mode of 1-MDT conjugation distinctly impacts the reducibility and thus activation of the prodrugs. This in turn affected ligand release, pharmacokinetic properties, efficiency of immunomodulation, and the anticancer activity in vitro and in a mouse model in vivo. Moreover, we could demonstrate that the design of albumin-targeted multi-modal prodrugs using platinum(IV) is a promising strategy to enhance the cellular uptake of bioactive ligands with low cell permeability (1-MDT) and to improve their selective delivery into the malignant tissue. This will allow tumor-specific anticancer therapy supported by a favorably tuned immune microenvironment.

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

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.

The Power of LC-MS Based Multiomics: Exploring Adipogenic Differentiation of Human Mesenchymal Stem/Stromal Cells.

The molecular study of fat cell development in the human body is essential for our understanding of obesity and related diseases. Mesenchymal stem/stromal cells (MSC) are the ideal source to study fat formation as they are the progenitors of adipocytes. In this work, we used human MSCs, received from surgery waste, and differentiated them into fat adipocytes. The combination of several layers of information coming from lipidomics, metabolomics and proteomics enabled network analysis of the biochemical pathways in adipogenesis. Simultaneous analysis of metabolites, lipids, and proteins in cell culture is challenging due to the compound's chemical difference, so most studies involve separate analysis with unimolecular strategies. In this study, we employed a multimolecular approach using a two-phase extraction to monitor the crosstalk between lipid metabolism and protein-based signaling in a single sample (~105 cells). We developed an innovative analytical workflow including standardization with in-house produced 13C isotopically labeled compounds, hyphenated high-end mass spectrometry (high-resolution Orbitrap MS), and chromatography (HILIC, RP) for simultaneous untargeted screening and targeted quantification. Metabolite and lipid concentrations ranged over three to four orders of magnitude and were detected down to the low fmol (absolute on column) level. Biological validation and data interpretation of the multiomics workflow was performed based on proteomics network reconstruction, metabolic modelling (MetaboAnalyst 4.0), and pathway analysis (OmicsNet). Comparing MSCs and adipocytes, we observed significant regulation of different metabolites and lipids such as triglycerides, gangliosides, and carnitine with 113 fully reprogrammed pathways. The observed changes are in accordance with literature findings dealing with adipogenic differentiation of MSC. These results are a proof of principle for the power of multimolecular extraction combined with orthogonal LC-MS assays and network construction. Considering the analytical and biological validation performed in this study, we conclude that the proposed multiomics workflow is ideally suited for comprehensive follow-up studies on adipogenesis and is fit for purpose for different applications with a high potential to understand the complex pathophysiology of diseases.

Anion-Exchange Chromatography Coupled to High-Resolution Mass Spectrometry: A Powerful Tool for Merging Targeted and Non-targeted Metabolomics.

In this work, simultaneous targeted metabolic profiling by isotope dilution and non-targeted fingerprinting is proposed for cancer cell studies. The novel streamlined metabolomics workflow was established using anion-exchange chromatography (IC) coupled to high-resolution mass spectrometry (MS). The separation time of strong anion-exchange (2 mm column, flow rate 380 µL min-1, injection volume 5 µL) could be decreased to 25 min for a target list comprising organic acids, sugars, sugar phosphates, and nucleotides. Internal standardization by fully 13C labeled Pichia pastoris extracts enabled absolute quantification of the primary metabolites in adherent cancer cell models. Limits of detection (LODs) in the low nanomolar range and excellent intermediate precisions of the isotopologue ratios (on average <5%, N = 5, over 40 h) were observed. As a result of internal standardization, linear dynamic ranges over 4 orders of magnitude (5 nM-50 µM, R2 > 0.99) were obtained. Experiments on drug-sensitive versus resistant SW480 cancer cells showed the feasibility of merging analytical tasks into one analytical run. Comparing fingerprinting with and without internal standard proved that the presence of the 13C labeled yeast extract required for absolute quantification was not detrimental to non-targeted data evaluation. Several interesting metabolites were discovered by accurate mass and comparing MS2 spectra (acquired in ddMS2 mode) with spectral libraries. Significant differences revealed distinct metabolic phenotypes of drug-sensitive and resistant SW480 cells.

Monitoring the production process of selenized yeast by elemental speciation analysis.

Elemental speciation analysis was implemented as an essential tool set addressing optimum fermentation conditions for the production of selenized yeast feed supplements. Accordingly, the study addressed intracellular levels of (1) total selenium and sulfur, (2) seleno methionine (SeMet), (3) cysteine (Cys) and methionine (Met) and (4) selenite and selenate. Dedicated sample preparation- and LC-ICP-MS methods were implemented and validated using the reference material Selm-1. Excellent repeatability precisions <10% (n = 4 biological replicates) could be obtained for all parameters. The study comprised fermentation monitoring over 72 hours (6 different time points) for a Saccharomyces cerevisiae strain under different selenite feed conditions. It was observed that for this strain an increase in the selenium concentration in the fermentation feed by 50% did not result in enhanced selenium accumulation. Fermentation monitoring of three different Saccharomyces cerevisiae strains under the same conditions showed strain specific selenium uptake after 72 hours. The strain with the lowest cell viability of 60% showed the lowest SeMet content. After 47 h of fermentation, all strains reached a critical point, at which seleno methionine accounted for approximately 100% of the total selenium and cell viability started to decrease. This could be explained by sulfur limitation and/or excess of the seleno methionine storage capacity. Strains showing cell viability of approx. 90% after 72 hours of fermentation revealed SeMet concentrations up to 3000 µg g(-1). In the final product, an apparent threshold level for Met/SeMet of approx. 1 was observed for all strains.