The copper transporter CTR1 and cisplatin accumulation at the single-cell level by LA-ICP-TOFMS.

More than a decade ago, studies on cellular cisplatin accumulation via active membrane transport established the role of the high affinity copper uptake protein 1 (CTR1) as a main uptake route besides passive diffusion. In this work, CTR1 expression, cisplatin accumulation and intracellular copper concentration was assessed for single cells revisiting the case of CTR1 in the context of acquired cisplatin resistance. The single-cell workflow designed for in vitro experiments enabled quantitative imaging at resolutions down to 1 µm by laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS). Cisplatin-sensitive ovarian carcinoma cells A2780 as compared to the cisplatin-resistant subline A2780cis were investigated. Intracellular cisplatin and copper levels were absolutely quantified for thousands of individual cells, while for CTR1, relative differences of total CTR1 versus plasma membrane-bound CTR1 were determined. A markedly decreased intracellular cisplatin concentration accompanied by reduced copper concentrations was observed for single A2780cis cells, along with a distinctly reduced (total) CTR1 level as compared to the parental cell model. Interestingly, a significantly different proportion of plasma membrane-bound versus total CTR1 in untreated A2780 as compared to A2780cis cells was observed. This proportion changed in both models upon cisplatin exposure. Statistical analysis revealed a significant correlation between total and plasma membrane-bound CTR1 expression and cisplatin accumulation at the single-cell level in both A2780 and A2780cis cells. Thus, our study recapitulates the crosstalk of copper homeostasis and cisplatin uptake, and also indicates a complex interplay between subcellular CTR1 localization and cellular cisplatin accumulation as a driver for acquired resistance development.

An Interlaboratory Comparison Study of Regulated and Emerging Mycotoxins Using Liquid Chromatography Mass Spectrometry: Challenges and Future Directions of Routine Multi-Mycotoxin Analysis including Emerging Mycotoxins.

The present interlaboratory comparison study involved nine laboratories located throughout the world that tested for 24 regulated and non-regulated mycotoxins by applying their in-house LC-MS/MS multi-toxin method to 10 individual lots of 4 matrix commodities, including complex chicken and swine feed, soy and corn gluten. In total, more than 6000 data points were collected and analyzed statistically by calculating a consensus value in combination with a target standard deviation following a modified Horwitz equation. The performance of each participant was evaluated by a z-score assessment with a satisfying range of ±2, leading to an overall success rate of 70% for all tested compounds. Equal performance for both regulated and emerging mycotoxins indicates that participating routine laboratories have successfully expanded their analytical portfolio in view of potentially new regulations. In addition, the study design proved to be fit for the purpose of providing future certified reference materials, which surpass current analyte matrix combinations and exceed the typical scope of the regulatory framework.

Achieving Absolute Molar Lipid Concentrations: A Phospholipidomics Cross-Validation Study.

Standardization is essential in lipidomics and part of a huge community effort. However, with the still ongoing lack of reference materials, benchmarking quantification is hampered. Here, we propose traceable lipid class quantification as an important layer for the validation of quantitative lipidomics workflows. 31P nuclear magnetic resonance (NMR) and inductively coupled plasma (ICP)-mass spectrometry (MS) can use certified species-unspecific standards to validate shotgun or liquid chromatography (LC)-MS-based lipidomics approaches. We further introduce a novel lipid class quantification strategy based on lipid class separation and mass spectrometry using an all ion fragmentation (AIF) approach. Class-specific fragments, measured over a mass range typical for the lipid classes, are integrated to assess the lipid class concentration. The concept proved particularly interesting as low absolute limits of detection in the fmol range were achieved and LC-MS platforms are widely used in the field of lipidomics, while the accessibility of NMR and ICP-MS is limited. Using completely independent calibration strategies, the introduced validation scheme comprised the quantitative assessment of the complete phospholipid sub-ome, next to the individual lipid classes. Komagataella phaffii served as a prime example, showcasing mass balances and supporting the value of benchmarks for quantification at the lipid species level.

Micro-droplet-based calibration for quantitative elemental bioimaging by LA-ICPMS.

In this work, a novel standardization strategy for quantitative elemental bioimaging is evaluated. More specifically, multi-element quantification by laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS) is performed by multi-point calibration using gelatin-based micro-droplet standards and validated using in-house produced reference materials. Fully automated deposition of micro-droplets by micro-spotting ensured precise standard volumes of 400 ± 5 pL resulting in droplet sizes of around 200 µm in diameter. The small dimensions of the micro-droplet standards and the use of a low-dispersion laser ablation setup reduced the analysis time required for calibration by LA-ICPMS significantly. Therefore, as a key advance, high-throughput analysis (pixel acquisition rates of more than 200 Hz) enabled to establish imaging measurement sequences with quality control- and standardization samples comparable to solution-based quantification exercises by ICP-MS. Analytical figures of merit such as limit of detection, precision, and accuracy of the calibration approach were assessed for platinum and for elements with biological key functions from the lower mass range (phosphorus, copper, and zinc). As a proof-of-concept application, the tool-set was employed to investigate the accumulation of metal-based anticancer drugs in multicellular tumor spheroid models at clinically relevant concentrations. Graphical abstract.

Cisplatin Uptake in Macrophage Subtypes at the Single-Cell Level by LA-ICP-TOFMS Imaging.

A high-throughput laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS) workflow was implemented for quantitative single-cell analysis following cytospin preparation of cells. For the first time, in vitro studies on cisplatin exposure addressed human monocytes and monocyte-derived macrophages (undifferentiated THP-1 monocytic cells, differentiated M0 macrophages, as well as further polarized M1 and M2 phenotypes) at the single-cell level. The models are of particular interest as macrophages comprise the biggest part of immune cells present in the tumor microenvironment and play an important role in modulating tumor growth and progression. The introduced bioimaging workflow proved to be universally applicable to adherent and suspension cell cultures and fit-for-purpose for the quantitative analysis of several hundreds of cells within minutes. Both, cross-validation of the method with single-cell analysis in suspension for THP-1 cells and with LA-ICP-TOFMS analysis of adherent M0 cells grown on chambered glass coverslips, revealed agreeing platinum concentrations at the single-cell level. A high incorporation of cisplatin was observed in M2 macrophages compared to the M0 and M1 macrophage subtypes and the monocyte model, THP-1. The combination with bright-field images and monitoring of highly abundant endogenous elements such as phosphorus and sodium at a high spatial resolution allowed assessing cell size and important morphological cell parameters and thus straightforward control over several cell conditions. This way, apoptotic cells and cell debris as well as doublets or cell clusters could be easily excluded prior to data evaluation without additional staining.

Mass spectrometry techniques for imaging and detection of metallodrugs.

Undoubtedly, metallomic approaches based on mass spectrometry have evolved into essential tools supporting the drug development of novel metal-based anticancer drugs. This article will comment on the state-of-the-art instrumentation and highlight some of the recent analytical advances beyond routine, especially focusing on the latest developments in inductively coupled plasma-mass spectrometry (ICP-MS). Mass spectrometry-based bioimaging and single-cell methods will be presented, paving the way to exciting investigations of metal-based anticancer drugs in heterogeneous and structurally, as well as functionally complex solid tumor tissues.

FI-ICP-TOFMS for quantification of biologically essential trace elements in cerebrospinal fluid - high-throughput at low sample volume.

In this work, we introduce a high-throughput quantitative multi-element method for biological fluids enabled by omitting sample preparation and an analysis time of a few seconds per sample. For the first time, flow injection of an undiluted cerebrospinal fluid (CSF) was combined to state-of-the-art ICP-TOFMS detection for multi-element analysis. Owing to the low sample volume and trace element concentrations of the CSF, flow injection methods with only 5 µL sample intake were used in combination with an icpTOF 2R TOF-based ICP-MS instrument. Due to the lack of certified reference materials for CSF analysis, a validated method employing open vessel digestion of the CSF material in combination with ICP-sectorfield-MS analysis was carried out and used as a reference. Additionally, the performance of the flow injection ICP-TOFMS was cross-validated by flow injection quadrupole-based ICP-MS/MS analysis using both external calibration and isotope dilution strategies. In the latter case, the sample had to be injected several times because of the need for tailored gas conditions for different elements. Overall, flow injection of biological fluids delivered quantitative values, which were in excellent agreement with the gold standard established by ICP-SFMS demonstrating the capability of ICP-TOFMS analysis in terms of resolution and sensitivity for the accurate quantification of trace elements in biological samples.

Laser Ablation-Inductively Coupled Plasma Time-of-Flight Mass Spectrometry Imaging of Trace Elements at the Single-Cell Level for Clinical Practice.

In this work, a combination of routine clinical practice and state-of-the-art laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) imaging is presented for multielement analysis of single cells on clinical samples. More specifically, routinely drawn blood thin films of a patient undergoing treatment with the anticancer drug cisplatin were studied. The presented label-free approach enabled rapid analysis of hundreds of cells at the single-cell level within a few minutes without additional tailored sample preparation. The employed low-dispersion LA setup is based on the tube-type COBALT ablation cell in combination with the aerosol rapid introduction system (ARIS) providing pixel-resolved imaging at 250-500 Hz for biological sample material. In order to cope with the short transient signals of only a few milliseconds delivered by the laser ablation setup, an icpTOF 2R TOF-based ICP-MS instrument was used for analysis, which has a mass coverage of m/ z = 14-256. Leukocytes and erythrocytes, imaged with a laser beam of 4 µm and pixel interspacing of 2 µm, were differentiated on the basis of their intrinsic trace-elemental pattern. Overall, red blood cells displayed high iron intensities, whereas individual white blood cells were characterized by their high phosphorus content and increased sulfur signal. Unsupervised multivariate statistical analysis was applied to the data set. Principal component plots showed a clear clustering of leukocytes versus erythrocytes. The approach allowed studying not only the drug distribution between plasma and cells but also, for the first time, the preferential accumulation of platinum in different blood cell types without the need of cell fixation and labeling. Extracellular hotspots of platinum were observed, whereas only a small fraction of platinum was associated with erythrocytes. The investigation demonstrates the potential of low-dispersion LA-ICP-TOFMS as a rapid and powerful tool for label-free single-cell imaging in the clinical context.