Beyond Single-Cell Analysis of Metallodrugs by ICP-MS: Targeting Cellular Substructures.

Platinum compounds such as cisplatin (cisPt) embody the backbone of combination chemotherapy protocols against advanced lung cancer. However, their efficacy is primarily limited by inherent or acquired platinum resistance, the origin of which has not been fully elucidated yet, although of paramount interest. Using single cell inductively coupled plasma mass spectrometry (SC-ICP-MS), this study quantifies cisPt in single cancer cells and for the first time in isolated nuclei. A comparison of cisPt uptake was performed between a wild type (wt) cancer cell line and related resistant sublines. In both, resistant cells, wt cells, and their nuclei, cisPt uptake was measured at different incubation times. A lower amount of cisPt was found in resistant cell lines and their nuclei compared to wt cells. Moreover, the abundance of internalized cisPt decreased with increasing resistance. Interestingly, concentrations of cisPt found within the nuclei were higher than compared to cellular concentrations. Here, we show, that SC-ICP-MS allows precise and accurate quantification of metallodrugs in both single cells and cell organelles such as nuclei. These findings pave the way for future applications investigating the potency and efficacy of novel metallodrugs developed for cancer treatment.

1H HR-MAS NMR Based Metabolic Profiling of Lung Cancer Cells with Induced and De-Induced Cisplatin Resistance to Reveal Metabolic Resistance Adaptations.

Cisplatin (cisPt) is an important drug that is used against various cancers, including advanced lung cancer. However, drug resistance is still a major ongoing problem and its investigation is of paramount interest. Here, a high-resolution magic angle spinning (HR-MAS) NMR study is presented deciphering the metabolic profile of non-small cell lung cancer (NSCLC) cells and metabolic adaptations at different levels of induced cisPt-resistance, as well as in their de-induced counterparts (cells cultivated in absence of cisPt). In total, fifty-three metabolites were identified and quantified in the 1H-HR-MAS NMR cell spectra. Metabolic adaptations to cisPt-resistance were detected, which correlated with the degree of resistance. Importantly, de-induced cell lines demonstrated similar metabolic adaptations as the corresponding cisPt-resistant cell lines. Metabolites predominantly changed in cisPt resistant cells and their de-induced counterparts include glutathione and taurine. Characteristic metabolic patterns for cisPt resistance may become relevant as biomarkers in cancer medicine.

Generation of Stable cisPt Resistant Lung Adenocarcinoma Cells.

Platinum compounds represent the backbone of combined chemotherapy protocols for advanced lung cancer. The mechanisms responsible for its frequent primary or acquired resistance to cisplatin (cisPt)-based chemotherapy remains enigmatic. The availability of two cell lines of the same origin, one resistant and the other sensitive, will facilitate research to reveal the mechanism of resistance formation. Lung adenocarcinoma cells, A240286S (A24), were cultivated in increasing cisPt concentrations over a prolonged time. After a significant increase in IC50 was measured, cultivation of the cells was continued in absence of cisPt and IC50s determined over a long period (>7 months). As a result, a cell line with lasting, high-level cisPt resistance, designated (D-)A24cisPt8.0, was obtained. The cells were cross-resistant to oxaliplatin and to pemetrexed at a low level. Previous publications have claimed that Leucine-rich repeat-containing protein 8 (LRRC8A and LRRC8D) of the volume-regulated anion channels (VRACs) affect cellular resistance to cisPt. Even though cisPt decreased LRRC8D expression levels, we showed by knockdown and overexpression experiments with LRRC8A and D that these proteins do not govern the observed cisPt resistance. The tumor cell sublines described here provide a powerful model to study the mechanisms of resistance to cisPt in lung cancer cells and beyond.