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

Anna Schoeberl,Walter Berger,Andreas Schweikert,Michael Gutmann,Sarah Theiner,Gunda Koellensperger,Martin Schaier

doi:10.1021/acs.analchem.1c03442

Abstract

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.

Highlights

Single-cell analysis based on inductively coupled plasma−mass spectrometry (ICP-MS) allows investigating heterogeneous cell populations with regard to cell numbers, cell types, and functionality.[1,2] Over the last decade, elemental single-cell analysis of cells in suspension[3−9] has evolved to the established strategy of mass cytometry, successfully applied in clinical routines
In comparison to primary human monocyte-derived macrophages isolated from peripheral blood, THP-1 cells exhibit no variation between different donors and are ideally suited to reproducibly investigate the characteristics of macrophage subtypes.[44]
All samples including the monocytic cell line THP-1 and the M0, M1, and M2 macrophages were incubated with the Rh intercalator prior to fixation and analyzed by laser ablation (LA)-ICP-TOFMS measurements (Figure S3), confirming the observation that dead cells could be identified based on microscopy only

Summary

■ INTRODUCTION

Single-cell analysis based on inductively coupled plasma−mass spectrometry (ICP-MS) allows investigating heterogeneous cell populations with regard to cell numbers, cell types, and functionality.[1,2] Over the last decade, elemental single-cell analysis of cells in suspension[3−9] has evolved to the established strategy of mass cytometry, successfully applied in clinical routines. For LA-ICP-TOFMS analysis, quantification was enabled by the use of gelatin-based microdroplet standards, as described previously.[29] The human monocytic THP-1 cell line upon cisplatin exposure was selected as it represents a widely used in vitro model to study macrophage differentiation and polarization toward different activation states. All samples including the monocytic cell line THP-1 and the M0, M1, and M2 macrophages were incubated with the Rh intercalator prior to fixation and analyzed by LA-ICP-TOFMS measurements (Figure S3), confirming the observation that dead cells could be identified based on microscopy only (exemplary dead cells are marked with a red circle). The established and validated single-cell LA-ICP-TOFMS workflow was applied to study the quantitative cisplatin uptake depending on the differentiation and polarization state of monocytes and macrophages. As DNA replication in the presence of cisplatin-DNA-cross-links is boosting double-strand breaks and apoptosis induction, the comparison of proliferative THP-1 with nonproliferative M0, M1, and M2 macrophages for drug-induced cell death induction is generally challenging

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES