Institute of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Waehringer Strasse 38, 1090 Vienna, Austria.
Institute of Cancer Research and Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8A, 1090 Vienna, Austria.
Anal Chem. 2021 Dec 14;93(49):16456-16465. doi: 10.1021/acs.analchem.1c03442. Epub 2021 Nov 30.
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.
建立了高通量激光烧蚀电感耦合等离子体质谱（LA-ICP-TOFMS）工作流程，用于细胞离心涂片后对单细胞进行定量分析。首次在体外研究顺铂暴露对人单核细胞和单核细胞衍生的巨噬细胞（未分化的 THP-1 单核细胞、分化的 M0 巨噬细胞，以及进一步极化的 M1 和 M2 表型）进行单细胞水平的研究。这些模型特别有趣，因为巨噬细胞构成了肿瘤微环境中存在的最大部分免疫细胞，并在调节肿瘤生长和进展方面发挥重要作用。所引入的生物成像工作流程被证明普遍适用于贴壁和悬浮细胞培养物，并且能够在几分钟内对数百个细胞进行定量分析。THP-1 细胞悬浮液中单细胞分析和腔室载玻片上生长的 M0 细胞的 LA-ICP-TOFMS 分析对该方法的交叉验证都表明，单细胞水平上的铂浓度一致。与 M0 和 M1 巨噬细胞亚型以及单核细胞模型 THP-1 相比，M2 巨噬细胞中顺铂的摄取量较高。与亮场图像结合，并以高空间分辨率监测高度丰富的内源性元素（如磷和钠），可以评估细胞大小和重要的形态学细胞参数，从而直接控制多种细胞条件。这样，在数据评估之前，可以轻松排除凋亡细胞和细胞碎片以及双细胞或细胞簇，而无需额外染色。
