Azienda Ospedaliera Universitaria Integrata Verona, Department of Pathology and Diagnostics - Unit of Immunology, Policlinico G. Rossi, P.le L.A. Scuro, 10, I-37134, Verona, Italy.
Analyst. 2013 Jul 21;138(14):3934-45. doi: 10.1039/c2an36393c.
We tested the ability of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) in combination with unsupervised Hierarchical Cluster Analysis (HCA) in identifying drug-resistance/sensitivity in leukemic cells exposed to tyrosine kinase inhibitors (TKIs). Experiments were carried out in a well-established mouse model of human Chronic Myelogenous Leukemia (CML). Mouse-derived pro-B Ba/F3 cells transfected with and stably expressing the human p210(BCR-ABL) drug-sensitive wild-type BCR-ABL or the V299L or T315I p210(BCR-ABL) drug-resistant BCR-ABL mutants were exposed to imatinib-mesylate (IMA) or dasatinib (DAS). MicroFTIR was carried out at the Diamond IR beamline MIRIAM where the mid-IR absorbance spectra of individual Ba/F3 cells were acquired using the high brilliance IR synchrotron radiation (SR) via aperture of 15 × 15 μm(2) in sizes. A conventional IR source (globar) was used to compare average spectra over 15 cells or more. IR signatures of drug actions were identified by supervised analyses in the spectra of TKI-sensitive cells. Unsupervised HCA applied to selected intervals of wavenumber allowed us to classify the IR patterns of viable (drug-resistant) and apoptotic (drug-sensitive) cells with an accuracy of >95%. The results from microFTIR + HCA analysis were cross-validated with those obtained via immunochemical methods, i.e. immunoblotting and flow cytometry (FC) that resulted directly and significantly correlated. We conclude that this combined microFTIR + HCA method potentially represents a rapid, convenient and robust screening approach to study the impact of drugs in leukemic cells as well as in peripheral blasts from patients in clinical trials with new anti-leukemic drugs.
我们测试了傅里叶变换（FT）红外（IR）微光谱（microFTIR）结合无监督层次聚类分析（HCA）在鉴定暴露于酪氨酸激酶抑制剂（TKI）的白血病细胞中的耐药/敏感性的能力。实验在建立良好的人类慢性髓性白血病（CML）小鼠模型中进行。用稳定表达人 p210（BCR-ABL）药物敏感野生型 BCR-ABL 或 V299L 或 T315I p210（BCR-ABL）耐药 BCR-ABL 突变体的小鼠源性前 B 细胞 Ba/F3 细胞转染并进行实验。用伊马替尼甲磺酸盐（IMA）或达沙替尼（DAS）处理暴露于 imatinib-mesylate（IMA）或 dasatinib（DAS）的 Ba/F3 细胞。MicroFTIR 在 Diamond IR 光束线上 MIRIAM 进行，在该光束线上，使用高亮度 IR 同步加速器辐射（SR）通过 15×15μm（2）的孔径获取单个 Ba/F3 细胞的中红外吸收光谱。使用常规 IR 源（globar）比较 15 个或更多细胞的平均光谱。通过对 TKI 敏感细胞的光谱进行有监督分析，确定药物作用的 IR 特征。无监督 HCA 应用于选定的波数间隔，使我们能够以>95％的准确性对存活（耐药）和凋亡（敏感）细胞的 IR 模式进行分类。通过免疫化学方法（即免疫印迹和流式细胞术（FC））验证 microFTIR + HCA 分析的结果，这些方法直接且显著相关。我们得出结论，这种组合的 microFTIR + HCA 方法可能代表一种快速，方便和强大的筛选方法，用于研究新的抗白血病药物在白血病细胞以及临床试验中患者外周血中的影响。
