Chandra Subhash
Cornell SIMS Laboratory, Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA.
Methods Mol Biol. 2010;656:113-30. doi: 10.1007/978-1-60761-746-4_6.
A detailed protocol for quantitative single cell mass spectrometry imaging (MSI) analysis is described in this chapter with examples of the treatment of cells with anticancer drug, cisplatin. Cisplatin, cis-diamminedichloridoplatinum ii (CDDP), is widely used for the treatment of many malignancies, including testicular, ovarian, bladder, cervical, head and neck, and small cell and non-small cell lung cancers. The possibility of renal injury by cisplatin treatment is a major dose-limiting factor in this cancer therapy. At present, the mechanisms of cisplatin-induced renal cytotoxicity are poorly understood. In this work, secondary ion mass spectrometry (SIMS) was used for investigating cisplatin-induced alterations in intracellular chemical composition in a well-established model (LLC-PK(1) cell line) for studying renal injury. The cells were cryogenically prepared by the sandwich freeze-fracture method for subcellular imaging analysis of chemical composition (total concentrations of K(+), Na(+), and Ca(2+)) in individual cells. The single cell analysis of these diffusible ions necessitates the use of reliable cryogenic sample preparations for SIMS. The sandwich freeze-fracture method offers a simple approach for cryogenically preserving diffusible ions and molecules inside the cells for SIMS analysis. A CAMECA IMS-3f SIMS ion microscope instrument capable of producing chemical images of single cells with 500-nm spatial resolution was used in the study. In cisplatin-treated cells, SIMS imaging showed the presence of detectable amount of platinum at mass 195, as (195)Pt(+) secondary ions in individual cells. SIMS observations also revealed that individual cells differed in their response to cisplatin. While the chemical composition of some cells was unaffected by cisplatin, others showed a reduction in cytoplasmic calcium stores that was not associated with changes in their intracellular K or Na concentrations. Another population of cells displayed an increase in cytoplasmic calcium concentration that was associated with higher levels of intracellular Na and a reduction in K concentration of the same cells. Since the loss of intracellular K and the gain of Na and Ca are typical symptoms of cell injury, it is plausible that the initial response of the cell to cisplatin treatment is the reduction in cytoplasmic calcium pool in stores. If, somehow, the calcium stores are compromised with cisplatin, then maintenance of free Ca(2+) homeostasis would become uncontrollable in the cell. These observations open new avenues of research for understanding of the mode of action of cisplatin in cell injury. This study also demonstrates the need and vast potential of single cell imaging mass spectrometry techniques in cell biology and medicine.
本章介绍了定量单细胞质谱成像（MSI）分析的详细方案，并列举了用抗癌药物顺铂处理细胞的示例。顺铂，即顺二氯二氨合铂（II）（CDDP），广泛用于治疗多种恶性肿瘤，包括睾丸癌、卵巢癌、膀胱癌、宫颈癌、头颈癌以及小细胞和非小细胞肺癌。顺铂治疗导致肾损伤的可能性是这种癌症治疗中的一个主要剂量限制因素。目前，顺铂诱导肾细胞毒性的机制尚不清楚。在这项研究中，二次离子质谱（SIMS）被用于研究顺铂在一个成熟的肾损伤研究模型（LLC-PK(1)细胞系）中诱导的细胞内化学成分变化。通过三明治冷冻断裂法对细胞进行低温处理，以对单个细胞内的化学成分（K(+)、Na(+)和Ca(2+)的总浓度）进行亚细胞成像分析。对这些可扩散离子进行单细胞分析需要使用可靠的低温样品制备方法用于SIMS。三明治冷冻断裂法为低温保存细胞内的可扩散离子和分子以供SIMS分析提供了一种简单的方法。本研究使用了一台能够以500纳米空间分辨率生成单细胞化学图像的CAMECA IMS-3f SIMS离子显微镜仪器。在顺铂处理的细胞中，SIMS成像显示在质量数195处存在可检测量的铂，以单个细胞中的(195)Pt(+)二次离子形式存在。SIMS观察还表明，单个细胞对顺铂的反应存在差异。虽然一些细胞的化学成分不受顺铂影响，但其他细胞显示细胞质钙储存减少，这与细胞内K或Na浓度的变化无关。另一群细胞表现出细胞质钙浓度增加，这与细胞内较高水平的Na以及同一细胞中K浓度降低有关。由于细胞内K的丢失以及Na和Ca的增加是细胞损伤的典型症状，因此细胞对顺铂治疗的初始反应可能是细胞质钙储存池的减少。如果顺铂以某种方式损害了钙储存，那么细胞内游离Ca(2+)稳态的维持将变得无法控制。这些观察结果为理解顺铂在细胞损伤中的作用方式开辟了新的研究途径。这项研究还证明了单细胞成像质谱技术在细胞生物学和医学中的必要性和巨大潜力。
