Quantitative imaging of chemical composition in single cells by secondary ion mass spectrometry: cisplatin affects calcium stores in renal epithelial cells.
Quantitative imaging of chemical composition in single cells by secondary ion mass spectrometry: cisplatin affects calcium stores in renal epithelial cells.
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DOI：
10.1007/978-1-60761-746-4_6
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发表时间：
2010
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本章以抗癌药物顺铂治疗细胞为例，描述了定量单细胞质谱学成像(MSI)分析的详细方案。顺铂，顺铂，顺铂(CDDP)，被广泛用于治疗许多恶性肿瘤，包括睾丸、卵巢、膀胱、宫颈、头颈部以及小细胞和非小细胞肺癌。顺铂治疗肾脏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。在这项工作中，二次离子质谱仪(SIMS)被用来研究顺铂对肾损伤模型(LLC-PK1细胞系)细胞内化学成分的影响。采用三明治冷冻破碎法冷冻制备细胞，对单个细胞的化学成分(K+、Na+、Ca~(2+)总浓度)进行亚细胞成像分析。对这些可扩散离子的单细胞分析需要使用可靠的低温样品制备用于SIMS。夹心冷冻-破碎法为SIMS分析提供了一种在细胞内低温保存可扩散离子和分子的简单方法。本研究使用Cameca IMS 3f SIMS离子显微镜仪器，能够产生500 nm空间分辨率的单细胞化学图像。在顺铂处理的细胞中，SIMS成像显示在质量为195的单个细胞中存在可检测到的数量的铂，即195个铂+二次离子。SIMS观察还显示，不同的细胞对顺铂的反应不同。虽然一些细胞的化学成分没有受到顺铂的影响，但另一些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。另一组细胞表现出胞浆钙浓度的增加，这与细胞内Na含量的升高和K浓度的降低有关。由于细胞内K的丢失和Na、Ca的增加是细胞损伤的典型症状，因此，细胞对顺铂治疗的最初反应可能是细胞内细胞质钙库的减少。如果顺铂以某种方式破坏了钙库，那么细胞内游离钙稳态的维持将变得无法控制。这些观察为了解顺铂在细胞损伤中的作用模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学技术在细胞生物学和医学中的必要性和巨大潜力。
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-PK1 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 Ca2+) 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 195Pt+ 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 increased 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 Ca2+ 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.