Development of next-generation ion conductance microscopy to unravel the phenotype and gene expression state of cancer cells

1.我们已经建立了一种新的SICM定量纳米级映射技术来测量乳腺癌MCF 7细胞和皮肤癌黑色素瘤A375 M细胞的刚度，而不损伤细胞。此外，我们在纳米移液管的尖端形成Pt官能化的碳纳米电极以测量单个癌细胞的ROS分布。现在我们可以在单个细胞水平上对乳腺癌MCF 7细胞和皮肤癌黑色素瘤A375 M细胞的刚度和ROS分布进行表型分析.越来越清楚的是，酸性pHe在癌细胞进展、侵袭性和对治疗的抗性中起着至关重要的作用。监测癌细胞的pHe变化将为理解病理过程中的肿瘤异质性提供关键信息，如上皮-间充质转化。基于我们先前对基于酶的pH传感纳米探针的研究，我们开发了非酶pH传感纳米探针，用于单个癌细胞的pH测量.关于基因表达评估，常规方法破坏细胞并导致其结果不稳定。了解单个癌细胞的细胞内基因表达状态一直是困难的。使用SICM纳米移液管的基因采样可以在不同时间从同一细胞进行重复采样，这允许动态基因表达评估。在SICM活检取样方法的帮助下，我们评估了单个癌细胞胞质中的mRNA表达。

1. We have established a new SICM quantitative nanoscale mapping technique to measure the stiffness of breast cancer MCF7 cells and skin cancer melanoma A375M cells without damaging the cells. In addition, we formed a Pt-functionalized carbon nanoelectrode at the tip of nanopipette to measure the ROS distribution of individual cancer cells. Now we can phenotype individual breast cancer MCF7 cells and skin cancer melanoma A375M cells with their stiffness and ROS distribution at single cell level.2. It is becoming clear that an acid pHe plays an essential role in cancer cell progression, invasiveness and resistance to therapy. Monitoring the pHe variation of cancer cells will provide critical information for understanding tumour heterogeneity during pathological processes such as epithelial-mesenchyme transition. Based on our previously studies on enzyme-based pH-sensing nanoprobes, we have developed non-enzyme pH-sensing nanoprobes for pHe measuring from individual cancer cells.3. Regarding gene expression evaluation, conventional methods disrupt cells and cause the instability in their results. It has been difficult to know the intracellular gene expression status of individual cancer cells. Gene sampling with SICM nanopipette can perform repeat sampling from the same cell at different time, which allows a dynamical gene expression evaluation. Under the help of SICM biopsy sampling methods, we have evaluated the mRNA expression in cytoplasm of individual cancer cells.