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

1. 我们建立了一种新的SICM定量纳米制图技术，在不损伤细胞的情况下测量乳腺癌MCF7细胞和皮肤癌黑色素瘤A375M细胞的刚度。此外，我们在纳米吸管的尖端形成了一个pt功能化的碳纳米电极，以测量单个癌细胞的ROS分布。现在我们可以从单个乳腺癌MCF7细胞和皮肤癌黑色素瘤A375M细胞的硬度和单细胞水平上的ROS分布进行表型分析。越来越清楚的是，酸性pHe在癌细胞的进展、侵袭性和对治疗的抵抗中起着至关重要的作用。监测癌细胞的pHe变异将为理解病理过程（如上皮-间质转化）中的肿瘤异质性提供关键信息。基于我们之前对基于酶的ph传感纳米探针的研究，我们开发了非酶ph传感纳米探针，用于测量单个癌细胞的pHe。在基因表达评估方面，传统的方法会破坏细胞，导致结果不稳定。了解单个癌细胞的细胞内基因表达状态一直很困难。利用SICM纳米吸管进行基因采样，可以在不同时间对同一细胞进行重复采样，从而实现基因表达的动态评估。在SICM活检取样方法的帮助下，我们评估了单个癌细胞细胞质中mRNA的表达。

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.