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定量纳米作图技术，在不破坏细胞的情况下测量乳腺癌MCF7细胞和皮肤癌黑色素瘤A375M细胞的硬度。此外，我们在纳米管的顶端形成了一个铂功能化的碳纳米电极来测量单个癌细胞的ROS分布。现在我们可以在单细胞水平上对单个乳腺癌MCF7细胞和皮肤癌黑色素瘤A375M细胞进行表型，并对其硬度和ROS分布进行研究。越来越清楚的是，酸性苯丙氨酸在癌细胞的发展、侵袭性和对治疗的抵抗力方面起着至关重要的作用。监测癌细胞的Phe变化将为了解肿瘤在上皮-间充质转化等病理过程中的异质性提供关键信息。在前期研究的基础上，我们开发了用于单个癌细胞苯酚测定的非酶pH敏感纳米探针。对于基因表达的评估，传统的方法会干扰细胞，导致结果不稳定。目前还很难了解单个癌细胞的细胞内基因表达状况。使用SICM纳米管的基因采样可以在不同时间对同一细胞进行重复采样，从而实现动态基因表达评估。借助于SICM活检取材方法，我们评估了单个癌细胞胞浆中的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.