2022 Fiscal Year Annual Research Report
Development of next-generation ion conductance microscopy to unravel the phenotype and gene expression state of cancer cells
| Project/Area Number |
21H01770
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| Allocation Type | Single-year Grants |
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| Research Institution | Kanazawa University |
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Principal Investigator |
Korchev Yuri.E. 金沢大学, ナノ生命科学研究所, リサーチ・プロフェッサー (10817349)
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| Co-Investigator(Kenkyū-buntansha) |
ZHANG YANJUN 金沢大学, ナノ生命科学研究所, 特任准教授 (70902807)
高橋 康史 名古屋大学, 工学研究科, 教授 (90624841)
周 縁殊 金沢大学, ナノ生命科学研究所, 特任助教 (60758556) [Withdrawn]
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| Project Period (FY) |
2021-04-01 – 2025-03-31
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| Keywords | 細胞の表現型 / 遺伝子 / 単一細胞解析技術 / イオンコンダクタンス顕微鏡 / 癌細胞 |
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| Outline of Annual Research Achievements |
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.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Although one of our co-PI, Professor Yasufumi Takahashi, moved his group to Nagoya University, we have set up our own nanoprobe fabrication and cell culture facilities to keep this project running well.
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| Strategy for Future Research Activity |
1. To phenotype the subgroups among breast cancer MCF7 cells and skin cancer melanoma A375M cells, we will continue detecting both stiffness and ROS from individual cancer cells this year. The relationship between stiffness, ROS production ability and gene expression status of breast cancer MCF7 cells and skin cancer melanoma A375M cells will also be investigated.
2. We will further develop non-enzyme pH-sensing nanoprobe for pHe measuring from individual cancer cells. This new pHe measuring will be combined with our newly developed ROS detecting method for MCF7 and melanoma A375M phenotyping this year. The relationship between pHe, ROS production ability and gene expression status of individual cancer cells will be investigated.
3. We will perform phenotyping and genotyping of individual human colorectal cancer Caco2 cells with our new-developed SICM quantitative nanoscale mapping technique and high-sensitivity ROS and pHe detecting nanoprobes. The relationship between stiffness, pHe, ROS production ability and gene expression status of individual colorectal cancer cells will be investigated.
4. We will continue improving SICM biopsy genotype method for dynamical detection of mRNA expression in individual cancer cells.
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