| Project/Area Number |
23K21070
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| Project/Area Number (Other) |
21H01770 (2021-2023)
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Multi-year Fund (2024)
Single-year Grants (2021-2023) |
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| Section | 一般 |
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| Review Section |
Basic Section 28040:Nanobioscience-related
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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)
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| Project Period (FY) |
2021-04-01 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2024)
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| Budget Amount *help |
¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000)
Fiscal Year 2024: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2023: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2022: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2021: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000)
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| Keywords | 癌細胞 / 細胞の表現型 / SICM / ナノプローブ / 表現型 / 遺伝子型 / 遺伝子 / 単一細胞解析技術 / イオンコンダクタンス顕微鏡 / 走査型プローブ顕微鏡 / 電気化学センサー / プローブ顕微鏡 |
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| Outline of Research at the Start |
癌組織の形成・単一細胞解析技術によるオミックス解析行われたが、個々の手法で得られた細胞の表現型・遺伝子発現状態の情報をリンクできない、細胞の情報とその環境の関係を特定できないという課題を抱えている。そこで、表現型と遺伝子発現をその場で計測可能な次世代イオンコンダクタンス顕微鏡を開発する。本手法は、多機能ナノピペットにより、単一細胞レベルで表現型をイメージングし、さらに、細胞質を直接回収して遺伝子解析を行う。この技術を用いて、癌細胞特有の微小環境の形成や、グルコース代謝に伴う活性酸素種の生成、癌の浸潤と密接に関係する細胞の硬さなど、癌細胞の表現型と遺伝子発現状態を単一細胞レベルで理解する。
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| Outline of Annual Research Achievements |
Single-cell genomics has emerged as a revolutionary technology, transforming nearly every field of biomedical research. However, there is a growing recognition that cell populations harbor far more heterogeneity than expected, making bulk population analysis inadequate for fully characterizing their biological complexity. It is well established that heterogeneity is one of the hallmarks of human cancer cells. Accurately defining cell types and states and understanding how they arise in disease demands functional single-cell measurements. To make progress in this area, we need more specific functional single-cell pheno-genotyping. Like Atomic Force Microscopy (AFM), scanning ion conductance microscopy (SICM) is a type of non-contact scanning probe microscopy, which we have pioneered for living cell imaging and phenotyping. In addition to high-resolution topographical imaging, SICM can perform accurate cell stiffness measurements and map cell mechanical properties. In this study, we utilized SICM and its based multifunctional nanopipette to image phenotypes at the single-cell level and directly collect cytoplasm for genetic analysis. With this technology, we can investigate the phenotype and gene expression state of cancer cells, including the formation of a cancer cell-specific microenvironment, the generation of reactive oxygen species, and cell stiffness, which is closely related to cancer invasion. This approach will enhance our understanding of cancer development and progression at the single-cell level.
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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
1. A cell’s mechanical properties have been linked to cancer development, motility and metastasis and are therefore an attractive target as a universal, scanning ion conductance microscopy (SICM) offers a nanoscale resolution, noncontact method of nanomechanical data acquisition for cell phenotyping.
2.We have established new methods with SICM and Pt-functionalized nanoelectrodes to measure dynamic extracellular to intracellular H2O2 gradients, cell morphology and stiffness of individual Caco-2 cells.
3. We report on single-molecule nanopore sensing combined with position-encoded DNA molecular probes, with chemistry tuned to simultaneously identify various antigen proteins and multiple RNA gene fragments of SARS-CoV-2 with high sensitivity and selectivity.
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| Strategy for Future Research Activity |
1. We will use SICM to measure the nanomechanical properties of melanoma cell lines from different stages with increasing metastatic ability. Then Young’s modulus changes following treatment with the anti-cancer drugs paclitaxel, cisplatin and dacarbazine will be measured for different melanoma phenotypes.
2. SICM and Pt-functionalized nanoelectrodes will be employed to measure dynamic extracellular to intracellular H2O2 gradients in individual Caco-2 cells. We plan to reveal the complex interplay between physical properties and biochemical signaling in cancer cells' antioxidant defense.
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