2020 Fiscal Year Research-status Report
Identification of early stage malignancy changes in the structural and biophysical proporties of exosomes by atomic force microscopy (AFM)-based nano-mechanical measurements
| Project/Area Number |
20K05321
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| Research Institution | Kanazawa University |
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Principal Investigator |
YURTSEVER AYHAN 金沢大学, ナノ生命科学研究所, 特任助教 (00761529)
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| Project Period (FY) |
2020-04-01 – 2024-03-31
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| Keywords | exosomes / 3D-AFM / Nanomechanics |
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| Outline of Annual Research Achievements |
As planned, we revealed the structural and mechanical properties of exosomes released from three different cell types. The 3D-AFM force maps enabled previously unidentified substructures of individual exosomes to be observed, indicating the presence of distinct nanodomains bulging out from the membrane surface. These protruding features were attributed to membrane associated proteins exposed on the outer surface. The nanomechanical properties of exosomes were determined from the 3D-force maps using the Sneddon (conical) model. We developed a Labview based software to process 3D-force mapping data and to extract Young's modulus maps from the acquired 3D-force data. We found a considerably high elastic modulus, ranging from 50 to 350 MPa, as compared to that obtained for synthetic liposomes. Moreover, malignancy-dependent changes in the exosome mechanical properties were revealed by comparing metastatic and nonmetastatic tumor cell-derived exosomes. We found a clear difference in their Young’s modulus values, suggesting differences in their protein profiles and other exosomal contents. Exosomes derived from a highly aggressive and metastatic k-ras-activated human osteosarcoma (OS) cell line (143B) showed a higher Young’s modulus than that derived from a nonaggressive and nonmetastatic k-ras-wildtype human OS cell line (HOS).
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
We have already achieved our goals that were planned for 2021.
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| Strategy for Future Research Activity |
[Experiment 1]: We will establish methods for the functionalization of AFM probes with specific biomolecules. This is an important step for resolving the compositional differences between normal and cancer-derive exosomes.
[Experiment 2]: The biochemical composition of exosomes will be determined using functionalized AFM probes with biomolecules specific to exosome surface receptors and proteins. Furthermore, we are planning to investigate the distribution of phosphatidylserine (PS) on the exosome surface by using PS-binding proteins.
[Experiment 3]: The internal composition content of exosomes, such as miRNA and DNAs, will be determined by high resolution AFM imaging and force mapping by rupturing the lipid membrane of exosomes.
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| Causes of Carryover |
We just changed our plan. To perform our experiments on exosomes and their parent cells, we need to have soft AFM cantilevers, such as AFM Cantilever, OMCL-ACS240TS-R3 Cantilever from OLYMPUS. We also need some chemical and antibodies.
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