News Release
Title
Spatial exosome analysis using cellulose nanofiber sheets reveals the location
heterogeneity of extracellular vesicles
Key Points⚫

Development of an innovative tool to capture and preserve exosomes frommicro-volume body fluids.

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Potential of cellulose nanofibers, a new material derived from wood, forcancer treatment applications.

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The new method of directly attaching to organs to collect exosomes for invivo spatial analysis.

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New characteristics of exosomes are revealed, leading to the developmentof therapeutic strategies.

Summary
Extracellular vesicles (EVs), including exosomes, are present in all human body
fluids and have attracted attention as an essential tool for cell-to-cellcommunication. Although various methods have been developed worldwide torecover EVs, recovering them from tiny samples has been considered extremely
difficult. We have developed an EV sheet with a nanopore structure suitable for
EV capture using cellulose nanofiber, a new material derived from wood, andestablished a method to recover and preserve EVs from extremely small
amounts of body fluids by taking advantage of its water absorbency andpore-closing property upon drying. The EV sheet can collect EVs from as little asten microliters of body fluid, and the collected EVs can be used to analyze thebioactive molecules contained in the EVs. EVs can also be recovered from tiny
volume of body fluids on the surface of moist organs by directly attaching EVsheets. We have validated the utility of the EV sheet in ovarian cancer. Thismalignant tumor is difficult to treat and has revealed new properties of EVs that
had not been shown before. The EV sheet enables spatial and functional
analysis of EVs in vivo, which was impossible to analyze before and is expected
to be used for future medical applications. The EV sheet also enable spatial andfunctional analysis of EVs in vivo, which has just been possible now and is
expected to have further medical applications.
Research Background
Ovarian cancer is a poor prognosis that affects about 13,000 people annually inJapan, and nearly half of them die. Because early screening is arduous, most1

cases are diagnosed at an advanced stage, and the 5-year survival rate is said tobe less than 45%. Our research group has been studying the function of
extracellular vesicles (EVs), including exosomes released by ovarian cancer
cells, to improve the prognosis of patients with ovarian cancer. In recent years,
EVs have become increasingly valuable in medical biology as an indispensable
tool for cell-to-cell communication. The heterogeneity of cells constituting
cancer tumor tissue has been well-recognized and studied recently. Still, thediversity of EVs in body fluid has not been well understood due to the difficultyof EV analysis. However, the importance of understanding this phenomenon isinternationally recognized. There have also been great expectations for
applying EVs as biomarkers to assist in the presence or absence of disease andin selecting future treatments.
The development of EV isolation methods from body fluids is still underway
worldwide. The trends are due to various barriers, such as the lack of an optimal
method for EV recovery, the difficulty in recovering EVs with a high degree ofaccuracy, and the fact that a certain amount of sample is required for recovery.
In particular, it is almost impossible to analyze EVs from body fluids with avolume of less than 100 microliters, and the development of a new method has
been desired.
Research Results
In this study, we first developed EV sheets, a novel exosome trapping tool, using
cellulose nanofibers, a new material derived from wood. Cellulose nanofibers
are a sustainable biomass material derived primarily from wood cell walls. They
have attracted significant attention due to their attractive properties, such as
being lightweight and high strength. We have developed a cellulose nanofiber,an "EV sheet," with a nanopore structure suitable for EV capture by applying
paper-making and solvent displacement technology. By taking advantage of its
water absorbency and pore-closing properties upon drying, we have establisheda method to collect and store efficiently EVs from tiny amounts of body fluids,
as little as ten microliters By taking advantage of its water absorbency and theability to close its pores upon drying, we have established a method for
efficiently recovering and preserving EVs from tiny amounts of body fluid, as
little as ten microliters (Figure 1).

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Fig.1 EV collection by EV sheetsFurthermore, by taking advantage of the EV Sheet's ability to collect EVs fromminute amounts of body fluid, we have devised a method to collect EVs directlyfrom the surface of moist organs (Fig. 2). When the EV sheet is attached to theorgan for a few seconds, body fluid is absorbed into the EV sheet by capillary
force, and the EVs in the fluid are selectively trapped in the nanopore structureof the EV sheet by subsequent drying treatment.

Fig.2 The EV sheet attachment method
The recovered EVs can be analyzed for bioactive molecules such as nucleic
acids and proteins. In this study, RNA was extracted from the EVs recovered by3

the EV sheet, and information on the microRNAs (miRNAs) contained in the EVs
was analyzed using a next-generation sequencer. MicroRNAs are small RNAs
about 22 nucleotides long. They are contained in cells and body fluids such as
blood, saliva, and urine. Recent studies have revealed that the types andamounts of microRNAs vary in the blood of patients with cancer and otherdiseases, and they are expected to become an entirely new diagnostic marker.
Ovarian cancer causes ascites fluid accumulation, and cancer cells and EVs
circulate in the fluid, resulting in metastasis, etc. By deciphering the miRNA
information carried by EVs in minute amounts of ascites fluid collected by EV
sheets, it was found that EVs in ascites fluid, which have been analyzed
centrally as ascites fluid, show changes based on the location of collection(Figure 3). (Fig. 3). Also, miRNAs different from those carried in the EVs ofascites fluid were present in the EVs on the tumor surface. Some of thesemiRNAs are decreased after tumor resection, indicating that these miRNAs may
be new biomarkers contributing to diagnosis and therapeutic drug selection.

Conventional
sampleFig.3 Spatial exosome analysis in patient bodyResearch Summary and Future Perspective
These results indicate that EV sheets can be used for spatial and functional
analysis of EVs in vivo, which has not been possible before. EV sheets havevarious advantages. Recently, the practical application of cellulose nanofibers,
a new material that originated in Japan and derived from wood, has been
progressing, and EV sheets open up entirely new functions and applications for4cellulose nanofibers. In the future, we will promote medical applications of EVsheets for various diseases.
Publication
Journal: Nature Communications(2023)

Title: Spatial exosome analysis using cellulose nanofiber sheets reveals thelocation heterogeneity of extracellular vesicles
Authors:

Akira Yokoi1,2,3*†, Kosuke Yoshida1,2, Hirotaka Koga3,4, Masami Kitagawa5,Yukari Nagao1, Mikiko Iida6, Shota Kawaguchi6, Min Zhang6, Jun
Nakayama7,8, Yusuke Yamamoto7, Yoshinobu Baba6,9,10, Hiroaki Kajiyama1
and Takao Yasui6.9.11, 12*†Affiliations:

1. Department of Obstetrics and Gynecology, Nagoya University
Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya,466-8550, Japan
2. Nagoya University Institute for Advanced Research, Furo-cho,
Chikusa-ku, Nagoya 464-8603, Japan.
3. Japan Science and Technology Agency (JST), FOREST, 4-1-8 Honcho,Kawaguchi, Saitama, Japan
4. SANKEN (The Institute of Scientific and Industrial Research), Osaka
University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan
5. Bell Research Center, Department of Obstetrics and Gynecology
Collaborative Research, Nagoya University Graduate School ofMedicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan
6. Department of Biomolecular Engineering, Graduate School ofEngineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya
464-8603, Japan
7. Laboratory of Integrative Oncology, National Cancer Center ResearchInstitute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045 Japan
8. Department of Oncogenesis and Growth Regulation, ResearchInstitute, Osaka International Cancer Institute, 3-1-69 Otemae,Chuo-ku, Osaka, 541-8567, +81-6-6945-1181, Japan.
9. Institute of Nano-Life-Systems, Institutes of Innovation for Future
Society, Nagoya University, Nagoya, Japan
10. Institute of Quantum Life Science, National Institutes for Quantumand Radiological, Science and Technology, Chiba, Japan
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11. Japan Science and Technology Agency (JST), PRESTO, Saitama, Japan
12. Department of Life Science and Technology, Tokyo Institute ofTechnology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, JAPAN
DOI: 10.1038/s41467-023-42593-9
Japanese ver.
https://www.med.nagoya-u.ac.jp/medical_J/research/pdf/Nat_231108.pdf

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