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大学・研究所にある論文を検索できる 「Implementation of highly sensitive small extracellular vesicle (sEV) quantification method in the identification of novel sEV production modulators and the evaluation of sEV pharmacokinetics」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。
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Implementation of highly sensitive small extracellular vesicle (sEV) quantification method in the identification of novel sEV production modulators and the evaluation of sEV pharmacokinetics

Yamamoto, Aki 京都大学 DOI:10.14989/doctor.k23473

2021.09.24

概要

Small extracellular vesicles (sEVs) are nano-sized, lipid membrane vesicles that are secreted from various cell types in the body. sEVs mediate intercellular communication by transferring their cargos including nucleic acids and proteins to the recipient cells, thereby modulating diverse biological and pathological processes in the body. Sensitive quantification of sEVs is crucial since sEVs have been reported to correlate with various disease progression. Small molecules that can control sEV production may become a useful tool to investigate the roles of sEV in the body. Thus, the development of an sEV quantification method that is both sensitive and rapid can facilitate the exploration of novel compounds that can directly target sEV secretion/biogenesis. Furthermore, since information regarding the specific mechanisms of sEV production during pathogenesis is still limited, the ability to quantify sEVs in vivo can further our understanding of the factors that influence their production and their in vivo fate. Therefore, I developed a rapid, highly sensitive sEV quantification method utilizing Gaussia luciferase (gLuc) reporter protein and screened for compounds that could modulate sEV secretion. Utilizing the gLuc protein quantification method, I then took a pharmacokinetic approach to elucidate the mechanism behind elevated sEV secretion during cancer pathogenesis and investigated the roles of surface glycans on sEV pharmacokinetics.

Chapter 1 Development of a highly sensitive sEV quantification method and the identification of novel sEV modulators via high throughput screening
The ability to regulate sEV production may become useful in sEV research. However, reports of pharmacological agents that potentially inhibit or induce sEV biogenesis/secretion is limited due to the lack of high-throughput method for sEV quantification. Therefore, I developed a rapid, highly sensitive, high-throughput sEV quantification method utilizing a fusion protein consisting of gLuc reporter protein and sEV marker CD63 (CD63-gLuc) to identify compounds that can modulate sEV production. A total of 480 compounds were screened, and two novel compounds were identified to be potent inducers or inhibitors of gLuc activity. The efficacy of the identified compounds in modulating sEV production was validated by protein quantification of the isolated sEVs. Altogether, a rapid, sensitive sEV quantification method was successfully developed, and two novel compounds were identified to modulate sEV production.

Chapter 2 Pharmacokinetic approach for the elucidation of elevated plasma sEV concentration during cancer pathogenesis
The mechanism behind elevated plasma sEV concentration during disease states is poorly understood due to the technical difficulty in tracking the in vivo fate of sEVs. By implementing a selective sEV labeling method utilizing gLuc reporter protein, which enabled the quantification of mouse plasma-derived sEVs (MP-sEVs) in vivo, I aimed to elucidate the mechanism of elevated plasma sEV concentration during cancer pathogenesis. Considering that the steady-state concentration of MP-sEVs is maintained by a balance between the rate of secretion (k0) and rate of elimination (kel), the elevated sEV concentration is due to either increased k0 or decreased kel. Intravenous administration of MP- sEVs isolated from healthy or tumor-bearing mice into either healthy or tumor-bearing mice showed comparable pharmacokinetic parameters, suggesting no changes in kel during cancer pathogenesis. To determine the k0 of tumor-derived sEVs, CD63-gLuc stably expressing B16BL6 tumor bearing mice model was utilized. gLuc activity of MP-sEV isolated from these tumor-bearing mice was below the limit of detection; furthermore, it was approximated that tumor-derived sEVs contributed to only 0.1% of the total sEV protein isolated. Thus, it was revealed that kel hardly changes, and that tumor-derived sEVs contribute very little to the overall increased MP-sEV concentration. These results indicate that cells other than tumor cells mainly contribute to the elevated blood concentration of sEV during disease states.

Chapter 3 Determination of the effect of surface glycans in sEV pharmacokinetics
Tumor cell-derived sEVs have been reported to exhibit altered glycosylation patterns and contribute significantly to its progression and metastasis. Since glycans are located on the outermost region of sEV surface, it can be expected that surface glycans participate in various sEV functions, which ultimately influence their in vivo pharmacokinetics. To determine the role of surface glycans in sEV pharmacokinetics, gLuc fusion protein consisting of Gag protein and gLuc (Gag-gLuc) was utilized to label the inner spaces of the sEVs. The inner labeling method preserves the chemiluminescence derived from the sEVs after the enzymatic deglycosylation of N- and O-glycans from the sEV surface, thus enabling the pharmacokinetic analysis of the deglycosylated sEVs. Deglycosylation had minimal impact on the physicochemical properties of the sEVs, however showed enhanced uptake by the peritoneal macrophages for N-glycan removed sEVs. Nonetheless, the increased uptake efficiency observed in vitro did not alter the sEV clearance of the glycosidase-treated sEVs in vivo, suggesting that the rate-determining process in sEV clearance at the whole-body level is blood-flow dependent, and not the uptake efficiency of macrophages. Thus, it was determined that surface glycans have minimal impact on sEV clearance rate.

In conclusion, I successfully developed a simple yet powerful quantification tool for sEVs and found two novel compounds that could induce or inhibit sEV production. Furthermore, I revealed that tumor-derived sEVs had minimal contribution to the elevated sEV concentration observed during cancer pathogenesis, and that surface glycans do not significantly influence sEV pharmacokinetics. The findings in this thesis contribute to our understanding on the biological and pathological roles of sEV and to the development of sEV-based therapies.

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