Life science
Medicine, Drug discovery

Exploring the potential of engineered exosomes as delivery systems fortumor-suppressor microRNA replacement therapy in ovarian cancer
Department of Obstetrics and Gynecology, Graduate School of Medicine
Associate Professor

Kenjiro Sawada

Researchmap https://researchmap.jp/gyne-3354

Life science

Abstract
MicroRNA (miRNA) plays a pivotal role in cancer biology. Exosomes are stable in circulation and selectively picked up by cancer cells, indicating that they can serve as a miRNA carrier. Theaim of this study was to explore the possibility of exosomes as acarrier for miRNA replacement therapy for ovarian cancer (OC).
First, exosomes were purified from primary-cultured omental fibroblasts of OC patients. miR-199a-3p was selected as a TS miRNA,and the synthesized miR-199a-3p was loaded into exosomes byelectroporation. Treatment with miR199a-3p-loaded-exosomes(M199-exosomes) drastically increased miR-199a-3p expression
level in OC cell lines. M199-exosomes suppressed c-Met expression, a direct target of miR-199a-3p, and thereby inhibited cellproliferation and invasion. In a xenograft study, M199-exosomes
also drastically inhibited peritoneal dissemination in OC mice model. These results suggest that miRNA replacement therapy using
exosomes shows promise for treatment of OC. Given that omental
fibroblasts can be obtained from most OC patients, patient-derived
exosomes can be utilized as a DDS for future molecular-targeted
therapies.

and thereby exosomes from omental fibroblasts can be obtained
from those, engineered exosomes can be utilized as a drug delivery carrier for a future molecular-targeted therapies for OC.

● negative control 1 :● negative control 2 :▍ Significance of the research and Future perspective
miRNA replacement therapy using engineered exosomes
showed promising treatment effects to ovarian cancer peritoneal
dissemination. Given that most OC patients undergo omentectomy; electroporation -）＋ （exo + miR199a-3p; electroporation -）● negative control 4 :（exo + control miRNA ; electroporation +）● transfection（exo + miR199a-3p ; electroporation +）

:

CaOV3

1.0

0.40

0.47

2.13

OVCAR3

SKOV38592

1.0

1.32

2.53

5.47 67188

1.0

0.94

0.68

0.512280

Figure1. miRNA
RT-PCR. miR-199a-3p
loaded exosomes
wereFigure 1. miRNA
RT-PCR.
miR-199a-3p
loaded exosomes
were efficiently
pickedefficiently picked up by ovarian cancer cells.
up by ovarian cancercells.

▍ Background & Results[Background] Considering pivotal roles of microRNA (miRNA) incancer biology, tumor suppressor (TS) miRNA remains to be an attractive molecular target; however, most of all clinical trials with TS
miRNAs have failed due to difficulties of miRNAs delivery to cancer
tissues efficiently, and therefore the development of a novel drugdelivery system for miRNA is warranted. Exosomes are known to
be stable in circulation and specifically picked up by cancer cells,indicating they can serve as a promising miRNA carrier. The aim of
this study is to pursue the possibility of engineered exosomes as acarrier for miRNA replacement therapy for ovarian cancer (OC).
[Methods & Results] Secreted exosomes were purified fromhuman fibroblasts primary cultured from omentum obtained during
gynecological surgery. miR-199a-3p was selected as TS miRNA.
Synthesized miR-199a-3p tagged with Alexa-488 was loaded
to exosomes by electroporation. Subsequentially, the effect of199a-3p-loaded exosomes (M199-exosomes) was analyzed invitro and in vivo. Treatment with M199-exosomes drastically increased the expression of miR-199a-3p in 3 different OC cell lines.
M199-exosomes suppressed c-Met expression, a direct target ofmiR-199a-3p, and thereby inhibited cell proliferation and invasiveness of OC cells significantly. In a xenograft study, intraperitoneal
treatment with M199-exosomes drastically inhibited peritoneal dissemination in mice.

（exo● negative control 3 :Scheme of staining exosomes

Time course in the accumulation of labeled exosomes

DIR-labelled exosomes
XenoLight DiR®
Wash and Ultracentrifugation
10,000g 1hr24min

Murine omentum
Reresection

Postresection.

Figure 2. Representative
takenby IVIS
imagingsystem of animals.
Figure 2. Representative
pictures pictures
taken byIVIS
imagingsystem of animals. DIR-labelled exosomes were selectively
DIR-labelled exosomes
wereselectively
pickedupbytumors.
picked up by tumors.
SKOV3-Luci Intraperitoneal Injection(2.0 × 106cells in 200μl PBS)

Supplemenloaded
tary Figure. exosomesmiR-199a-3p loainhibit
ded exosoovarian
mes inhibit cancer
ovarian cancperitonealer
Figure 3. miR-199a-3p
disperitoneal dissemination in xenograft ovarian cancer model mice. Luciferase
semination in xenograft
activities ofactivities of peovarianritoneal tumcancer
ors of mice model
injected intmice.
ravenouslLuciferasey w i t h P BS ,ntromicel miRNAinjected
loaded exosintravenously
omes, or miR-199a-with
3p loadPBS,
ed exosocontrol
mes were miRNA loaded
peritoneal tumorscoof
measured weekly. **; P<0.01.
exosomes, or miR-199a-3p loaded exosomes were measured weekly. **; P<0.01.

P a t e n t
Kobayashi, Masaki; Sawada, Kenjiro; Miyamoto, Mayuko et al. Exploring the potential of engineered exosomes as delivery systems for tumor-suppressor
T r e a t i s e microRNA replacement therapy in ovarian cancer Biochemical and Biophysical Research Communications, 2020; 527(1): 153-161. doi:10.1016/
j.bbrc.2020.04.076
URL
K e y w o r d exosome, ovarian cancer, peritoneal dissemination, microRNA