Intraperitoneal transfer of microRNA‐29b‐containing small extracellular vesicles can suppress peritoneal metastases of gastric cancer

Abstract Small extracellular vesicles (sEV) contain various microRNAs (miRNAs) and play crucial roles in the tumor metastatic process. Although miR‐29b levels in peritoneal exosomes were markedly reduced in patients with peritoneal metastases (PM), their role has not been fully clarified. In this study, we asked whether the replacement of miR‐29b can affect the development of PM in a murine model. UE6E7T‐12, human bone marrow‐derived mesenchymal stem cells (BMSCs), were transfected with miR‐29b‐integrating recombinant lentiviral vector and sEV were isolated from culture supernatants using ultracentrifugation. The sEV contained markedly increased amounts of miR‐29b compared with negative controls. Treatment with transforming growth factor‐β1 decreased the expression of E‐cadherin and calretinin with increased expression of vimentin and fibronectin on human omental tissue‐derived mesothelial cells (HPMCs). However, the effects were totally abrogated by adding miR‐29b‐rich sEV. The sEV inhibited proliferation and migration of HPMCs by 15% (p < 0.005, n = 6) and 70% (p < 0.005, n = 6), respectively, and inhibited adhesion of NUGC‐4 and MKN45 to HPMCs by 90% (p < 0.0001, n = 5) and 77% (p < 0.0001, n = 5), respectively. MicroRNA‐29b‐rich murine sEV were similarly obtained using mouse BMSCs and examined for in vivo effects with a syngeneic murine model using YTN16P, a highly metastatic clone of gastric cancer cell. Intraperitoneal (IP) transfer of the sEV every 3 days markedly reduced the number of PM from YTN16P in the mesentery (p < 0.05, n = 6) and the omentum (p < 0.05, n = 6). Bone marrow mesenchymal stem cell‐derived sEV are a useful carrier for IP administration of miR‐29b, which can suppress the development of PM of gastric cancer.


| INTRODUC TI ON
Peritoneal metastases frequently occur in patients with gastric cancer, which is associated with an extremely poor prognosis. [1][2][3] Although PM are likely to develop from free IP tumor cells exfoliated from the serosal surface of a primary tumor, 4,5 the detailed mechanisms leading to the formation of PM have not been fully elucidated.
Emerging evidence suggests that tumor-secreted EV contains various functional molecules such as proteins, DNA, and various types of RNAs, [6][7][8] conveying potential biological information to target organs and creating a premetastatic niche to establish organotypic metastases. 9,10 In particular, miRNAs in sEV circulating in the blood have been shown to be dysregulated in the circulating blood of patients with cancer 11 and are used as noninvasive biomarkers for early diagnosis and evidence of tumor progression in various types of malignancies. [12][13][14] In comparison to exosomes in serum, the molecular composition and functions of sEV in the peritoneal cavity are less understood, presumably due to the difficulty of obtaining samples from patients. However, previous studies have shown that sEV derived from gastric [15][16][17][18] or ovarian 19 cancer cells as well as from malignant ascites from patients 20 are efficiently incorporated in MCs and induces MMT, which facilitates the adhesion of tumor cells to MC and enhances development and growth of PM from gastric cancer.
In addition, inhibition of sEV secretion by Rab27b knockdown in tumor cells suppressed PM formation in a murine model. 21 Based on these results, it is supposed that sEV in the peritoneal cavity also play key roles in the formation of metastases on peritoneal surfaces. 22,23 In previous studies, we undertook a comprehensive analysis of miRNA in sEV contained in the IP fluid (ascites/peritoneal lavage fluid) from patients with gastric cancer and found that the miR-29 family, especially miR-29b, is markedly reduced in peritoneal sEV in patients with PM. 24 Among the patients with advanced gastric cancer with serosal exposure (T4) who underwent curative gastrectomy, miR-29b was markedly downregulated in patients who developed peritoneal recurrence as compared with those without recurrence. 25 As miR-29b is well known to have suppressive effects on tumor progression, [26][27][28] we hypothesized that exogenous replacement of miR-29b in sEV might suppress PM. In a previous study, we found that IP transfer of miR-29b mimics together with atelocollagen can partially suppress PM partly though the effects of MC. 29 In the present study, therefore, we sought to determine how the antitumor effects of miR-29b are modified if administered in the form of encapsulation in sEV. As a source of EVs, we used BMSCs, because MSCs are known to be well-suited for mass production of sEV for drug delivery. 30 We obtained DAPI from Dojindo. Anti-mouse CD31, CD34, CD44, CD49d, CD73, and CD90 were obtained from BioLegend. Antihuman CD9 and CD63 were obtained from BD Biosciences. The lentivirus plasmid of miR-29b precursor and negative control miRNA as well as the pLV-miRNA Expression Vector System were purchased from Biosettica. The sequence of the oligonucleotides used for miR-29b precursor (hsa-mir-29b-1) is as follows:

| Cell culture
NUGC-4 human gastric cancer cells were obtained from Riken, and a BMSC line, UE6E7T-12, was obtained from JCRB cell bank. The cells were cultured in DMEM supplemented with 10% FBS (Sigma), 100 U/ mL penicillin, and 100 mg/mL streptomycin (Life Technologies) at 37°C in a 5% CO 2 cell culture incubator.

| Isolation and culture of HPMCs
Human greater omentum (3-5 cm 3 ) was obtained from patients who underwent sleeve gastrectomy with written informed consent.
Human omental tissue-derived mesothelial cells were isolated as described elsewhere. 32 In brief, cells were collected from omental samples, placed in a half TrypLE Express (Thermo Fisher Scientific) with pure PBS, and incubated at 37°C for 2 h. The supernatants were collected after filtration through 100 μm-pore nylon mesh, then centrifuged at 420 g at 4°C for 5 min. Explants were cultured in DMEM with 20% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin into collagen-coated 10 cm 2 tissue culture dishes. All procedures were carried out in accordance with guidelines and regulations of the

| Lentiviral transfection of human/mouse BMSCs and isolation of sEV
The lentivirus incorporating miR-29b precursor and RFP was created using the pLV-miRNA Expression Vector System according to the manufacturer's instructions. 33 UE6E7T-12 and mouse BMSCs were harvested at a density of 1 × 10 5 cells/well in 6-well plates and then incubated at 37°C in DMEM and 5% CO 2 . After 12 h, cells were transfected with recombinant lentiviral vector integrated with miR-29b or miR-NC vector with 10 μg/mL polyacrylamide (Sigma-Aldrich) to assist the internalization of virus particles.
The MOI was 10. After 20 h, the medium was replaced with fresh culture medium. Three days after transfection, more than 90% of BMSCs were identified to be positive for RFP. The BMSCs were additionally cultured for 48 h, and EVs were extracted from culture supernatants by ultracentrifugation. In brief, supernatants

| Immunofluorescence observation
The BMSC-derived sEV were stained with PKH26 using Cell Linker Kits for General Cell Membrane Labeling (Sigma-Aldrich) as described previously. 34 The HPMCs or gastric cancer cells (1 × 10 4 ) were plated in 24-well plates at 70%-80% confluence and incubated with labeled sEV for 24 h.
To examine the effects on MMT, the HPMCs (5 × 10 4 ) were incubated with 10 ng/mL TGF-β1 in 24-well collagen-coated plates and transfected with miR-29b or NC-containing sEV for 48 h. Cells were washed with PBS, fixed in 4% paraformaldehyde for 10 min at 37°C, and permeabilized with 0.5% Tween-20 in PBS for 20 min.

| Statistical analysis
Data are represented as mean ± SD. The significance of the differences between groups was assessed with one-way ANOVA using GraphPad Prism 8. Differences were considered significant when the p value was less than 0.05.

| MicroRNA-29b-containing sEV derived from human BMSCs suppress MMT of HPMCs
UE6E7T-12 was transfected with lentiviral vector integrated with miR-29b or miR-NC, and the EV were purified from the supernatant by ultracentrifugation. A scanning electron microscope and nanoparticle tracking analysis showed that most of the vesicles were 100-200 nm in diameter ( Figure 1A,B). Flow cytometry revealed that EVs highly expressed CD9 and CD63 ( Figure 1C). These data  290.2 ± 45.1 counts/HPF, n = 5, p < 0.0001; Figure 4A,B).
As FN was reported to be an important molecule mediating the adhesion between tumor cells and mesothelial cells, the effect on the expression of FN on HPMCs was next examined. Although FN was faintly detected on the surface of resting HPMCs, the expression was markedly enhanced after stimulation with TGF-β1 ( Figure 5).
However, when Exo-miR-29b was present with TGF-β1 stimulation, the expression of FN was not increased significantly ( Figure 5).

| Isolation of miR-29b-containing sEV derived from murine BMSCs
After three passages of primary cultures of murine bone marrow, a large of number of spindle-shaped cells was obtained ( Figure 6A) Most of the cells were positive for CD44, CD73, and CD90 but negative for CD31, CD34, and CD49d ( Figure 6B). As these phenotypes were consistent with BMSCs, we undertook transfection of miR-29b-or miR-NC-integrated lentiviral vector, and isolated sEV extracted from their supernatants by ultracentrifugation. A scanning electron microscope and nanoparticle tracking analysis showed that the peak size of the vesicles was approximately 130 nm with positive expression of CD9 and CD63 ( Figure 7A-C). The sEV derived from miR-29b-transfected BMSCs contained markedly higher levels of miR-29b than in the NC ( Figure 8A). As in the human system, PKH26-stained sEV derived from mouse BMSCs were incorporated in mouse peritoneal mesothelial cells more efficiently than YTN16P ( Figure 8B). F I G U R E 2 MicroRNA (miR)-29bcontaining small extracellular vesicles (sEV) suppresses phenotypic changes of human omentum tissue-derived mesothelial cells (HPMCs) stimulated with transforming growth factor-β1 (TGF-β1). HPMCs were cultured with 10 ng/mL TGF-β1 for 48 h. In some wells, HPMCs were transfected with miR-29b in sEV (Exo-miR-29b) or negative control miR (Exo-Control) and cultured with TGF-β1. HPMCs were immunostained with mAbs to E-cadherin, calretinin, and vimentin and their expressions were observed with fluorescein microscopy. No trtmt, without stimulation with TGF-β1. Magnification, 400×.

| DISCUSS ION
The peritoneum is composed of a single layer of flat peritoneal MCs; it functions as the first barrier against bacterial invasion and tumor attachment under physiological conditions. 36,37 In the process of PM formation, however, these MCs lose their intercellular junctions, increase their migratory capacity, and produce substantial amounts of ECM components as well as inflammatory and angiogenic factors.
The phenotypic and functional change of MCs is referred to as MMT and considered to be a crucial component in the development of a premetastatic niche for disseminated tumor cells to form PM. 5,[38][39][40] Recent studies have suggested that specific miRNAs contained in exosomes are critically involved in the induction of MMT and PM formation from gastric cancer. 17,18 In the present study, we focused on miR-29b based on the results of previous translational studies from this laboratory 24 However, in vivo results showed marked differences. In the previous study, atelocollagen-mixed miR-29b was IP injected in a murine model, which reduced the number of PM on the greater omentum but not on the mesentery or parietal peritoneum. Although the reason is unclear, it is speculated that miR-29b transferred with atelocollagen was degraded before suppressing PM in the whole abdominal cavity, as miRNAs are generally degraded rapidly by RNA-degrading enzymes that are abundant in the body. 43 In this study, however, IP transfer of Exo-miR-29b clearly suppressed the development of PM not only in the omentum but also on the mesentery. These results indicate that sEV are more suitable than atelocollagen as the carrier of miR-29b for delivery to the peritoneal space.
In recent years, sEV have attracted a great deal of attention not only for diagnostic but also for therapeutic purposes. 44,45 For example, exosome-mediated delivery of inhibitors for specific miR-NAs could possibly antagonize the effect on growth, migration, and chemoresistance of gastric cancer cells to retrieve drugs. 46,47 In particular, MSCs are known to be ideal candidates as producers of exosomes for drug delivery. 30 There is an increasing body of clinical evidence demonstrating safe transplantation of MSCs, which suggests that transplanting MSC-derived sEV would be unlikely to lead to serious adverse effects. 48  The results of the present study clearly suggest that IP administration of miR-29b when encapsulated in sEV potently inhibits the development of PM partly though the inhibition of MMT induced by TGF-β1. In particular, miR-29b reduced the expression of FN1 on F I G U R E 8 MicroRNA (miR)-29bcontaining small extracellular vesicles (sEV) derived from murine bone marrowderived mesenchymal stem cells (BMSCs). (A) miR-29b expression in mouse BMSCs and corresponding sEV was quantified with 3D-digital PCR. **p < 0.01; ***p < 0.005; **** p < 0.001. Data show mean ± SD in one of three different experiments. (B) Mouse omentum-derived mesothelial cells (mouse PMCs) and YTN16P were cultured with PKH26stained sEV derived from mouse BMSCs for 24 h and observed with fluorescein microscopy. Magnification, 400×. mesothelial cells and inhibited the tumor cell attachment to mesothelial cells, which is the initial and critical step in the process of PM. This is consistent with the results of previous studies showing that blocking the TGFβ signal inhibits MMT, leading to the inhibition of PM from gastric cancer. 52,53 The precise molecular mechanisms of miR-29b to suppress the process of PM remain unclear, which is the limitation of this study.
However, numerous studies have already shown that miR-29b suppresses the proliferation and migration of tumor cells through the inhibition of CKD, CDC42, PI3K/AKT signal, and the p53-mediated apoptotic pathway. 28,54 MicroRNA-29b is also shown to suppress EMT though the direct inhibition of a number of EMT-related genes such as β-catenin (CTNNB1), integrin β1 (ITGB1), lysyl oxidase like 2 (LOXL2), and mucin-1 (MUC1) as well as many ECM proteins (e.g., COL1A1, COL3A1, and LAMA2). 28,54 We confirmed that Exo-miR-29b also suppressed the migration and tended to reduce the proliferation of human (NUGC-4) and murine (YTN16P) gastric cancer cells, possibly though the inhibition of these molecules ( Figure S2).
Given that these EMT-related genes are largely shared with mesothelial cells, these molecular mechanisms are considered to be involved in MMT. In addition, it is well known that miR-29 can suppress fibrosis, angiogenesis, and immune responses. 28,55 Taken together, miR-29b is supposed to suppress the PM through the effects both on tumor and host cells through multiple molecular mechanisms.
The results of this study strongly suggest that miR-29b might be clinically useful to prevent PM in patients with advanced gastric cancer. The results also suggest that sEV are a hopeful tool for drug delivery of miRNA drugs. There are still few clinical application examples of nanomedicine therapeutic drugs using sEV, especially targeting cancer. 56 However, modification of the surface structure of sEV could further enhance the targeting efficiency to specific cell types. 57,58 Therefore, induction of MC-specific molecules on sEV might further increase the inhibition on PM. Further studies are warranted to develop stable production and preservation methods to maintain the biological quality of sEV. F I G U R E 9 Intraperitoneal (IP) transfer of microRNA (miR)-29b-containing small extracellular vesicles (sEV) suppresses peritoneal metastasis in a mouse syngeneic model. C57BL6/N mice were IP injected with YTN16P (1.0 × 10 5 cells, 500 μL) and then IP injected with PBS (500 μL) or sEV (40 μg/500 μL) isolated from murine bone marrow-derived mesenchymal stem cells transfected with lentivirus integrating miRNA-29b (Exo-miR-29b) or negative control (Exo miR-NC) every 3 days from day 0 to day 18. Mice were killed on day 21 and peritoneal metastases were evaluated based on the number of macroscopic nodules on the mesentery and the omental surface. Data show mean ± SD in one of the two different experiments. *p < 0.05. No trtmt, without treatment.

ACK N OWLED G M ENTS
We thank N. Nishiaki, J. Shinohara, H. Hatakeyama, and I. Nieda for technical and clerical work.

FU N D I N G I N FO R M ATI O N
This work was supported by the Japan Society for the Promotion of Science (20K07704) and subsidized by JKA through its promotion funds from KEIRIN RACE.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors have no conflict of interest.

E TH I C S S TATEM ENT
Approval of the research protocol by an Institutional Review Board: