Mesenchymal Stem Cell-Derived Extracellular Vesicles: Roles in Tumor Growth, Progression, and Drug Resistance

Mesenchymal stem cells (MSCs) are ubiquitously present in many tissues. Due to their unique advantages, MSCs have been widely employed in clinical studies. Emerging evidences indicate that MSCs can also migrate to the tumor surrounding stroma and exert complex effects on tumor growth and progression. However, the effect of MSCs on tumor growth is still a matter of debate. Several studies have shown that MSCs could favor tumor growth. On the contrary, other groups have demonstrated that MSCs suppressed tumor progression. Extracellular vesicles have emerged as a new mechanism of cell-to-cell communication in the development of tumor diseases. MSCs-derived extracellular vesicles (MSC-EVs) could mimic the effects of the mesenchymal stem cells from which they originate. Different studies have reported that MSC-EVs may exert various effects on the growth, metastasis, and drug response of different tumor cells by transferring proteins, messenger RNA, and microRNA to recipient cells. In the present review, we summarize the components of MSC-EVs and discuss the roles of MSC-EVs in different malignant diseases, including the related mechanisms that may account for their therapeutic potential. MSC-EVs open up a promising opportunity in the treatment of cancer with increased efficacy.

MSC-EVs could also be involved in the effects of MSCs on tumor growth and behavior. Several studies describing the influence of MSC-EVs on tumor growth have been reported. Thus, it is reasonable to postulate that MSC-EVs transport key MSC-associated molecules which change the physiology of target cells in a specific manner. MSC-EVs have emerged 2 Stem Cells International as a new mechanism of cell-to-cell communication in the development and growth of human malignancies.
In this article, first we will review the composition of MSC-EVs which will be classified based on their molecular contents into four groups: proteins, messenger RNAs (mRNAs), microRNAs (miRNAs), and others. Then the effects of MSC-EVs on cancer development and progression will be highlighted. Finally, we will address the possible molecular mechanisms underlying MSC-EVs-mediated therapeutic effects.

Cargoes of MSC-EVs
Several studies have revealed that MSC-EVs contain proteins, lipids, and genetic materials, such as mRNAs and miRNAs [65] (Figure 1). Transfer of these biological materials into adjacent or distant cells may influence the behavior of the recipient cells [32,36,66].

mRNA.
Besides proteins, one of the most distinct features of MSC-EVs is that they also contain nucleic acids, including mRNAs and miRNAs [65]. mRNAs and miRNAs can be transferred into a recipient cell located in the tumor microenvironment or at distant sites via fusion of MSC-EVs with the target cell membrane.
In EVs from porcine adipose tissue-derived MSCs, researchers found distinct classes of RNAs were selectively expressed using high-throughput RNA sequencing [48]. EVs preferentially express mRNAs for angiogenesis, adipogenesis, Golgi apparatus, and transcription factors associated with alternative splicing, apoptosis, and chromosome organization. EVs also express genes involved in TGF-signaling (TGFB1, TGFB3, FURIN, and ENG).
Likewise, some other miRNAs, such as miRNA-15a [50], miRNA-16 [52], miRNA-21, miRNA-34a, and miRNA-191 [41,72], have been identified in MSC-EVs and shown to prevent apoptosis, promote cellular growth [73], reduce cardiac fibrosis [74], and inhibit tumor growth [75] by regulating their target genes in recipient cells. While these miRNAs are not randomly sorted into the MSC-EVs, some miRNAs are present only in the original cells, but not in the MSC-EVs. However, some certain miRNAs are selectively sorted into the MSC-EVs, which are undetectable in the original MSCs, such as miRNA-564, miRNA-223, and miRNA-451. The specific mechanism of MSC-EVs content sorting is not clear.

Lipid and Other Contents of MSC-EVs.
Our knowledge on the lipid composition of MSC-EVs is quite limited. Only a few studies confirmed high level of bioactive lipids such as diacylglycerol and sphingomyelin but trace amounts of dihydroceramide and -hydroxy-ceramide in MSC-EVs. Furthermore, small molecule metabolite assays have demonstrated the presence of lactic acid and glutamic acid in EVs [41].

MSC-EVs Inhibit Proliferation and Promote the Apoptosis of Tumor Cells
The role of MSC-EVs in tumor proliferation has been well documented. However, the mechanisms by which MSC-EVs Human adipose tissue-derived MSCs Neprilysin Degrade intracellular and extracellular -amyloid peptide in neuroblastoma cell lines [42] Human bone marrow-derived MSCs TIA, TIAR, HuR T cell internal antigen [43] Human bone marrow-derived MSCs Stau1, Stau2 Involved in the transport and stability of mRNA [43] Human bone marrow-derived MSCs

Ago2
Involved in the miRNA transport and processing [43] Human umbilical cord-derived MSCs
A similar effect was observed in EVs derived from human cord blood Wharton's jelly MSCs (hWJMSC-EVs) [55]. hWJMSC-EVs abolished T24 bladder tumor proliferation via G0/G1 phase arrest in a dose-dependent manner and induced apoptosis in T24 cells in vitro and in vivo. The antiproliferative and proapoptotic effects were mainly mediated by restraining phosphorylation of Akt, upregulation of p-p53, and activation of caspase cascade (caspase-3 cleavage).
Another recent paper described the effect of murine MSC-EVs on the expression of VEGF in mouse breast cancer cell line (4T1). It demonstrated that murine MSC-EVs significantly downregulated the expression of VEGF in a dose-dependent manner, causing inhibition of angiogenesis in vitro and in vivo. Additionally, miRNA-16 shuttled by MSC-EVs was partially responsible for the antiangiogenic effect of MSC-EVs [52].
In addition, it was reported that in hematological malignancies normal BMSC-EVs inhibited the growth of multiple myeloma (MM) cells, while MM BMSC-EVs promoted MM tumor growth [50]. Further study found that normal and MM BMSC-EVs differed in their protein and miRNA contents, with higher expression of cytokines, oncogenic proteins, and protein kinases in MM BMSC-EVs, but lower level of miRNA-15a. On the basis of this information, MSC-EVs could therefore exert either antiproliferation or proapoptotic effects on tumor cells (Table 4).

MSC-EVs Promote the Growth and Metastasis of Tumor Cells
The tumor growth promoting effects of MSC-EVs have also been suggested by various reports. For instance, researchers have found that MSC-EVs could increase tumor growth in BALB/c nu/nu mice xenograft model by enhancing VEGF expression through activation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 MAPK pathway [56]. Inhibition of ERK1/2 activation could reverse the increase of VEGF level by MSC-EVs. However, the proproliferative effect on cancer cells was not observed in vitro, and there were no differences in the percentage of cells in the G0/G1, S, and G2/M phases between EV-treated and untreated cells. These findings suggest that MSC-EVs do not directly stimulate proliferation of cancer cells in vitro but instead induce activation of an angiogenesis program that could favor tumor engraftment and growth. MSC-EVs can also promote the metastasis of the breast cancer cell line MCF7 by activating the Wnt pathway. In a study on MM, researchers found that BMSC-EVs could promote proliferation, survival, and metastasis of myeloma cells. p38, p53, c-Jun N-terminal kinase, and Akt pathways in MM cells were influenced by BMSC-EVs [57].
In addition, Du et al. have reported that hWJMSC-EVs promoted the growth and migration of human renal cell carcinoma (RCC) cells both in vitro and in vivo. EVs facilitated the progression of cell cycle from G0/G1 to S. The mechanisms underlying this effect were suggested to be transfer of RNA material by EVs to induce hepatocyte growth factor (HGF) expression in RCC and activate Akt and ERK1/2 signaling pathways. Use of c-Met inhibitors can abrogate the activation of AKT and ERK1/2 signaling in 786-0 cells [58]. Interestingly, the same group has demonstrated the antiproliferative and proapoptotic effects of hWJMSC-EVs on bladder cancer cells [74].
Taken above findings together, the same EVs can have opposite effects on different tumors (Figure 2). The specific mechanism is not precisely known.

MSC-EVs Promote Dormancy of Tumor Cells
Some researchers have found that BMSC-EVs could decrease the proliferation of BM2 cells and reduce the abundance of stem cell-like surface markers. Further studies showed that dormant phenotypes were induced by overexpression of miR-23b in BM2 cells which suppressed MARCKS gene [51]. Another study has also indicated that stroma-derived exosomes contributed to breast cancer cells quiescence. The transfer of miRNAs might be involved in the dormancy of BM metastases [59]. Thus, targeting miRNA may be a valid therapeutic tool to reduce breast cancer metastasis.

MSC-EVs Promote Drug Resistance of Tumor Cells
It has been reported that BMSC-EVs not only increase MM cells growth but also induce resistance to bortezomib (BTZ), a proteasome inhibitor [57]. BMSC-EVs could inhibit the reduction of Bcl-2 expression caused by BTZ and reduce the cleavage of caspase-9, caspase-3, and PARP. Researchers also found BMSC-EVs could decrease the sensitivity of BM2 cells to docetaxel, a common chemotherapy agent [51]. In addition, the EVs derived from rat bone marrowderived MSCs (rBMSC-EVs) can protect the rat pheochromocytoma PC12 cells against the excitotoxicity induced by glutamate. In this study it was also revealed that rBMSC-EVs reduced the expression of Bax and Bcl-2. Inhibition of PI3K/Akt pathway could partially abrogate the protective effects [60].

Conclusion
MSC-EVs could mimic the effects of mesenchymal stem cells in tumor therapies. Compared with cells, MSC-EVs are much smaller and have a lower possibility of immune rejection and formation of tumor. Therefore, MSC-EVs represent a promising alternative that could overcome the limitations of cell-therapy approaches. Besides being therapeutic agents, MSC-EVs have been advocated as "natural" drug delivery vehicles [76][77][78]. These lipid vesicles could be engineered to deliver therapeutic agents to target sites. For instance, it has been reported that the EVs secreted by SR4987 cells primed with paclitaxel (SR4987PTX) delivered active drugs and inhibited human pancreatic adenocarcinoma cells proliferation in a dose-dependent manner [79]. However, several questions have to be answered before clinical application of MSC-EVs. Firstly, it is very important to carefully evaluate the safety issues. For MSC-EVs have been reported to promote tumor growth, it is necessary to verify what kind of tumors may benefit from the treatment and to which extent MSC-EVs contribute to the beneficial effects. Secondly, researchers should thoroughly characterize the content of MSC-EVs and identify what molecules shuttled by MSC-EVs would function. Thirdly, the technologies for the isolation, detection, characterization, and engineering of MSC-EVs need to be standardized for their clinical application. Meanwhile, MSC-EVs dose, optimal timing of MSC-EVs administration, and schedule of administration also need to be developed for effective usage of MSC-EVs.
In conclusion, although MSC-EVs open up a promising opportunity to develop new "biotech drugs" in malignant diseases, further investigation is still required in some areas.