The Role of Exosomal MicroRNAs in the Tumor Microenvironment of Breast Cancer

Breast cancer, ranking first among women’s cancers worldwide, develops from the breast tissue. Study of the breast tissue is, therefore of great significance to the diagnosis and treatment of breast cancer. Exosomes, acting as an effective communicator between cells, are in the ascendant in recent years. One of the most important cargoes contained in the exosomes is microRNAs, belonging to the non-coding RNA family. When the exosomal microRNAs are absorbed into the intracellular location, most of the microRNAs will act as tumor promoters or suppressors by inhibiting the translation process of the target mRNA, thus affecting the behavior of other stromal cells in the tumor microenvironment. At present, growing research focuses on the different types of donor cell sources, their contribution to cancer, miRNA profiling, their biomarker potential, etc. This review aims to state the function of diverse miRNAs in exosomes medicated cell–cell communication and the potency of some specific enriched miRNAs as molecular markers in clinical trials. We also describe the mechanism of anti-cancer compounds through exosomes and the exploration of artificially engineered techniques that lead miRNA-inhibitors into exosomes for therapeutic use.


Breast Cancer
Breast cancer is one of the most common female malignant tumors and threatens the physical and psychological health of women around the world. About 1.3 million cases of mammary cancer worldwide are diagnosed and 450,000 people's lives are being taken every year [1]. Moreover, it was reported by the American Cancer Society that breast cancer ranked first in the incidence rate among American women s cancer between 1975 and 2014 [2], ranking second in mortality (after lung cancer) [3]. According to the latest report, there were approximately 268,670 new breast cancer patients in 2018 in the United States, which resulted in 40,000 deaths [2].
Breast carcinoma is a complex disease of morphological and molecular heterogeneity, characterized by three morphological grades and over four different molecular subtypes (at the gene expression level) [4]. According to the consensus reached at the St. Gallen International Expert Conference of breast cancer in 2015 and 2017 [5,6], breast cancer was clinically classified into four major subtypes: triple negative, hormone receptor (HR)-negative and human epidermal growth factor receptor 2 (HER2)-positive, HR-positive and HER2-positive, HR-positive and HER2-negative (Table 1). For different types of breast tumors, there are great differences in the treatment options.
As known to us all, the constant growth of tumor metastasis is responsible for most cancer deaths [9]. Since Paget first proposed the famous 'seed and soil' hypothesis (1989), the relationship between the microenvironment and the tumor has caused widespread concern that tumor metastasis was not an accidental event, it happened only when those cancer cells with potential to metastasize (the 'seed') were compatible and familiar with proper organ microenvironment (the 'soil') [9][10][11]. The TME often refers to an area that is close to the existence of the solid tumor. Apart from breast cancer cells, the TME also contains plenty of other different types of cells including vascular endothelial cells (VECs), cancer-associated fibroblasts (CAFs), immune cells like tumor-associated macrophages (TAMs), myeloid-derived suppressor cell (MDSCs), T lymphocytes, B lymphocytes, as well as myoepithelial cells, adipocytes, etc. Moreover, some non-cellular components are also involved, covering the extracellular matrix (ECM), exosomes, soluble cytokines or signaling molecules [12,13]. It is worth noting that the physical characteristics of the tumor microenvironment are also different from normal tissues, such as hypoxia, acidity, high interstitial fluid pressure [13,14].
As described by Kalluri et al. [15], tumors can also be seen as a wound, accompanying inflammatory reactions. Different immune cells in the tumor microenvironment have different effects, thus creating a balance between carcinogenesis and tumor suppressor. Tumor-associated macrophages (TAMs) belong to bone marrow-derived cells with important roles in innate and adaptive immunity [25]. They are very abundant and highly infiltrating in the tumor microenvironment, and the richer density the macrophages, the worse the prognosis of patients [26]. TAMs can be derived from the following types of cells: blood monocytes, blood monocyte-related myeloid-derived suppressor cells, tissue-resident macrophages [27]. They can be recruited to tumor sites by cytokines (colony-stimulating factor-1(CSF1), chemokine (C-C motif) ligand 2 (CCL2), CCL5, etc.), and differentiate into TAMs [27]. Generally speaking, there are two subtypes of TAMs-classically (M1)-and alternatively-activated (M2) macrophages [12]. M1 macrophages have antineoplastic effect with the function of secreting tumor necrosis factor-α (TNF-α), IL-23, IL-12, etc., while M2 phenotype will express IL-10, CCL20, transforming growth factor-β (TGF-β), etc., to promote tumor [28]. In addition, TAMs can excrete cytokines such as epidermal growth factor (EGF), PDGF, VEGF, CCL2, CXCL8 to promote angiogenesis [29]; participate in CSF1 (secreted from breast cancer cell) and EGF (contributed by activated macrophages) feedback loop to cause metastasis [29]; and accumulate in hypoxic area [26].
In brief, it must be emphasized that tumor microenvironment is critical for tumor behaviors: occurrence, progression, invasion and metastasis, prognosis and drug resistance [29,30].

Exosomes
Exosomes, a branch of extracellular vesicles (EVs), are encased in cell membranes made up of lipid bilayers with a diameter of 30-100 nm and cup-shape appearance [31,32]. Almost all eukaryotic cells can secrete exosomes, including tumor cells and normal stromal cells, but it is reported that cancer cells express more exosomes than normal proliferation cells [32,33].
Recently, exosomes have come under increasing interest from researchers, mainly because they have been found to wrap many biomolecules, such as DNAs, mRNAs, non-coding gene family (microRNA, lncRNA), proteins, and lipids [38]. Carrying these active biomolecules, exosomes have also been found to circulate in body fluids, such as blood (plasma or serum), urine, feces, breast milk and saliva, so that exosomes are able to transmit intercellular regulatory information to the both surrounding and distant sites, which indicates the potential to be non-invasive biomarkers [39]. Increasing articles of exosomes reported extensive involvement in physiological and pathological processes, such as placental physiology [40], angiogenesis and increased heart function [41], endometrial-embryo interactions [42], and pituitary tumors [43]. Here, we mainly focus on the role of exosomes in breast cancer.

MicroRNAs
In 1993, Lee et al. reported the first discovery of miRNA that gene lin-4 encoded small RNA molecules to regulate the expression of protein lin-14 in Caenorhabditis elegans [44]. MicroRNAs (miRNAs) are small RNA molecules with single chains that do not encode proteins. They usually have a length of about 19 and 25 nucleotides, most of which negatively regulate the post-transcriptional level of target messenger ribonucleic acids (mRNAs) by binding to the 3 -untranslated region (UTR) [45]. Three possible mechanisms may be involved in the process: repression of translation initiation, post-initiation inhibition and target mRNA destabilization, resulting in the degradation of mRNA or translation inhibition [46].
In recent years, numerous studies have shown that varieties of microRNAs function in direct or indirect interactions between breast cancer cells and components of TME [47,48]. For example, miR-373 and miR-520c can raise the invasion and metastasis of tumor by inhibiting metastasis-related gene CD44 [49]. Moreover, miRNAs can be circulated in body fluid and the expression of miRNAs in peripheral blood may be used as a biological marker for the differential diagnosis and prognosis of breast tumors [7,39]. Exosomal miRNA profiling analyses were reported and compared, such as between drug-resistant and non-drug-resistant cells [50,51], normal mammary epithelial cells (MCF-10A) and MCF-7 [52], ER-positive (MCF-7) and triple negative (MDA-MB-231) cancer cell lines [53], HER2-positive and triple negative cancer patients [54], from canine mammary cells [55]. Liu et al. [56] integrated small RNA sequencing information of 17 diseases and built up an open EVmiRNA database for public searching of miRNA profiles.
This review mainly focuses on the bioactivity and underlying mechanism of exosomal microRNAs in the change of tumor microenvironment during the initiation and development of breast cancer. The synthesis of microRNAs may occur in extracellular microvesicles. Melo et al. [57] reported that exosomes derived from mammary tumor cells (MDA-MB-231, MCF-7, 4T1) can specifically aggregate pre-miRNAs, as well as other proteins of the RISC complex, and then generate the mature miRNAs (up-regulated: miR-10a, -10b, -21, -27a, -155, -373) inside. When non-tumorigenic mammary epithelial cells (MCF-10A) and exosomes of tumor cells (MDA-MB-231) were co-cultured, the cancerization of normal cells was promoted, and Dicer enzyme (an important instrumental enzyme in the formation of mature miRNAs) could be seen as a controlling factor of this process. When MCF-10A was co-injected with exosomes derived from serum of breast cancer patients or healthy controls into nude mice, the former could form tumors with higher level of Dicer while the latter could not. Furthermore, exosomal miR-210 from tumor cells could transfer to endothelial cells to promote angiogenesis and metastasis and the secretion process of exosomes was also proved to be dependent on neutral sphingomyelinase 2 (nSMase2) enzyme (regulate ceramide biogenesis) [58,59]. Singh et al. [59] proved that nSMase2 or ceramide could promote the level of exosomal miR-10b, resulting in enhanced invasion ability of non-malignant cells by inhibiting the expression of homeobox D10 (HOXD10) [60] and KLF4. Other miRNAs such as miR-1246 [61] were also found to be secreted from breast cancer cells and change the behaviors of normal epithelial cells (Table 2).

The Role of miRNAs in Exosomes in the Intercellular
Some highly metastatic/drug-resistant tumor cells can affect other tumor cell lines with low metastatic/non-drug resistance by secreting miRNAs in the exosomes, which is more convenient for their excessive growth. Different chemotherapeutic drugs-resistant MCF-7 cell lines were reported to secrete different miRNAs such as miR-222 [62,63], -23a [64], -100 [65], and -149 [66], etc., to non-drug-resistant cells to improve their resistance, leading to treatment failure ( Table 2). In addition, some in vitro experiments also showed that after incubating purified exosomes from the metastatic cell line MDA-MB-231, the mobility and anchorage-independent ability (metastatic behaviors) of low metastatic MCF-7 cells were all increased and the underlying mechanism was related to exosomal miR-9 and miR-155 [67,68]. Genetically manipulated cells can also increase the level of specific microRNAs in exosomes and be absorbed by the original cell lines [69,70], which prompts a therapeutic direction.
Not only breast-related cells, but other stromal cells in the microenvironment can also uptake the external exosomes derived from the tumor cells, and then the contained miRNAs will be transported into them and affect their behavior, providing a favorable environment for the growth of the tumor ( Figure 1). Exosome-mediated transfer of miR-105 [71] and miR-939 [72] could increase the permeability and destroy the integrity of vascular endothelial cells, leading to enhanced metastasis by targeting the zonula occludens 1 (ZO-1) gene and VE-cadherin, respectively. From data of clinical trials, exosomal miR-105 was proved to be associated with the incidence of metastasis [71]. Yan's study [73] also demonstrated that the level of miR-105 increase in the exosomes secreted from breast cancer cells was induced by oncogene MYC, and then transferred to CAFs. They also showed that miR-105 targeted at MAX-interacting protein 1 (MXI1) to activate MYC signaling and reprogram CAFs. Interestingly, whether under the circumstances of efficient nutrition or not, reprogrammed CAFs would ultimately promote tumor growth by leading to different metabolic pathways (glucose and glutamine metabolism with sufficient or metabolic waste decomposition, such as lactic acid and ammonium) and regulating the components of TME to provide energy to fuel cancer cells [73], which was similar to exosomal miR-122 transferred to lung fibroblasts and brain astrocytes [74]. In a recent study, after overexpressing miR-940 in breast cancer cells, treatment of human mesenchymal stem cells (MSCs) with exosomes contained culture medium-was shown to promote osteogenesis in vitro by acting on Rho GTPase-activating protein 1 (ARHGAP1) and family with sequence similarity 134, member A (FAM134A) [75]. The well-designed in vivo experiment also demonstrated that exosomes (labelled with CD63 (exosomes' marker) fused red fluorescent) were derived from miR-940 overexpressed cancer cells and absorbed by host cells (labelled with GFP) under confocal microscopy, which caused osteoblastic lesions by implanting cancer cells onto the calvarial bones/tibial sites of immunodeficient mice [75]. The growing expression of miR-770 (acting as a tumor suppressor, direct targets at STMN1) from TNBC cells to tumor-associated macrophages (TAMs) through exosome delivery modulated more differentiation to M1 phenotype instead of M2 phenotype and inhibited drug-resistance [76]. Cancer-produced exosomes may also be regulated by the physical conditions of microenvironment like hypoxia, an unignored feature of solid tumors that motivates tumor deterioration [77]. By using a miR-210 specific reporter system, elevated miR-210 was visually proved to be transferred from hypoxic cancer cells to proximal endothelial cells via exosomes both in vitro and in vivo, which may be interceded by hypoxia-inducible factor-1α (HIF-1α) and restrain the expression of vascular remodeling related genes, like Ephrin A3 and PTP1B, to support angiogenesis and tumor growth [78,79]. Cancer-produced exosomes may also be regulated by the physical conditions of microenvironment like hypoxia, an unignored feature of solid tumors that motivates tumor deterioration [77]. By using a miR-210 specific reporter system, elevated miR-210 was visually proved to be transferred from hypoxic cancer cells to proximal endothelial cells via exosomes both in vitro and in vivo, which may be interceded by hypoxia-inducible factor-1α (HIF-1α) and restrain the expression of vascular remodeling related genes, like Ephrin A3 and PTP1B, to support angiogenesis and tumor growth [78,79]. Exosomes are effective in transmitting drug resistance and the delivery of miR-222 via exosomes may be a mechanism.
[63] [69]   (Table 3) In turn, as the reports accumulated, stromal cells can also express exosomes to function in cancer cells (Figure 2). For instance, miR-9 can be secreted in exosomes by both breast cancer cells and CAFs [80]. When miR-9 is transferred from tumor cells, it can enhance the transformation of normal fibroblasts (NFs) to CAFs and its migration and invasion abilities. Conversely, CAFs can secrete miR-9 to tumor cells and NFs to promote tumor growth [80]. Dioufa et al. [81] observed that the tumor-derived exosomes helped to transfer the tumor cells to the liver and then remain in the dormancy state in the early stage of metastasis, which was characterized in slow proliferation, insensitivity to chemotherapy and difficulty in discovery, resulting in recurrence and poor prognosis [82]. However, exosomes from hepatic niche cells led to an increased mesenchymal to epithelial reverting transition (MErT) of cancer cells, which was explained as the adaptation process of tumor cells in heterogeneous organs [81]. Uen et al. [83] demonstrated that miR-122-5p, which was found in the human hepatoma cells' exosomes, would target at syndecan-1 (SDC-1) and promote breast cancer cell mobility.

Exosomes from Stromal Cells Can Transfer miRNAs to Cancer Cells and Contribute to Cancer Progression
A relatively higher level of miR-155 was detected in exosomes of breast cancer stem cells (CSC) and chemo-resistant cancer cells. When sensitive cancer cells were co-cultured with resistant cells' exosomes, the migration ability and chemoresistance was promoted [84]. Exosomal miR-23b was reported to transit from bone marrow mesenchymal stem cells (BM-MSCs) to metastatic breast cancer cells which were homing to bone marrow (BM2), and the BM2 cells could be induced to enter into dormancy by inhibiting myristoylated alanine-rich C kinase substrate (MARCKS) [82]. Similarly, miR-222/223 [85], miR-127 and miR-197 [86], miR-21 and miR-34a [87], miR-126a [88] in exosomes from BM-derived cells also supported tumor growth in different ways such as drug resistance, dormancy, metastasis, and angiogenesis (Table 3).
However, there are some reports that exosomal anti-cancer miRNAs would also be secreted by stromal cells to fight with the malignancies (Table 3). MSC-derived exosomes shuttled miR-16 [92] and miR-100 (inhibiting the mTOR/HIF-1α/VEGF pathway) [93] to the nearby cancer cells and decreased their VEGF expression, subsequently resulting in inhibition of the endothelial cells' vascular behavior. After chemotherapy, the upregulation of miR-503 via exosomes in endothelial cells reduced cancer cells' proliferation and invasion through targeting cyclin D2 (CCDN2) and CCDN3 [94].   Increased plasmatic miR-503 in breast cancer patients after neoadjuvant chemotherapy, which could be partly due to increased miRNA secretion state in the early stage of metastasis, which was characterized in slow proliferation, insensitivity to chemotherapy and difficulty in discovery, resulting in recurrence and poor prognosis [82]. However, exosomes from hepatic niche cells led to an increased mesenchymal to epithelial reverting transition (MErT) of cancer cells, which was explained as the adaptation process of tumor cells in heterogeneous organs [81]. Uen et al. [83] demonstrated that miR-122-5p, which was found in the human hepatoma cells' exosomes, would target at syndecan-1 (SDC-1) and promote breast cancer cell mobility. A relatively higher level of miR-155 was detected in exosomes of breast cancer stem cells (CSC) and chemo-resistant cancer cells. When sensitive cancer cells were co-cultured with resistant cells' exosomes, the migration ability and chemoresistance was promoted [84]. Exosomal miR-23b was reported to transit from bone marrow mesenchymal stem cells (BM-MSCs) to metastatic breast cancer cells which were homing to bone marrow (BM2), and the BM2 cells could be induced to enter into dormancy by inhibiting myristoylated alanine-rich C kinase substrate (MARCKS) [82]. Similarly, miR-222/223 [85], miR-127 and miR-197 [86], miR-21 and miR-34a [87], miR-126a [88] in exosomes from BM-derived cells also supported tumor growth in different ways such as drug resistance, dormancy, metastasis, and angiogenesis (Table 3).

Circulating MicroRNAs in Exosomes Imply Their Potential Biomarker Value
Some circulating microRNAs contained in exosomes can potentially be regarded as predictive, diagnostic and prognostic biomarkers for breast cancer ( Table 4). The advantages of exo-miRs over miRs are obvious: (1) Exo-miRs act as cell-cell communicator and circulate in the body fluids, such as peripheral blood andbreast milk. Thus, the collection method is non-invasive, or simply a common method like blood collection. When it comes to miRs, it is not easy to get tumor tissues and pathological diagnosis is still the gold standard. (2) It has also been reported that the expression level of some special exo-miRs have shown significant differences in early stages of cancer, even during dormancy period. In this case, miRs in tumor tissues cannot be detected. (3) Post-operative monitoring is even more important because recurrence and metastasis are still the greatest cause of breast cancer death. Additionally, exo-miRs can be easily detected and constantly monitored. Lower levels of exosome-derived miR-130a-3p are associated with lymph node metastasis (p = 0.0019) and advanced TNM stage (p = 0.0014).    Plasma-and serum-derived miR-106a-5p and miR-20b-5p, belonging to miR-106a-363 cluster on chromosome X, showed an increasing trend in tissues and exosomes, indicating their role as potential diagnostic biomarkers [95]. In addition, a combination of multiple markers may also be a possible way to indicate the presence of breast cancer. For example, exosomal miR-12 and miR-1246 were proved to be a potential combined indicator [96]. Cell-free miR-101 and miR-373 in blood serum exhibited significant differences between malignant and benign tumors, and it is also worth noting that the higher content of exosomal miR-373 was associated with malignant breast cancer like TNBC [97]. Gao et al. [98] also reported that high level of miR-155, which may be derived from blood cells exosomes, was found in the early stage in cancer patients. Additionally, miR-130a-3p (tumor suppressor) [99], miR-16, miR-30b and miR-93 [100], miR-200c and miR-141 [101], and miR-223-3p [102] in exosomes also exhibited valuable functions to classify cancer stages (Table 4).
Recurrence often happens in breast cancer patients even after a mastectomy, which is totally different from patients never being treated, so the development of molecular markers for recurrence and treatment monitoring is very essential. High level of exosomal miR-21, -105, and miR-222 in serum samples [103] could possibly become a complementary tool for prognostic and monitoring use clinically because the level of miR-21 was proved to link with tumor size and Ki-67 expression during the treatment. The miRNA profile was detected in clinical patients with or without recurrence, and the level of miR-340-5p, miR-17-5p, miR-130a-3p, and miR-93-5p exhibited close relation with recurrence rate in their logistic regression analysis [104].
When the sample (e.g., plasma, serum) are obtained, the usual method is to use ultracentrifugation or commercial kits to extract and purify exosomes, and then use the transmission electron microscope (TEM) to detect their diameter size (30-100 nm), western blot (WB) to determine surface biomarkers (such as CD63, CD81, CD9), and other methods to confirm that the isolated EVs are exosomes, and to preserve in low temperature afterwards. When testing microRNAs, experimental methods such as RNA sequencing or qRT-PCR will be used. Exosomes uptake can be observed by PKH67 staining or confocal microscopy detection of fluorescence. Furthermore, since miRNAs can easily get degraded with time, different in situ and quantitative methods were also explored in order to detect the content of exosomal miRNAs in a faster and better manner. A direct method without RNA isolation or purification procedures, which was also evaluated in the terms of specificity, accuracy and efficiency, was used to prove miR-106a in plasma as a potential diagnosis biomarker [105]. Zhai et al. [106] synthesized an in situ probe which can quantitatively evaluate the level of miR-1246 in plasma exosomes with high degree of sensitivity and specificity, which is a promising translation for clinical use. Likewise, other reported methods for detecting exosomal miRNAs are mainly based on biosensors [107,108]; fluorescent probes [109,110]; DNA enzyme probes [111]; green fluorescent protein (GFP) tag technology [112]; weight-dependent molecular sieves [113], etc.
It is necessary to develop more effective, specific, sensitive biomarkers and detection methods to accelerate detection speed and improve patients' compliance. In the future, this kind of reagent will make it possible for a single drop of blood to verdict breast cancer, different from subtypes and treatment response monitoring.

Therapeutic Method Targeting at the Exosomal microRNAs
The important role of exosomal miRNAs in cancer progress cannot be ignored, so it is also significant (1) to develop new drugs targeting at vital miRNAs and (2) to achieve a novel treatment of the exosomes with therapeutic microRNAs.
The mechanisms of some clinical anticancer drugs from natural herbs or synthetic sources are reported to be partly dependent on miRNAs as exosomal cargoes (Table 5). D Rhamnose β-hederin (DRβ-H), a triterpenoid saponin, could reduce the growth and apoptosis of cancer cells by inhibiting the secretion process of exosomes. Subsequently, some specifically encapsulated miRNAs, including miR-130a and miR-425, were also downregulated [114]. Shikonin (SK) could also suppress exosome release accompanying with reduced miR-128 [115] and restrain the cancer-promoting effects of preadipocytes through disturbing miR-140/SOX9 signaling [116]. Epigallocatechin gallate (EGCG) is being pointed out to express anti-cancer activity by inhibiting the macrophages infiltration in TME through upregulating miR-16 in exosomes [117], and chemo-susceptibility could be elevated by β-elemene through affecting the expression of resistance-related genes such as miR-34a, miR-452, Pgp, and PTEN [118]. Hannafon et al. [119] reported that after docosahexaenoic acid (DHA) administration, exosomes secreted from breast cancer cells mediated the increase of miR-23b and miR-320b in recipient endothelial cells and decreased the expression of target genes and corresponding proteins (PLAU, AMOTL1, NRP1, ETS2, respectively), thus inhibiting their tube formation and angiogenesis capability.
Increasing evidence indicated that exosomes are important carriers and they probably can be manipulated to deliver tumor suppressor-miRNA or oncomiR-inhibitors to express their potential therapeutic effects ( Table 5). As described, exosomal let-7a from donor cells was specifically delivered to epidermal growth factor receptor (EGFR)-expressing tumor sites for therapeutic use [120]. Recently, O'Brien et al. [121] genetically modified MSCs by lentiviral transduction to enrich miR-379 in derivative extracellular vesicle (EV), which showed significant anti-cancer activity in vivo by direct delivery. Meanwhile, tumor-derived exosomes could also be engineered to overexpress miR-155, -142, and let-7i by electroporation to mature dendritic cells and to trigger the immunity process [122], to load siRNAs or miRNAs by sonication to knockdown oncogene like HER2 [123]. Co-transfection of antagomiR-222/223 into MSCs reversed tumor dormancy and drug resistance [85]. Gold nanoparticle (AuNP)-based gene silencing technology could also load anti-gene/miRNA oligonucleotides [124], and locked nucleic acid (LNA)-modified BM-MSCs could secrete anti-miR-142-3p oligonucleotides in exosomes and exert inhibitory effects in vitro and in vivo [125]. β-elemene effectively sensitizes drug-resistant BCA cells to Doc and Adr through a signaling pathway that involves miRNA and gene regulation MSCs-derived exosomes could be used as a feasible nano-vehicle to deliver drug molecules like LNA-anti-miR-142-3p in both in vitro and in vivo studies. [125]

Conclusions and Future Perspectives
In the tumor microenvironment of breast cancer, the powerful regulatory effect of the exosomal miRNA is obvious and true. Tumor cells, especially with the phenotype of malignancy and drug-resistance, would secrete exosomes containing specific miRNAs to non-drug-resistant cancer cells, and even to normal stromal cells. Conversely, macrophages, fibroblasts, and other stromal cells could also transmit the exosomes to the cancer cells, which encouraged the erosion of cancer cells.
Although some progress has been made in this field, and some advanced understandings have arrived, we still face difficulties. For example, there is only little research on the response of exosomes to the changes of physical conditions during the carcinogenesis such as acidity and hypoxia in TME. Additionally, no systematic and accepted method has been found in the separation of the exosomes from plasma or serum yet, which may result in a non-reliable outcome. It is recognized that the clinical use of molecular markers is very demanding. The conversion of exosomal miRNA in the peripheral blood, which is clearly enriched and differentiated in basic scientific research, into a clinical biomarker for diagnosis and detection requires more detailed screening and more support of clinical data. A greater effort needs to be taken to exploit the deeper potential of miRNAs in exosomes, such as revealing their mechanisms in depth, the modification of exosomes, and the development of new clinical treatments.
Author Contributions: This review was conceptualized by all the authors; Q.L. conceived ideas about the microRNAs contained in exosomes and drafted the article; F.P. and J.C. critically reviewed the content; The project and funding were administrated by J.C. All the authors approved to publish the final version.