Breast cancer (BC) is one of the most common malignant tumors among females. As a highly heterogeneous cancer, BC can be divided into various distinct subtypes according to gene expression profiling, such as luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-positive and basal-like (triple-negative) BC (1). Despite there are tremendous advances in early detection, diagnosis, and therapy strategies, BC is still one of the primary reasons for cancer-related deaths in females due to the poor prognosis caused by tumor metastasis and recurrence (2). Over the past decades, numerous studies have attempted to illuminate the underlying mechanisms leading to BC oncogenesis, proliferation, and metastasis (3). However, the accurate mechanisms remain elusive by now. The establishment of identifying the key molecules and mechanisms is a prerequisite for the development of predictive and diagnostic biomarkers and innovative treatments for overcoming BC.

Exosomes are specifically defined as membrane-enclosed extracellular vesicles (EVs) with particle diameter sizes of 40 nm to 160 nm, which are released from cells upon fusion of the multivesicular body with the plasma membrane (4). Exosomes are widely distributed in the body fluids, such as blood, urine, saliva, amniotic fluid, cerebrospinal fluid, lymph, and bile, under both healthy and pathological conditions (5). The common exosomal biomarkers are associated with the exosome biogenesis, release, and fusion events, including a conserved set of proteins (caveolins, clathrin, transferrin receptors), tetraspanins (CD63, CD81, CD9) (6). Exosomes can deliver cargos into the extracellular space mainly containing soluble proteins (enzymes, cytokines, chemokines) and membrane-bound proteins, DNAs, messenger RNAs (mRNAs), non-coding RNAs (ncRNAs), lipids, and chemical messengers, which can reflect their cell of origin (7). Understanding the carried molecular and cellular properties of exosome cargos is important to offer benefits for identifying the key molecular events in disease initiation, occurrence, and progression. Exosome secretion is one of the emerging significant mechanisms for tumor cells to reciprocal communicate with surrounding cells. Recent studies have shown that tumor-released exosomes could shuttle and interplay between seeds and soil to induce tumor cell malignancy and organotypic metastasis (8). It is worth mentioning that the exosomes can transfer a variety of photogenic factors to the target organ to induce inflammation foci formation and thus lead to the recruitment of bone marrow precursors and the remodeling of the pre-metastatic niches (9). The metastasizing process is closely related to the surface-specific integrin on tumor exosomes. Therefore, the tumor exosomes are the important link in coordinating the complex multi-stage process of tumor metastasis. Due to their stability and disease-specific cargos, tumor-derived exosomes in blood circulation and in blood cell-surface-associated origination emerging as valuable targets for monitoring tumor progression (10). For example, Laktionov et al. analyzed the plasma-separated exosome from healthy females and BC patients by ultracentrifugation and found that the plasmic exosomal miR-103, miR-191, miR-195 that was associated with the fraction of red blood cells were more precisely to discriminate the BC in comparison to the cell-free exosomes circulating in plasma, confirming that better diagnostic value of isolated miRNA from cell-derived exosomes (11). Gonzalez et al. confirmed that the concentration of exosomes in BC patients with stages I, III, and IV was significantly higher compared with healthy donors (12). Determining the molecular properties of these cargos in exosomes remains a fundamental milestone toward understanding the molecular heterogeneity in BC as well as improving the application in theranostic strategies in BC ( Figure 1 ).

A large number of ncRNAs and their functionalities have generated lots of interest in BC. ncRNAs make up the vast majority of the eukaryotic transcriptome. Although with no or only limited protein-coding ability, ncRNAs can be transcribed into various RNA species to exert vital functions mainly responsible for post-transcriptional regulation (13). Dysregulation of ncRNAs has been proven to orchestrate BC progress by inducing proliferation, invasion, metastasis, cachexia of tumor cells (14). Importantly, a wide range of ncRNAs including microRNA (miRNAs), long-noncoding RNA (lncRNA), circular RNAs (circRNAs), piwi-interacting RNAs (piRNAs), small nucleolar RNA, can be synergistically packaged into tumor-derived exosomes (15). The most abundant ncRNAs within exosomes are miRNA, lncRNA, and circRNA. miRNAs, belonging to a class of small ncRNAs ranging from 19-24 nt in length, can regulate gene expression posttranscriptionally via either translation repression or mRNA degradation by binding to their complementary sites in the 3’-untranslated region (UTR) of mRNAs (16). lncRNAs are broadly defined as a class of ncRNAs transcripts for gene regulations with a length longer than 200 nucleotides (17). CircRNAs, are a new type of functional ncRNAs characterized by a covalent single-strand loop, accompanied by several notable characteristics, including high abundance, species diversity, structural stability, evolutionary conservation, localization, and specificity (18). Tumors are regulated by different genetic and epigenetic events of endogenous and exogenous sources. As a new way of cellular communication, exosomes are capable of carrying arrays of these oncogenic proteins and ncRNAs and then are internalized by the neighbor cells. As a consequence, the RNA-containing exosomes can then relay cellular signals from the donor cells to regulate the activities of the recipient cells. Especially, the exosomal ncRNAs are crucial players in the establishment of the metastatic niche by communication between cancer cells and normal cells (19).

As discussed above, tumor-derived exosomal ncRNAs are essential to modify the cancer cell phenotypes locally or systemically through the exosome-dependant exchange of genetic information. Thus, this article has deciphered the recent existing research on the roles and mechanisms of tumor-derived exosomal miRNA, lncRNA, circRNA in BC, especially in aspects of BC cell proliferation, metastasis, and immunoregulation. It also emphasizes the clinical implication of tumor-derived exosomal miRNA, lncRNA, circRNA in BC diagnosis, and drug resistance. Overall, an in-depth understanding of tumor-derived exosomal ncRNAs in BC will provide profound insight into the BC oncogenesis and progress and screen novel strategies for therapeutic interventions of BC.

Importantly, the miRNA carried by exosomes was shown to transfer to recipient cells through direct uptake, playing a gene silencing effect to fine-tune target expression like endogenic miRNAs. Moreover, exosomal miRNAs can even impact the environment surrounding the tumor, influence the extracellular matrix (ECM) as well as immune system activation and recruitment.

lncRNAs are a category of cellular RNAs with various secondary structures to specifically bind to proteins and nucleic acids, based on the principle of complementary base pairing (48). Exosomal lncRNAs commonly function as competing RNA sponges or endogenous RNAs (ceRNAs) to regulate gene expression. Aberrant expression of tumor-derived lncRNA served as a momentous part in BC cell proliferation, invasion, and metastasis.

circRNAs are highly conserved and relatively stable compared with other linear counterparts (56). Based on the composition, circRNAs can be generally classified into 4 types: exonic circRNAs, exon-intron circRNAs, circular intronic RNAs, and tRNA intronic circRNAs (57). Most circRNAs can function as a miRNA sponge or RNA binding protein, and can also regulate alternative splicing and modulate the expression of their parental genes. It has been reported that circRNAs are highly abundant and stable in exosomes, particularly in tumor-derived exosomes. Notably, circRNAs are capable of longer half-life resulted in more frequent incorporation into exosomes than linear RNAs (57). It is expectable that crucial regulatory functions of exosomal circRNAs in various behaviors of cancer, such as tumorigenesis, differentiation, proliferation, and metastasis.

By RNA sequence profiling, Yang et al. found that 646 circRNAs were significantly upregulated and 157 circRNAs were significantly downregulated in serum-exosomes of BC patients. It also identified the higher expression of hsa-circRNA-0088088 and hsa-circRNA-00000751 and lower expression of hsa-circRNA-00005795 by reverse transcription-polymerase chain reaction (RT-PCR) (58). Predicted potential circRNA-miRNA interaction networks showed that hsa-circRNA-0088088 and hsa-circRNA-0005795 might function as ceRNAs and were associated with several cancer-related pathways, such as the Wnt, estrogen, TGF-β, and FoxO signaling pathways (58). Amorim et al. presented a next-generation sequencing (NGS)-based transcriptome view of the miRNA and circRNA landscape of two breast-derived cell lines, HB4a and C5.2, in the context of HER2 upregulation, demonstrating that identification of hundreds of circRNAs in these cells as well as their exosome-containing EVs (59). Wang identified 5842 DE circRNAs in the MDA-MB-231 cells compared with the MCF-7 cells by cluster analysis (60). Compared with healthy controls, 1061 and 1084 exosomal circRNAs were upregulated, while 86 and 301 exosomal circRNAs were downregulated in patients with metastatic disease and patients with localized disease, respectively. They also obtained 432 pairs of circRNA/miRNA interactions to construct circRNA/pathway and circRNA/miRNA networks. However, these circRNA studies have some potential limitations, mainly lacking a larger sample confirmed by RT-PCR. In addition, the circRNA-miRNA interactions should be confirmed by using the luciferase reporter assay and the enriched pathways should also be evaluated by experimental methods, but not only by bioinformatic methods (60).

Exosomes are considered to be important mediators of cellular communication between the immune cell and cancerous cells (61). Tumor-immune cell interactions shape the immune cell phenotype, with miRNAs, lncRNAs, and circRNAs being crucial components of this crosstalk ( Figure 2B ). Jiang et al. proposed a novel interplay mode between cancer cells and immunocytes (62). BC tumor exosomal miR-9 and miR-181a activated the JAK/STAT signaling pathway by targeting SOCS3 and PIAS3 respectively, and thus consequentially promoting the development and immunosuppressive function of mice early-stage myeloid-derived suppressor cells (eMDSCs), resulted in T-cell immunity inhibition and tumor progress (62). Xing et al. demonstrated loss of XIST also augmented secretion of BC exosomal miRNA-503, which triggered M1-M2 polarization of microglia to upregulate immune-suppressive cytokines in microglia to suppress T-cell proliferation. Thus, the lncRNA XIST played a critical role in BCBM in a gender-specific manner by influencing both tumor cells and the TME (54).

miR-27a-3p has been acknowledged as an oncogenic RNA in various cancers and is one of the highly enriched miRNAs in BC-derived exosomes. Under endoplasmic reticulum stress, BC cells could produce exosomes containing a high level of miR-27a-3p, which up-regulated PD-L1 in macrophages and thereby promoted immune evasion of BC cells by activating the PTEN/AKT/PI3K axis (63). Jang et al. showed that exosomal miR-16 from 4T1 cells treated with epigallocatechin gallate (EGCG), could be transferred to tumor-associated macrophages (TAMs) via exosome, ultimately leading to reduced tumor growth by inhibiting TAMs infiltration and M2 polarization (64). Exosomes secreted by macrophages shuttle invasion-potentiating miRNAs into BC cells. In the BC cell and macrophage co-culture system, Yang et al. concluded that macrophages exosomal miR-223 promoted the invasion of BC cells via the Mef2c-β-catenin pathway (65). These results have also emphasized the importance of the exosome shuttled between BC cells and TAMs.

Additionally, Ni et al. revealed that BC could modify the CD73 expression on γδT cells in a non-contact manner. BC-derived exosomal lncRNA SNHG16 served as a ceRNA by harboring miR-16-5p to derepress SMAD5, resulted in the conversion of γδ1 T cells into the CD73+ immunosuppressive subtype for favoring BC progress (66). lncRNA BCRT1 was markedly up-regulated in BC tissues, which was associated with poor prognosis in BC patients. lncRNA BCRT1 competitively harbored with miR-1303 to inhibit the degradation of target gene PTBP3, resulted in promoted BC progression (67). Moreover, lncRNA BCRT1 could be transported to macrophages via BC cell exosomes, thereby promoting M2 polarization and enhancing tumor progression.

Early detection, therapy, and metastasis monitoring are of great importance to a favorable prognosis. Conventional diagnostic programs, such as breast X-ray mammography, ultrasound imaging, and positioning biopsy, usually cause radioactive or invasive damage to patients with limited accuracy. As a non-invasive method, liquid biopsy is convenient for repeated sampling in clinical cancer prognostic, metastatic assessment, and recurrence monitoring. Aberrant expression of ncRNAs profiles in exosomes released into circulating systems especially in plasma and serum are promising candidate biomarkers for BC liquid biopsy.

Lots of studies showed that there were differential expressions of exosomal miRNAs in BC patients and healthy individuals. As miR-155 was up-regulated in the tumor cells of BC patients, particularly the patients with early-stage and TNBCs, the plasma miR-155 could serve as a non-invasive biomarker for non-invasive detection of early-stage BC (68). Given that approximately 20 to 25% of women diagnosed with localized BC (LBC) were subjected to neoadjuvant therapy, it was significant to screen effective biomarkers of liquid biopsy for the diagnosis and prediction of treatment response in BC patients with neoadjuvant therapy. By detecting the circulating tumor cells (CTCs) and serum exosomal miRNAs isolated from blood samples before or after neoadjuvant therapy, a study found that higher levels of exosomal miRNA-21, miRNA-222, and miRNA-155 were significantly correlated with the presence of CTCs, indicating that exosomal miRNAs and CTCs could be a complementary strategy for improving diagnosis and prognosis of patients with neoadjuvant therapy (69). However, in another study, miR-145, miR-155, and miR-382 in exosomes were isolated from the serum of BC patients and healthy donors but were not in a preferential manner in BC patients for detection (12). Although these studies both detected miR-155 as biomarkers in BC by PCR, it seemed to be a potential limitation with certain variations of results, which might attribute to sample differences, exosome abundance, and separation efficiency.

The gene information exchange between cells by exosome-mediated transfer may produce different miRNA patterns. Panels or combinations of specific exosomal miRNAs, combined with other conventional clinical biomarkers or pathological examinations, might improve the BC diagnostic efficiency. Based on a case-control study, Hirschfeld et al. confirmed a panel of 4-variable miRNA signature (miR-424, miR-423, miR-660, and let7-i) as a highly specific combinatory biomarker tool for distinguishing BC patients from healthy controls, with 98.6% sensitivity and 100% specificity (70). Similarly, Li et al. used qRT-PCR to identify 4 plasma miRNAs and 4 serum miRNAs from the miR-106a-363 cluster for BC diagnoses, such as miR-106a-3p, miR-106a-5p, miR-20b-5p, and miR-92a-2-5p, indicating the biomarker potential of exosomal miRNAs (71). By comparing the serum levels of exosomal miRNAs of 50 BC patients and 12 healthy donors, Eichelser et al. also indicated that exosomal miR-101 and miR-373 were linked to differentiation of BC from benign breast diseases (BBDs) or healthy individuals, and the expression level of exosomal miR-373 was higher in TNBC and more aggressive breast carcinomas (70).

Serum exosomal miR-148a might be a promising biomarker for the diagnosis and prognosis prediction for BC. Li et al. reported that serum exosomal miR-148a level was significantly reduced in patients with BC and the decreased exosomal miR-148a was associated with an unfavorable prognosis of BC (72). Li et al. profiled that exosomal miR-122-5p expression was distinctly up-regulated in BC plasma-derived exosomes compared with the adjacent normal tissue samples (73). Exosomal miR-122-5p might be useful as a supplement for the traditional BC diagnostic strategies. Plasma exosome-encapsulated miR-223-3p level was significantly associated with the malignancy of BC, showing the potential function for the early detection of invasive BC (22). Based on the nucleic acid-functionalized Au nanoflare probe, Zhai developed an in the situ detection method of miR-1246 level in human plasma exosomes (74). By using the exosomal miR-1246 as a marker, they successfully differentiated 46 BC patients from 28 healthy controls with 100% sensitivity and 92.9% specificity at the best cutoff (74). Lee et al. also constructed a method of in situ simultaneous detections of exosomal miR-21, miR-27a, and miR-375 by competitive strand displacement (75). This method of clinical serum samples detection could effectively distinguish BC patients from healthy donors. The exosomal miRNA profiles with high levels of cancer-associated sensitivity and specificity are hopeful indicators for prediction and early detection of BC, ultimately reflecting disease information of development, tumor burden, malignant progression towards metastatic recurrence, and drug resistance.

For lncRNA detection, Tang et al. showed that BC patients expressed higher serum exosomal HOTAIR than that in healthy individuals (76). Interestingly, the serum exosomal HOTAIR levels were also markedly decreased 3 months after surgery compared with the levels before surgery. In addition, high expression of exosomal HOTAIR caused a worse disease-free survival and overall survival (76). Zhong et al. detected the levels of lncRNA H19 in serum-derived exosomes by using quantitative RT-PCR (77). This work revealed that exosomal H19 levels were up-regulated in BC patients compared to that in patients with BBDs and healthy controls and that the median serum exosomal H19 levels were significantly decreased in post-operative than that in the pre-operative patients. Moreover, exosomal H19 expression levels were related to lymph node metastasis, distant metastasis, and TNM stages, all indicating the H19 potential as a non-invasive biomarker for BC diagnosis (77).

In conclusion, the exosomal ncRNAs for BC detection are mainly in tumor lesions and blood. Notably, most reports about detection are associated with miRNA, but the lncRNA and circRNAs are rarely studied. Compared with the other biomarkers, exosomal ncRNAs cargoes can avoid degradation by external proteases and other enzymes to keep the quantity and activation. Thus, as exosomal ncRNAs are of high stability, low complexity, and less invasive acquisition, the quantification and characterization of these ncRNAs can serve as a supplementary tool of the non-invasive detection for BC, ranging from early detection, recurrence prediction, metastasis assessment, and therapeutic outcomes.

Currently, chemotherapeutic treatments based on hormones, cytotoxic agents, and targeted antibodies are frequently effective in controlling tumor growth and progression. The BC patients who initially respond to these therapies can develop resistance at a later stage. Chemoresistance is the most common obstacle for BC (78). Treatment resistance is not entirely determined by the intrinsic characteristics of tumor cells, but also by the simultaneous actions of many local microenvironmental factors. Emerging evidence emphasized that tumor cells, especially with the phenotype of malignancy and drug resistance, would secrete exosomes containing specific miRNAs, lncRNAs, and circRNAs to non-drug-resistant recipients like cancer cells, immune cells, and even normal stromal cells. Conversely, these nonneoplastic cells could also transmit the exosomes to the cancer cells, which encourages the erosion of cancer cells (79). Therefore, the gene modifications and information exchanges by tumorous exosomal ncRNAs are critical to therapeutic resistance for BC. Meanwhile, it would be very valuable to identify specific exosomal miRNAs involved in resistance to chemotherapeutic agents to make an early prediction warning to chemotherapy possible.

Functionally, as vital mediators of cell-cell communication, ncRNAs-containing exosomes are pluripotent carriers with intriguing and elaborate roles in BC progression. After being released by parental cell exosomes, ncRNAs shuttle and transfer to neighboring or distant cells for reprogramming TME. In this review, it is highlighted that exosomal miRNAs, lncRNAs, and circRNAs participate in signaling pathways implicated in proliferation, apoptosis, invasiveness, EMT as well as in angiogenesis of recipients, thereby facilitating BC oncogenesis, proliferation, metastasis, and drug resistance ( Table 1 ). Additionally, based on specific de-regulated expression verified by RNA sequencing, bioinformatic analyses, and PCR experiments, these tumor-derived exosomal ncRNAs that existed in blood samples are verified to be excellent candidates for improving early diagnosis, personalized prognosis, and therapeutic monitoring.

Up to date, there are still some limitations and challenges in this subject. Firstly, the complex role of exosomal ncRNAs in BC progress remains to be further deeply explored. As TME is a complex entity, different types of cells secrete differential types of exosomal ncRNAs information, it would be an interesting angle that the specific factor is secreted by which cell type to exert the maximum or dominant impact in BC. Given the high level of genetic heterogeneity of BC tumors, understanding the pathogenesis of the key exosomal ncRNAs is meaningful to the development of new therapeutic strategies. Secondly, as the overwhelming priorities of BC-caused deaths result from metastasis, it is of great value to excavate new ncRNAs or exosomal ncRNAs for combating metastasis. It has been proved that BC-derived exosomes are involved in the key steps of primary tumor metastasis and spread, from the oncogenic reprogramming of malignant cells to the formation of the pre-metastasis niche. These effects are achieved by mediating crosstalk between cells and then modifying the local and remote microenvironment in an autocrine and paracrine manner. Identifying the key exosomal ncRNAs in the metastasis process will benefit the precision medicine-based strategies for diagnosis and therapy of BC metastasis (105). Thirdly, studies have emphasized that ncRNAs present an obvious asymmetric distribution in parent cells and their exosomes (106). It means that these regulatory RNAs are selectively encapsulated into exosomes during biogenesis. Coincidentally, Ni et al. suggested a selective and wavelike packaging of miR-16 into exosomes in the different BC subtypes possibly during tumor development and progression (21). Therefore, the DE ncRNAs in exosomes might be of the relatively slight or large difference in expression levels over their parental cells, but still reflecting the different pathological status of BC. However, the mechanism of selective packaging ncRNAs into exosomes needs to be elucidated more clearly. Fourthly, although numerous studies have addressed the characteristics of circRNAs in BC, there are still few reports regarding exosomal circRNAs. Most available circRNA biomarkers are currently not sensitive or specific enough to be applied clinically. The abundance, biogenesis and biological functions, and possible sorting mechanisms and potential roles of exosomal circRNAs in promoting or inhibiting cancer are worth further explorations.

It is also very important for utilizing tumor-derived exosomal ncRNAs as newly-developing diagnostic and therapeutic targets. The difficult part is that it is challenging to analyze candidate exosomal biomarkers in a routine clinical setting, mainly due to the lack of standardization for rapid assays, sources of variability, and methodological issues. Most blood-based biomarker studies are retrospective case-control studies, with small sample sizes, and different sample handling and storage methods due to lack of standardization and large-scale testing, lack of method validation, and non-exosomal contaminants like proteins existence (78). In addition, as sensitivity and specificity are two major standards for an accurate diagnostic tool, the optimal plasma/serum miRNA cut-off levels should be confirmed. In order to systematically prove the clinical utility as diagnostic, prognostic, and predictive biomarkers, it is necessary to quantify miRNAs in large-scale prospective multicenter studies and independent patient cohorts of different tumor stages.

Of particular, the ncRNAs, acting as regulators in shaping cellular activity, are believed to boost the clinical translations. Nevertheless, the promising strategies based on exosomal ncRNA in the field of personalized oncology are still in their infancy. Considering that exosomes are bioavailable, well-tolerable, targetable, and membrane-permeable, they are ideal candidates for delivering miRNAs, proteins, drugs, and other molecules to tumors. The suitability of exosomes as delivery vehicles for BC treatment has been investigated in recent years. For instance, Gong et al. yielded a novel exosome loaded with a chemotherapeutic adriamycin and cholesterol-modified miRNA, which exhibited effective anti-tumor function in MDA-MB-231 cell-represented cells and xenograft (107). Another treatment strategy may be to block the secretion of tumor cell-derived exosomes or to deliver therapeutic ncRNAs to the primary tumor through exosomes. Zhao et al. identified exosomes with lung targeting ability that derived from autologous BC cells accordingly constructed biomimetic nanoparticles CBSA/siS100A4@Exosome. These particles significantly inhibited the growth of malignant BC cells, showing a promising strategy to suppress postoperative BC metastasis (108). Ohno et al. also verified that exosomes were excellent candidates to therapeutically target EGFR-expressing cancerous tissues by efficiently delivering nucleic acid drugs, such as let-7a miRNA (109). Limoni et al. considered that exosomes were the best options for gene targeting and they established modified exosomes to target HER2-positive BC cells by loading siRNA (110). Up to the present, almost all the existing studies are still in the preliminary preclinical stage, including bioinformatics, sample sequencing, and testing, cell and animal experiments. That means that there are no clinically approved exosome-based therapeutics. The continuous exploration in fundamental researches and further clinical trials are urgent for the era of precision oncology in BC patients.

In conclusion, we offer the latest elucidation on the roles and mechanisms of tumor-derived exosomal ncRNAs in tumor growth, metastasis, detection, and drug resistance in BC ( Figure 3 ). Further studies focusing on the tumor-derived exosomal ncRNAs will not only undoubtedly deepen the comprehensive understanding of their behavior, but also contribute to better clinical outcomes for BC-bearing individuals.

YY, MW, and HZ performed a literature search and wrote the manuscript. YW, QZ, and PD conceived the project and revised the manuscript. WH, CZ, MX, and WL edited the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by China Guanghua Science and Technology Foundation (grant number 2019JZXM001) and Wuhan Science and Technology Bureau (grant 2020020601012241).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.