Roles and clinical application of exosomal circRNAs in the diagnosis and treatment of malignant tumors

The occurrence and development of malignant tumors are multifactorial and multi-linked intricate processes. The most important characteristic of malignant tumors is their ability to grow, infiltrate, and diffuse to surrounding tissues without restraint. The morbidity and mortality of malignant tumors are high worldwide and are increasing markedly, representing a non-negligible threat to human health. Most patients are diagnosed at an advanced stage owing to the insidious onset of the disease, which leads to deterioration of their condition and causes them to miss the best time for treatment. Although there has been some progress recently in the diagnosis and treatment of malignant tumors, early diagnosis and effective precision treatment to improve the survival rate remain major challenges in world public health and are key to precision medicine and individualized treatment of malignant tumors. At present, early diagnosis methods and effective treatments are very limited, and the mechanisms of occurrence and progression have not yet been clearly specified. Therefore, it is crucial to explore new non-invasive biomarkers for early diagnosis and treatment of malignant tumors and apply them in clinical practice.

Exosomes, which have an average diameter of 30–150 nm and a double-layered membrane structure, are extracellular vesicles secreted by a variety of cells by exocytosis. Exosomes are important carriers for signaling between cells and are present in body fluids such as saliva, blood, urine, cerebrospinal fluid, and breast milk. Their diverse constituents include almost the entire cell-derived molecular spectrum: circular RNA (circRNA), DNA, mRNA, microRNA (miRNA), proteins, and lipids [1]. Under physiological or pathological conditions, most cells can promote tumor occurrence, development, and metastasis by releasing exosomes containing the above substances into the microenvironment and transferring materials and information between cells.

CircRNAs are important non-coding RNA molecules (ncRNAs) that are produced by alternative splicing of premature mRNAs and usually do not encode proteins. Compared with linear RNAs, circRNAs have stronger stability owing to their characteristic covalently closed loops without 5′ head or 3′ end structures. In recent years, circRNAs have become a hotspot in RNA research. Widely present in eukaryotic cells, they exert specific biological functions by regulating miRNAs and proteins [2, 3] and are closely involved in the occurrence, development, and prognosis of a variety of malignant tumors. Owing to their structural stability, wide distribution, and tissue specificity, circRNAs have advantages over other ncRNAs as molecular diagnostic markers and drug therapy targets. The expression of circRNAs in tumor tissues has been shown to differ significantly from that in normal tissues [4], indicating that circRNAs could serve as new tumor biomarkers for guiding clinical diagnosis. Known circRNAs can affect the biogenesis of cancers in diverse ways [4], such as acting as molecular sponges for miRNAs or competing endogenous RNAs (ceRNAs) to regulate the expression of target genes; they can also combine with RNA-binding proteins (RBPs) to regulate the expression of specific proteins, participate in protein translation and other biological functions, and indirectly participate in the occurrence and development of malignant tumors.

Large numbers of circRNAs stably exist in exosomes, which are involved in the composition and regulation of the microenvironment of malignant tumors. The circRNAs are delivered to normal cells via exosomes and have a significant impact on the progression and metastasis of malignant tumors. Recent exosome-related studies have further promoted the clinical translation of circRNAs in oncology, owing to the close connections between circRNAs and exosomes. Here, we summarize the biological functions, roles in diagnosis and treatment, possible mechanisms, and potential applications of exosome-derived circRNAs in malignant tumors.

Exosomes were first identified in sheep reticulocytes and named by Johnstone. Transmission electron microscopy images show that they have a double-layer phospholipid structure with a “spherical” or “cup-like” shape and obvious heterogeneity, and they can be enriched in a concentration gradient of 1.13–1.19 g/ml sucrose solution. Exosomes are biologically active extracellular vesicles that are rich in lipids including cholesterol, ceramide, and sphingomyelin. They contain a variety of functional proteins [e.g., CD9, TSG101, and heat shock protein (HSP70)], DNA, miRNA, lncRNA (long non-coding RNA), and other biologically active substances, which are protected from destruction by being encapsulated in the exosome. The continuous formation process of exosomes mainly involves endocytosis, fusion, and exocytosis, that is, the invagination of the membrane to form a dynamic subcellular structure of multivesicular bodies. These multivesicular bodies have two outcomes: (1) they fuse with lysosomes, and the luminal vesicles are degraded; (2) they fuse with cell membranes, and the tubular vesicles inside depress again to form granular vesicles by endogenous budding and are then released into the extracellular environment in the form of exosomes [5, 6].

Exosomes can be detected in almost all normal and tumor cells and are present in a wide variety of body fluids including saliva, blood, urine, ascites, and breast milk [7, 8]. Their main function is to mediate intercellular communication and material exchange of their contents under physiological and pathological conditions [6]. By transmitting genetic and molecular messages from tumor cells to other cells located at near or distant sites to alter their functional properties and phenotypes [9], exosomes are also important carriers of specific signals for tumor proliferation, metastasis, immune response, angiogenesis, and drug resistance [10‐12]. Studies have shown that tumor cells secrete more exosomes whose contents are different than normal cells. Tumor-cell-derived exosomes can provide a suitable microenvironment for tumor development and regulate cell proliferation, angiogenesis, extracellular matrix degradation, metastasis, drug resistance, and the formation of a pre-metastatic microenvironment [13, 14]. Tumor-derived exosomes are mainly composed of ncRNAs, including circRNAs, lncRNAs, and miRNAs. They are considered to be extracellular organelles that play essential parts in remodeling the tumor microenvironment, and represent a new type of non-invasive diagnostic biomarker for tumors [8]. Exosomes have been proved to promote tumor angiogenesis and metastasis during tumorigenesis [15], contributing to the formation of pre-metastatic ecological niches and the establishment of a suitable microenvironment at distant metastatic sites through stimulating neovascularization, activating mesenchymal stromal cells, and remodeling the extracellular matrix [16, 17]. Tumor-derived exosomes can participate in tumor microenvironment remodeling by releasing cytokines and other bioactive components that act on endothelial cell surface receptors, leading to angiogenesis and promoting tumor metastasis [18].

Studies have shown the main mechanisms of exosomes function are as follows [19]: direct interaction between exosomes and surface receptors on target cells; cleavage of surface receptors on exosomes and subsequent direct interaction of receptor fragments with receptors on target cells; fusion of exosome membranes with the plasma membrane of target cells, with release of informational material carried by exosomes into the cytoplasm and internalization of the entire exosome through phagocytosis.

Both circRNAs and linear RNAs originate from precursor mRNAs; however, circRNAs are formed by reverse splicing, in contrast to the production of linear RNAs by classical splicing. CircRNAs originate from all regions of the genome, including intergenic, intron, antisense, and untranslated regions. According to their origin, circRNAs can be classified into three major groups: exonic circRNAs, exon–intron circRNAs, and intronic circRNAs. CircRNAs are more stable and less susceptible to degradation by nucleic acid exonucleases than linear RNAs because of their chemical structure of covalent closed loops [2].

CircRNAs were initially thought to be non-functional byproducts resulting from splicing errors. However, with the development of high-throughput sequencing technology and bioinformatics research, large numbers of circRNAs have been found in mammalian cells, and their structural stability, high conservation, and abundant expression have been demonstrated [20]. The main biological functions of circRNAs: (1) competitive binding with miRNAs to regulate target gene expression; (2) interactions with RBPs to regulate gene expression; (3) regulation of gene transcription or splicing; (4) translation of proteins; and (5) regulation of epigenetic inheritance. Research has shown that circRNAs are related to the pathogenesis of many human diseases and have key roles in tumorigenesis, development, and metastasis; thus, they can be used as effective biomarkers for early diagnosis and prognostic prediction in tumor patients, as well as being ideal targets for early treatment [21].

Exosomes are involved in the formation of the tumor microenvironment before metastasis. By mediating immunosuppression and immunosurveillance, exosomes are associated with tumor recurrence and tumor cell survival [22] Li et al. [3] found that large numbers of circRNAs were stably present in exosomes derived from various carcinoma cell lines [including laryngeal cancer, oral squamous cell carcinoma (OSCC), thyroid cancer, liver cancer, colon cancer, lung cancer, stomach cancer, and breast cancer] and could be detected in patients’ saliva, serum, urine, and tumor tissues. Various studies have reported that exosomal circRNAs may be related to the occurrence and development of malignant tumors and may regulate tumor cell proliferation, invasion, metastasis, chemotherapy drug resistance, and radiosensitivity.

A certain amount of progress has been made with respect to the treatment of malignant tumors, for example, in early diagnosis, surgery, and radiotherapy; however, the early symptoms of most tumors are not typical and final diagnosis is difficult as it requires specialized clinicians to obtain samples for processing and to perform complex histopathological biopsies. There is still a lack of rapid, accurate, and non-invasive early diagnostic biomarkers for use in clinical practice. Liquid biopsy based on exosomal circRNAs is a novel method, taking advantage of the fact that circRNAs are enriched in exosomes and have good stability, and that the expression profile of exosomal circRNAs in patients with malignant tumors is different from that in the healthy population [22]. Liquid biopsy can be easily performed using samples of saliva, serum, urine, and other body fluids, avoiding the harm caused to patients by surgery or puncture. As well as its advantages in terms of safety, this method is easier to perform [58]; it also enables monitoring of the whole process of malignant tumor diagnosis and prognosis evaluation, with strong real-time and timeliness. Thus, exosomal circRNAs have important diagnostic value in a variety of malignant tumors and have great potential as molecular markers for non-invasive early diagnosis and prognostic assessment, thereby providing a new direction for exploration of powerful diagnostic markers for different tumors. Although exosomal circRNAs have great potential to become novel tumor markers, the current understanding of exosomal circRNAs is still in its infancy. Further studies are needed to verify whether the proposed circRNAs have clinical value as tumor markers.

Studies have shown that exosomes are rich in circRNAs with potential biological functions, and human serum exosomes have been proven to contain more than 1000 types of circRNA. This phenomenon is caused by the entry of circRNAs from tumors into the bloodstream [59]. CircRNAs are stably enriched in malignant exosomes, and the transport of circRNAs and other types of molecules by exosomes protects them from degradation and dilution in the cellular extracellular space, resulting in long-distance distribution of circRNAs via the bloodstream or tissue fluid [60]. To detect whether exosomal circRNAs enter the blood circulation and can be quantified for malignancy diagnosis, Xu et al. [61] analyzed the expression of exosomal circRNAs in serum of endometrial cancer patients and healthy individuals by an RNA sequencing technique. They found that the number of circRNAs upregulated in exosomes of endometrial cancer patients was higher than the number of downregulated circRNAs, indicating that human malignant circRNAs can enter the blood and be easily quantified in serum, and that exosomal circRNAs can affect target cells. These findings may help to identify new mechanisms of malignant tumor development. Li et al. [3] constructed a mouse tumor model and detected human circRNA-CDYL in serum exosomes; the amount of circRNA-CDYL was closely related to tumor development, further confirming that circRNAs can be used as early diagnostic biomarkers of malignant tumors.

Han et al. [62] showed that plasma circ_0019201, circ_0011773, and circ_0122790 are potential biomarkers for predicting LSCC. Circ_0019201, circ_0011773, and circ_0122790 were found to be consistent with the training set. The receiver operating characteristic (ROC) curves also showed high diagnostic power, as did the area under the ROC curve (AUC) for both single and combined circRNAs. Circ_0019201, circ_0011773, circ_0122790, and their combinations had AUCs of 0.933, 0.908, 0.965, and 0.990 in the training set and 0.766, 0.864, 0.908, and 0.951 in the validation set.

CircRNAs have a significant role in the development of esophageal squamous carcinoma (ESCC). The expression of serum exosomal hsa_circ_0026611 was found to be significantly upregulated in ESCC with lymph node metastasis [63]; exosomal hsa-circ-0048117 may have key roles in remodeling the ESCC microenvironment and regulating the progression of ESCC [64]; and hsa_circ_0001946 shows potential as a good prognostic biomarker [65]. Thus, circRNAs are expected to emerge as new markers for the diagnosis and prognostic assessment of ESCC patients.

In CRC studies, exosomal circRNA levels have been shown to be significantly upregulated in K-ras wild-type CRC DKs-8 cells, suggesting that exosomal circRNA could be used as a biomarker for CRC [66]. Serum exosomal circ_0004771 was found to be significantly upregulated in CRC patients with different TNM stages compared with the control group (P = 0.017), and its AUC for the diagnosis of CRC was 0.88, with a sensitivity of 80.91% and specificity of 82.86%; serum exosomal circ_0004771 in patients with stage I-II CRC was significantly higher than that in normal controls (P < 0.001), and the AUC for the diagnosis of stage I-II CRC was 0.86, with a sensitivity of 81.43% and specificity of 80.00% [67]. Barbagallo et al. [68] found that serum exosomal circHIPK3 was significantly upregulated in patients with CRC, and the AUC for the diagnosis of CRC was 0.771, with a sensitivity of 71% and a specificity of 80%.

Xie et al. [69] showed that exosomal circSHKBP1 regulates the miR-582-3p/HUR/VEGF pathway, inhibits HSP90 degradation, and promotes GC progression. CircSHKBP1 is thus a promising circulation biomarker for GC diagnosis and prognosis. Circ-RanGAP1 is significantly upregulated in both GC tissues and plasma exosomes of GC patients and is closely associated with advanced TNM stage, lymph node metastasis, and low survival; therefore, it could be used as a prognostic biomarker and therapeutic target for GC [39]. Shao et al. [70] found that plasma exosomal circ_0065149 levels in patients with early GC (EGC) were significantly lower than those in healthy controls (P < 0.001), and the AUC for screening for EGC was 0.64, with higher sensitivity (48.7%) and specificity (90.2%) than traditional tumor markers (CEA, CA199, and CA125).

Studies in HCC have confirmed [71] seven upregulated and five downregulated circRNAs, hsa_circ_0004001, hsa_circ_0004123, and hsa_circ_0075792, which among the upregulated circRNAs, the combination of which can be used as a valuable diagnostic biomarker for HCC. Exosome-derived circ_0051443, which is downregulated in the plasma of HCC patients, inhibited the progression of HCC and had an AUC of 0.8089 for the diagnosis of HCC [72]. Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive and lethal malignancies, with a 5-year survival rate of only 5% owing to the high risk of metastasis and recurrence. Microarray analysis [39] showed that circ-PDE8A is a highly expressed circRNA in PDAC and may play a critical part in PDAC invasion; thus, exosomal circ-PDE8A may be an ideal biomarker for early diagnosis or assessment of progression of PDAC. Tumor-derived exosomal circRNA_102481 may promote resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in NSCLC via the miRNA-30a-5p/ROR1 axis. Exosomal circRNA_102481 therefore has potential as a novel diagnostic biomarker and therapeutic target in EGFR-TKI-resistant NSCLC [73]. Exosomal circFARSA plays a crucial part in the interaction between macrophages and NSCLC cells through the PTEN/PI3K/AKT signaling pathway and is a promising diagnostic/prognostic biomarker for NSCLC [74].

Li et al. [42] found that expression of serum exosomal FLI1 exonic circRNA 1 (FECR1) was abnormally upregulated in patients with small-cell lung cancer; moreover, higher levels of exosomal FECR1 expression were observed in patients with distant metastases and serum FECR1 levels were closely related to patients’ response to chemotherapy, indicating that FECR1 could be used as a biomarker to assess the progression of small-cell lung cancer. Wang et al. [75] found that serum exosomal circ_002178 was significantly upregulated in lung adenocarcinoma (LUAD) patients and enhanced expression of programmed cell death ligand 1 (PDL1) by adsorbing miRNA-34 and miRNA-28-5p; circ_002178 showed potential as a non-invasive diagnostic marker for LUAD with an AUC of 0.9967.

Chen et al. [76] comprehensively analyzed the expression profiles of plasma exosomal circRNAs in LUAD groups and healthy controls and found that the expression levels of circ_0001492 and circ_0001346 were upregulated in early-stage LUAD but almost undetectable in plasma exosomes of healthy controls, suggesting that circ_0001492 and circ_0001346 could be used as novel early diagnostic markers for LUAD. Lin et al. [77] extended current knowledge regarding the differential expression of circRNAs in plasma exosomes of advanced LUAD, offering prospects for further research on the development and use of biomarkers for diagnosis and prognosis of LUAD. Circ_0102537 was proved to be an effective diagnostic biomarker for advanced LUAD. Chen et al. reported for the first time the altered expression of exosomal circRNAs in plasma samples from LUAD patients, supporting the necessity to explore its usage as a biomarker and its pathological function in lung cancer [76]. Xu et al. [78] found that circSETDB1 promoted tumor formation through the miR-7/Sp1 pathway. Tumor-secreted circSETDB1 can be released into the circulation via exosomal transport under hypoxic stress, and plasma exosomal circSETDB1 is associated with lymphatic metastasis and T-stage in LUAD patients; thus, it may be an important biological factor in the development of LUAD and is expected to be an ideal biomarker for early diagnosis.

Bioinformatics analysis and real-time fluorescence quantitative polymerase chain reaction technology have been used to show that circ_00005795 and circ_0088088 can compete for endogenous RNAs, and both of these circRNAs were shown to have important diagnostic value in breast carcinoma by comparing the expression of serum exosomal circRNAs in breast carcinoma patients and healthy controls. These results provide a theoretical basis for the occurrence, progression, and metastasis of breast carcinoma [79, 80]. Circ-0001068 is introduced into T cells and induces PD1 expression via exosomes as a ceRNA for miR-28-5p; it represents a novel ovarian cancer biomarker and inducer of PD1 expression in T cells [81]. Targeting exosomal circ-CYP24A1 was found to inhibit the progression of cutaneous squamous cell carcinoma by attenuating the malignant behavior of the tumor [82]. It is thus a promising therapeutic target and non-invasive diagnostic biomarker for cutaneous squamous cell carcinoma.

Luo et al. [83] showed that circulating exosomal circMYC has great potential as a diagnostic and prognostic biomarker for multiple myeloma. Chemotherapy resistance limits the durability of therapeutic effects and is a major constraint to clinical treatment of oncology. Timely identification of potential drug-resistant patients, monitoring of resistance to chemotherapeutic agents, and finding alternative treatment options are extremely important strategies, enabling treatment to be adjusted and prognosis improved. Exosome-mediated circNFIX enhances temozolomide (TMZ) resistance in glioma by sponging of miR-132 and shows potential as a prognostic biomarker to improve the clinical benefit of TMZ treatment in glioma patients [84]. Exosome-mediated circ-HIPK3 enhances TMZ resistance via modulating the miR-421/ZIC5 axis and promotes tumorigenesis in vitro and in vivo; its expression in serum exosomes can be used as a biomarker for diagnosis of TMZ-resistant glioma [85].

The roles of exosomal circRNAs in diagnosis and prognostic assessment of malignant tumors are shown in Table 1.

The therapeutic delivery of exosomal circRNAs and related genes and proteins as targets can inhibit the proliferation, invasion, and metastasis of malignant tumor cells. This approach can also provide new ideas for therapy of tumors including OSCC [86], CRC [87, 88], GC [89], PC [90], cervical cancer [91, 92], gallbladder cancer [93], breast cancer [94], and OS [95, 96]. Exosomes can transport RNA or deliver therapeutic drugs to cancer cells and act as drug carriers for targeted therapy owing to their unique advantages, such as their nano nature, double lipid membrane, ability to serve as multiple carriers, good histocompatibility, high bioavailability, low cytotoxicity and immunogenicity, long life span, and high cargo capacity [97‐99]. Exosomes can further target tumor cells through surface receptors, reducing side effects on healthy tissue. The stability of circRNAs and their function as miRNA molecular sponges suggest that circRNAs could be used as drug therapy targets. A variety of circRNAs have been proved to promote cancer progression. Moreover, whereas drugs such as specific small interfering RNAs (siRNAs) targeting specific circRNAs are inherently unstable and easily degraded, their encapsulation in exosomes can maintain the stability of siRNAs and reduce the expression of circRNAs in cancer cells. Exosomal circRNAs can also promote the expression of tumor suppressor genes through sponge-like uptake of miRNAs and indirectly inhibit the damage caused by circRNAs. These facts provide potential new approaches to tumor therapy using exosomal circRNAs. Tumor suppressor circRNAs are downregulated in tumors, and overexpression of circRNAs in tumor cells through exosomes may inhibit tumor progression; cancer-promoting circRNAs are upregulated in tumors, and siRNAs targeting circRNAs delivered by exosomes may downregulate the expression of cancer-promoting circRNAs and thus inhibit tumor progression. Given the important biological functions of exosomal circRNAs in tumor chemoresistance, the therapeutic use of RNAs targeting exosomal circRNAs has the potential to reverse tumor chemoresistance. Exosomal circRNAs have promising clinical applications as targets for tumor therapy. However, circRNAs are still in the research stage, and the clinical applications of exosomal circRNAs require deeper exploration.

Upregulation of circ_0000199 in circulating exosomes of OSCC patients is positively correlated with poor survival; thus, circulating exosomal circ_0000199 could be used as a diagnostic biomarker and potential therapeutic target in OSCC patients [100]. By combining the in vitro and in vivo studies, exosomal circGDI2 can regulate the malignant behavior of OSCC cells by targeting the miR-424-5p/SCAI axis and is expected to serve as an exosome-based biomarker and therapeutic target for OSCC [101]. The circulating exosomes were obtained from 210 patients with nasopharyngeal carcinoma (NPC); the result suggested that circMYC is an oncogene in NPC cells that enhances the resistance of NPC cells to radiotherapy; circMYC in circulating exosomes could be a potential therapeutic target for NPC [102]. CircFNDC3B has been shown to regulate progression of papillary thyroid cancer (PTC) progression in tissues and cell lines through the miR-1178/TLR4 pathway [103]; it may thus be an effective therapeutic target for the treatment of PTC.

A series of in vitro and in vivo assays were applied to elucidate that circLONP2 acts as a key metastasis initiator in CRC progression by regulating intracellular maturation and intercellular transfer of miR-17, promoting the ability to initiate metastasis at the primary site and the formation of metastatic foci in other tissues. Therefore, it could serve as an effective prognostic predictor and as a new target for anti-metastatic therapy in CRC treatment [104]. Transfecting specific siRNAs in CRC cells was shown to inhibit the expression of circRNA-ACAP2 and circCCDC66 and significantly reduced cell proliferation, invasion, and metastasis in the treatment group compared with the control group [87, 88]. Exosomal circ_IFT80 promotes tumorigenesis and reduces radiosensitivity by regulating the miR-296-5p/MSI1 axis in vitro and in vivo, potentially indicating a new strategy for the treatment of CRC [105].

Bone marrow mesenchymal stem cell (BM-MSCs) exosomal circ_0030167 promotes progression and acquisition of stem cell properties in PC cell lines through the miR-338-5p/wif1/Wnt 8/β-catenin axis, offering new prospects for PC treatment [106]. Ye et al. [107] showed that hsa_circ_0000069 gene downregulation could inhibit the development of pancreatic carcinogenesis and the malignant transformation of HPDE cells, indicating that it may be an effective therapeutic target for PC therapy. The interplay network of hsa_circ_0002130–hsa_miR_4482-3p–NBN suggests the possible existence of sponge-attracting miRNAs and target mRNAs, and indicates a role for circRNAs in exploring the molecular mechanisms of PC cell reaggregation after radiotherapy, as well as their potential function as therapeutic targets [108].

Exosomal circ_0061395 promotes the growth of HCC cells by competitively binding to miR-877-5p and increasing the expression of PIK3R3, and could therefore be an effective target for HCC therapy [109]. Exosomal circUHRF1, which is mainly secreted by HCC cells, can promote immunosuppression by inducing natural killer cell dysfunction, which may lead to resistance to anti-PD1 immunotherapy and provides a potential therapeutic strategy for HCC patients [110]. Data have shown that circRNAs are abundant and stable in exosomes and are not only novel participants in the pathogenesis of cholangiocarcinoma but also good noninvasive diagnostic biomarkers in vitro and in vivo [111].

In renal cell carcinoma, circ_400068 has an important regulatory role, and the circ_400068/miR-210-5p/SOCS1 axis may be a candidate therapeutic target for renal cell carcinoma treatment in vitro and in vivo [112]. Li et al. [113] showed that circPRRC2A can competitively bind miR-514a-5p and miR-6776-5p and reduce the degradation of the tissue-specific oncogene TRPM3, which in turn promotes tumor growth and invasion. High expression of circPRRC2A is positively correlated with clinical stage and decreased survival in patients with advanced renal cell carcinoma and is expected to become a therapeutic target for renal cell carcinoma. Tumor-derived exosomal circPSMA1 promotes development, invasion, and metastasis of triple-negative breast cancer (TNBC) via the miR-637/Akt1/β-catenin (cyclin D1) axis, providing a new perspective from which to explore new potential biomarkers and immunotherapeutic strategies for TNBC in vitro and in vivo [114]. CircHIF1A from the exosomes of hypoxic cancer-associated fibroblasts (CAFs) has an important role in maintaining the stem cell properties of breast cancer cells, and circHIF1A may serve as an effective target molecule for breast cancer therapy [115].

Han et al. [116] showed that exosomal circRNA_0001445 promotes glioma progression through the miRNA-127-5p/SNX5 signaling pathway in vitro, providing new insights that could be used to explore the molecular mechanisms of this process. CircRNA_0001445 is thus a promising therapeutic target for glioma. CircSMARCA5 acts as a potent pharmacological tumor suppressor in glioblastoma and functions by bundling RBP SRSF1 in vitro and in vivo [117]. Li et al. [118] found that the transfer of circ-0000190 encapsulated by extracellular vehicles (EVs) from normal cells to OS cells could reduce the proliferation, invasion, and migration ability of OS cells, suggesting that EVs encapsulating circ-0000190 could be constructed and used to affect intercellular communication between normal cells and OS cells, thereby inhibiting the progression of OS.

The clarification of biological functions forms a basis for probing ideal tumor markers, and identifying the regulatory factors of exosomal circRNAs and the mechanisms by which they affect malignant tumor cell growth is the basis for deciding whether exosomal circRNAs can become novel diagnostic markers and therapeutic targets for malignant tumors. The causes and mechanisms of tumor growth are very complex and include regulation of tumor cells apoptosis and proliferation, invasion and metastasis, and modulation of tumor chemotherapy resistance and radiosensitivity.