Recent advances of exosomal circRNAs in cancer and their potential clinical applications

CircRNA is a type of non-coding RNA formed by back-splicing in which a downstream splice donor site is joined with an upstream splice acceptor site to form a covalently closed, uninterrupted loop [31, 32] (Fig. 1). It was first reported by Dr. Hsu, and it was thought to have no valuable biological functions [33]. However, some recent studies revealed that more than 180,000 circRNAs are present in human transcriptomes and that their expression is associated with both normal cellular biological processes and disease progression [34, 35]. Based on their origin, circRNAs are classified into three major types: circular intronic RNAs, exon-intron circRNAs and exonic circRNAs [12, 36]. CircRNA were confirmed to play multiple roles in the biological processes through acting microRNAs or RNA binding proteins sponges to regulate target gene expression, regulating gene transcription or splicing and acting as templates for protein translation [37‐39]. Research has shown that dysregulated circRNAs are associated with the pathogenesis of many human diseases, particularly cancer. Such as, circRNAs has been reported contribute to cancer metastasis and immune escape [40, 41].

Recently, circRNAs were found to be localized to exosomes and capable of being transferred between cells via exosomes, thereby affecting tumor progression. For example, exosome-derived circ-TFDP2 promotes the proliferation of prostate cancer (PC) cells by inhibiting caspase-3-dependent cleavage of PARP1 and DNA damage [42]. Furthermore, Zhao et al. reported that exosome-mediated transfer of circ_0000338 enhances 5-fluorouracil resistance in CRC by regulating microRNA-217/485-3p [43]. Exosomal circ-GSE1 promoteS immune escape of hepatocellular carcinoma (HCC) by inducing the expansion of regulatory T cells via the regulation of miR-324-5p/TGFBR1/Smad3/Tregs axis [44]. Importantly, circRNAs have the potential to serve as biomarkers for cancer diagnosis due to their exosome localization and enrichment. Such as, exosomal circ_0004771 has been reported to be overexpressed in CRC, with area under the curve (AUC) values of 0.86 and 0.88 used to differentiate stage I/II CRC patients and CRC patients from healthy controls, respectively [45].

Various exosomal circRNAs have been reported to regulate the proliferation of cancer cells. For example, exosomal circ-PDK1 promotes pancreatic cancer (PCa) cell proliferation by sponging miR-628-3p to activate the BPTF/c-Myc axis during hypoxia [46]. Furthermore, exosomal circ-PRRX1 promotes cell proliferation in vitro and tumor growth in vivo by sponging miR-596 and activating the NF-κB signaling pathway in gastric cancer (GC) [47]. According to a previous study, cancer-derived exosomal circ-SERPINE2 is shuttled to tumor-associated macrophages (TAMs), and it enhances IL-6 secretion, leading to increased proliferation of breast cancer cells [48]. TAM-secreted exosomal circ_0020256 promotes the proliferation and progression of cholangiocarcinoma by modulating the miR-432-5p/E2F3 axis [49]. In renal cell carcinoma (RCC), tumor-derived exosomal circ-PPKCI increases tumor cell proliferation via the miR-545-3p/CCND1 signaling pathway [50]. In HCC, adipocyte-derived exosomal circ-DB promotes tumor growth by suppressing miR-34a and activating the USP7/Cyclin A2 signaling pathway [51]. Furthermore, hepatic stellate cell-derived exosomal circ-WDR25 facilitates HCC cell proliferation by regulating the miR-4474-3p/ALOX15 axis [52]. Exosomal circ-RACGAP1 recruiteS PTBP1 to induce RIF1 deacetylation, which then activates the Wnt/β-catenin pathway and prmotes the proliferation of non-small cell lung cancer (NSCLC) cells [53]. Interesting, multiple myeloma (MM)-derived exosomal circ-HNRNPU encodes a novel 603-aa peptide, which regulates the bone marrow microenvironment and promotes cell proliferation [54].

However, Circ-LPAR1 expression in plasma exosomes was decreased in CRC and it suppressed the tumor cell proliferation by suppressing the translation of oncogene BRD4 [55]. Exosomal circ-PTPRA induced CRC cell cycle arrest and inhibited cell proliferation by enriching the level of SMAD4 via competitively binding to miR-671-5p [56]. Chen et al. reported that circ_0051443 was transmitted from normal cells to HCC cells via exosomes and suppressed the cell proliferation and malignant biological progression [57]. In oral squamous cell carcinoma (OSCC), exosomal circ-GDI2 was downregulated and its upregulation weakened the cell proliferation by regulating miR-424-5p/SCAI axis [58]. In addition, Chen et al. reported that tumor-suppressive circ-RHOBTB3 could be excreted out of CRC cells via exosomes and circ-RHOBTB3 suppressed cell growth and metastasis [59]. Besides, exosomal circ-BTG2 or circ_0004658 secreted from RBP-J overexpressed-macrophages inhibited glioma or HCC progression by regulating miR-25-3p/PTEN or miR-499b-5p/JAM3 pathway, respectively [60, 61].

Exosomal circRNAs also have crucial function in regulating tumor metastasis. Circ-PACRGL is secreted by CRC cells, and acts as a miR-142-3p/ miR-506-3p sponge to activate the TGF-β-related signaling and promote metastasis [62]. In HCC, exosome-transmitted circMMP2 induced metastasis by sponging miR-136-5p and increasing MMP2 expression [63]. Moreover, exosomal circRAPGEF5 promoted the metastasis of lung adenocarcinoma through the miR-1236-3p/ZEB1 axis [64]. Tumor-derived exosomal circPSMA1 facilitated the metastasis in triple-negative breast cancer through the regulation of miR-637/Akt1/β-catenin regulatory axis [65]. Furthermore, exosomal circ_0081234 promoted the epithelial-mesenchymal transition (EMT) of PC cells [66]. Circ_0003028 induced EMT of HCC cells by exosome pathway via microRNA-498/ODC1 signaling [67]. And exosomal circ_007293 promoted EMT of papillary thyroid carcinoma cells via the regulation of the miR-653-5p/PAX6 axis [68]. In addition, the metastatic ability of HCC cells could be enhanced by transferring exosomal circRNA-100,338 to human umbilical vein endothelial cells (HUVECs), and promoting angiogenesis [69]. In GC, tumor-derived exosomal circ_0044366 promoted tube formation of HUVECs and enhanced cancer migration [70]. In ovarian cancer, exosomal circ-NFIX increased angiogenesis via miR-518a-3p/TRIM44/JAK/STAT1 pathway [71]. In esophageal squamous carcinoma, exosomal circ_0026611 contributed to LNM by interacting with N-α-acetyltransferase 10 (NAA10) to inhibit NAA10-mediated PROX1 acetylation [72].

However, Chen et al. reported that CAFs directly transferred circ-IFNGR2 into ovarian cancer cells and suppressed metastasis by activating miR-378/ST5 [73]. Moreover, bone marrow mesenchymal stem cell-derived exosomal circ_0006790 suppressed metastasis of pancreatic ductal adenocarcinoma by binding to CBX7 and regulating S100A11 DNA methylation [74]. Lin et al. found that exosomal circ_0072088 suppressed migration and invasion of hepatic carcinoma cells by regulating miR-375/MMP-16 [75]. In GC, the expression of exosomal circ-ITCH and circ-STAU2 were significantly downregulated, they suppressed the metastasis of GC by regulating miR-199a-5p/Klotho axis or miR-589/ CAPZA1 respectively [76, 77].

Exosomal circRNAs were associated with the drug resistance of cancers. Exosomal circ_0076305 promoted cisplatin (DDP) resistance of non-small cell lung cancer cell (NSCLC) by enhancing ABCC1 expression [78]. Circ-VMP1 and circ_0014235 were elevated in DDP-resistant NSCLC exosomes, they facilitated DPP resistance by regulating miR-524-5p/METTL3/SOX2 or miR-520a-5p/CDK4 axis, respectively [79]. In osteosarcoma, exosomal circ_103801 conferred DDP resistance by increasing the expression of MRP1 and p-glycoprotein [80]. Warburg effect promoted temozolomide (TMZ) resistant glioma cells releasing exosomal circ_0072083, which induced TMZ resistance of sensitive cells by regulating miR-1252-5p/NANOG [81]. Circ-ZNF91 was remarkably increased in exosomes of PCa under hypoxia condition and promoted gemcitabine resistance of normoxic PCa cells via regulating miR-23b-3p/SIRT1 and enhancing glycolysis [82]. In neuroblastoma, exosomal circ-DLGAP4 enhanced glycolysis and doxorubicin resistance via miR-143-HK2 axis [83]. Oxaliplatin-resistant CRC cells delivered exosomal circ_0005963 to sensitive cells, promoted drug resistance by miR-122 sponging and PKM2 upregulation [84]. Furthermore, exosomal circ_0091741 promoted oxaliplatin resistance of GC cells via the miR-330-3p/ TRIM14/Dvl2/Wnt/β-catenin pathway [85]. Exosomal circ-SFMBT2 and circ-XIAP were upregulated in docetaxel-resistant PC cells, their knockdown enhanced docetaxel sensitivity by regulating miR-136-5p/TRIB1 or miR-1182/TDP52 axis [86, 87]. Pan et al. reveled that exosomal circATG4B induced oxaliplatin resistance in CRC by encoding a novel protein to increase autophagy [88].

However, Xu et al. found that exosomal circ-FBXW7 led resistant cells sensitive to oxaliplatin and suppressed oxaliplatin efflux via sponging miR-18b-5p in CRC [89]. Moreover, circRNA-CREIT could be packaged into exosomes and disseminate doxorubicin sensitivity among TNBC cells by destabilizing PKR [90]. In liver cancer, transarterial chemoembolization increased the expression of exosomal circ-G004213, which promoted DDP sensitivity by regulating miR-513b-5p/PRPF39 axis [91].

We summarized exosomal circRNAs and their function in tumorigenesis in Table 1.

Exosomal circRNAs mediate the communication between tumor cells and immune cells (Fig. 2). In bladder cancer, exosome-derived circ-TRPS1 promotes CD8 + T cell exhaustion and the malignant phenotype by sponging miR-141-3p [143]. In NSCLC, upregulated plasma exosomal circ-USP7 inhibites CD8 + T cell function by sponging miR-934 and increasing SHP2 expression [144]. In LUAD, exosomal circ_002178 can be delivered to CD8 + T cells to induce PD1 expression and T cell exhaustion [145]. In ovarian cancer, exosomal circ-0001068 can be delivered to T cells and induced PD1 expression by sponging miR-28-5p [146]. In HCC, exosomal circ-CCAR1 promotes CD8 + T cell dysfunction by stabilizing the PD1 protein [147]. In OSCC, the transfer of circ_0069313 to Treg cells promotes immune escape by inhibiting miR-325-3p-induced Foxp3 degradation [148]. Moreover, CAF-derived exosomal circ-EIF3K increases the PD-L1 expression in CRC [149].

In NSCLC, exosomal circ-SHKBP1 or circ-FARSA promotes M2 polarization and cancer progression via the miR-1294/PKM2 or PTEN/PI3K/AKT pathway [150, 151]. In glioma, exosomal circ-NEL3 induces macrophage immunosuppressive polarization by stabilizing the oncogenic protein IGF2BP3 [152]. In LUAD, exosomal circ-ZNF451 restrains anti-PD1 treatment by polarizing macrophages and complexing with TRIM56 and FXR1 [153]. In breast cancer, exosomal circ_0001142 is released by cancer cells under endoplasmic reticulum stress, and it induces M2 polarization of macrophages [154]. In RCC, exosomal circ-SAFB2 reshapes the tumor environment, mediates M2 macrophage polarization, and promotes tumor progression [155]. In esophageal squamous cell carcinoma, tumor-derived exosomal circ_0048117 facilitates M2 macrophage polarization by regulating microRNA-140/TLR4 axis [156].

In HCC, cancer cells secrete exosomal circ-UHRF1, which induces natural killer cell exhaustion and promotes immune therapy resistance by regulating the miR-449c-5p/TIM3 axis [157]. CRC-derived exosomal circ-PACRGL regulates the differentiation of N1/N2 neutrophils [62]. Wang et al. reported that upregulated expression of plasma exosomal circ-ADAMTS6 is positively related to neutrophil extracellular traps in cholangiocarcinoma [158].

CircRNAs have a special stable tertiary structure, and it has been reported that their expression is not significantly altered after 24 h of incubation at room temperature [14]. Furthermore, circRNAs were found to be dysregulated under pathological conditions and enriched in exosomes, which could be detected in body fluids such as blood, serum, urine, saliva, and cerebrospinal fluid [14, 15]. These features indicate that exosomal circRNAs can serve as biomarkers for cancer diagnosis. Xu et al. found that the expression of circ_0109046 and circ_0002577 were higher in exosomes isolated from serum samples of patients with stage III endometrial adenocarcinoma compared to healthy controls [159]. Xu et al. reported that circ-SHKBP1 is a promising circulating biomarker for GC diagnosis and prognosis due to its upregulation in serum and positive relationship with advanced TNM stage and poor survival [160]. Deng et al. reported that oral squamous cell carcinoma patients with higher expression of exosomal circ_047733 showed a lower risk of LNM [161]. Plasma exosome-derived circ_0055202, circ_0074920, and circ_0043722 are upregulated in glioblastoma multiforme and associated with tumor progression [162]. Furthermore, Hong et al. revealed that circ_0006220 and circ_0001666 are highly expressed in exosomes in the plasma of PCa patients compared to healthy controls and that they are associated LNM and tumor size. The AUC values were 0.7817 for circ_0006220, 0.8062 for circ_0001666, and 0.884 for the combined diagnosis [163]. The expressions of circ_0001492, circ_0001439, and circ_0000896 were significantly higher in the serum exosomes of LUAD patients, and the combination of these exosomal circRNAs had diagnostic sensitivity and specificity with an AUC value of 0.805 [164]. Furthermore, circ_0028861 was identified as a novel biomarker for HCC diagnosis, with an AUC of 0.79, and was capable of detecting small (AUC = 0.81), early-stage (AUC = 0.82), and AFP-negative (AUC = 0.78) tumors [165]. What’s more, exosomal circ_0015286 has an oncogenic function in GC, and its expression is closely associated with tumor size, TNM stage, LNM, and overall survival of GC patients [166]. Besides, clinical data have shown that exosomal circ_0000437 is enriched in the serum of GC patients and associated with LNM [167]. In addition, Wang et al. identified circ-SLC38A1 in the serum exosomes of bladder cancer patients, which could distinguish bladder cancer patients from healthy individuals with a diagnostic accuracy of 0.878 [30].

Other exosomal circRNAs that could serve as potential biomarkers for cancer diagnosis are summarized in Table 2.

Exosomes can transport RNA molecules and deliver therapeutic drugs to cancer cells with good histocompatibility, high efficiency, and low cytotoxicity. Researchers have reported that some circRNAs have tumor suppressor functions, and the therapeutic delivery of exosomal circRNAs could suppress the proliferation, metastasis, drug resistance and progression of malignant tumors. Circ-EPB41L2 is downregulated in the exosomes of CRC patients, and exosome-mediated circ-EPB41L2 suppresses tumor progression by regulating the PTEN/AKT signaling pathway [191]. Zhang et al. reported that exosome-delivered circ-STAU2 inhibites the progression of GC by targeting the miR-589/CAPZA1 axis [77]. Moreover, Sang et al. reported that the exosomal transmission of circ-RELL1 suppresses the proliferation, invasion, and migration of GC cells [192]. Circ-DIDO1 is downregulated in GC, and circ-loaded, RGD-modified engineering exosomes significantly inhibit the proliferation, migration, and invasion of GC cells both in vivo and in vitro [193]. Furthermore, Circ-CREIT is aberrantly downregulated in doxorubicin-resistant TNBC cells and is associated with a poor prognosis. The exosomal transmission of circ-CREIT could disseminate doxorubicin sensitivity among these cells by destabilizing PKR [90]. Circ_0094343 is significantly downregulated in CRC, and exosome-carried circ_0094343 playes a tumor suppressor role and improves the chemosensitivity of tumor cells to 5-fluorouracil, oxaliplatin and doxorubicin [194].

Tumor microenvironment-associated cells also play tumor suppressor roles by delivering exosomal circRNAs to cancer cells. For example, CAF-derived exosomes deliver circ-IFNGR2 to ovarian cancer cells and inhibit malignant tumor progression by regulating the microRNA-378/ST5 axis [73]. Moreover, RBP-J-overexpressed- macrophage-derived exosomal circ-BTG2 or circ_0004658 inhibit glioma or HCC progression [60, 61]. Furthermore, Yao et al. reported that exosomal circ_0030167 derived from bone marrow-derived mesenchymal stem cells (BM-MSCs) exhibit significant tumor suppressor function in PCa by sponging microRNA-338-3p and targeting the Wif1/Wnt8/β-catenin axis [195]. BM-MSC-derived exosomal circ_0006790 inhibits growth, metastasis, and immune escape in pancreatic ductal adenocarcinoma [74].

Besides, Nanoparticles or exosomes mediated circRNAs silencing also a potential strategy for cancer treatment. For example, nanoparticles delivery si-circ-ROBO1 to hepatocellular carcinoma cells circ-ROBO1 inhibited tumor progression by modulating circ-ROBO1/miR-130a-5p/CCNT2 Axis[196]. And natural compound matrine blocked circ-SLC7A6 exosome secretion from CAFs, and then inhibited CRC cell proliferation and invasion[197]. These studies indicate that exosomal delivery of tumor-suppressing circRNAs or exosomal circRNAs-based engineering of exosomes or exosome circRNAs release inhibition may be novel cancer therapies.

The recent data reporter about “exosome-based circRNA delivery for cancer therapy” were summarized in Table 3.