Extracellular vesicles circular RNA: A new perspective and clinical application potential for mediating chemoresistance in colorectal cancer

Abstract

Colorectal cancer (CRC) is a common and deadly malignancy worldwide, causing high morbidity and mortality rates. Chemoresistance continues to be the major barrier to the effective treatment of CRC. Circular RNA (circRNA), a recently identified class of non-coding RNA, has become an emerging focus in CRC research due to its abundant presence and stability in extracellular vesicles (EVs). The circRNA of EVs (EVs-circRNA) is closely associated with CRC progression and plays a pivotal role in chemoresistance mechanisms. This paper investigates the circRNA mechanism underlying different chemoresistance scenarios in CRC and explores the dual role of EVs-circRNA in mediating chemotherapy resistance. Furthermore, EVs-circRNA, a non-invasive biomarker for liquid biopsies, holds significant promise in clinical applications, ranging from early CRC diagnosis to monitoring disease progression and assessing prognosis. These studies provide a new perspective for uncovering CRC pathogenesis and lay the foundation for precision therapy and personalized treatment strategies for CRC. In the future, therapeutic strategies targeting EVs-circRNAs are anticipated to revolutionize CRC treatment, leading to improved therapeutic outcomes and enhanced quality of life for patients.

Key Words: Extracellular vesicles; Circular RNA; Colorectal cancer; Chemotherapy resistance; Clinical application

Core Tip: Colorectal cancer (CRC) chemoresistance remains a major therapeutic challenge. Circular RNA (circRNA) is enriched and stable in extracellular vesicles (EVs), drives CRC progression, and mediates drug resistance. The circRNA of EVs as a non-invasive biomarker for diagnosis, monitoring, and prognosis offers a promising strategy for precision therapy of CRC.

INTRODUCTION

Cancer has become a major global health concern in the 21^st century. The latest data from the International Agency for Research on Cancer reported 18.74 million new cases of malignant tumors and around 9.67 million deaths globally in 2022. Of these, colorectal cancer (CRC) accounts for 1.92 million new cases (10.2%) and 904000 deaths (9.3%), making it the third most common cancer by incidence and the second leading cause of cancer-related deaths globally. It has the highest morbidity and mortality among gastrointestinal cancers[1]. The incidence of CRC is projected to rise further by 2050 due to an aging population and increasing risk factors[2]. Surgery remains the mainstay of treatment for CRC, but metastatic recurrence occurs in approximately 30% of patients after surgery, and certain individuals present with advanced disease stages upon initial diagnosis and are unable to undergo radical surgery[3,4]. The effectiveness of chemotherapy, the core treatment for advanced CRC, is frequently hindered by chemoresistance. Resistance not only reduces drug sensitivity but also constitutes a major obstacle in improving patient survival[5]. The mechanisms underlying resistance to CRC chemotherapy are complex, involving increased drug efflux, altered drug targets, and DNA damage repair, among others[6-8]; In addition to the characteristics of tumor cells themselves, drug resistance also spreads to the microenvironment through cell-to-cell signaling and epigenetic remodeling, in which extracellular vesicles (EVs) play a key role as information carriers. Therefore, an in-depth exploration of resistance mechanisms is urgently needed to optimize therapeutic strategies.

EVs have garnered significant attention in recent years for their crucial role in intercellular communication[9]. According to minimal information for studies of EVs of the International Society for Extracellular Vesicles[10], the term "EVs" in this study refers to lipid bilamellar vesicles (including exosomes) with a diameter of 30-200 nm. These vesicles are produced by the endocytic pathway and released by exocytosis and are found in blood, urine, saliva, and other bodily fluids[11,12]. EVs consist of a variety of substances, including specific lipids, proteins, DNA, microRNAs (miRNAs), non-coding RNAs, and other biologically active molecules, which can be involved in tumorigenesis and metastasis through intercellular communication[13]. Additionally, EVs play a pivotal role in tumor drug resistance and are closely linked to its development[14]. EVs have been found to act as "molecular pumps" that actively excrete chemotherapeutic drugs[15], thereby reducing intracellular drug concentration, while also enhancing tumor cell plasticity by triggering epithelial-mesenchymal transition (EMT)[16].

Circular RNAs (circRNAs) are non-coding RNAs that have gained attention in cancer research due to their unique covalently closed-loop structure, which offers superior stability and conservation compared to linear RNAs[17]. Initially, circRNAs were thought to be “useless structures” for RNA splicing. However, with the advancement of high-throughput sequencing, their roles in tumor proliferation and invasion have been uncovered, including functions as miRNA sponges, regulation of protein interactions, and translation of functional peptides[18-20]. Moreover, circRNAs are involved in resistance against various chemotherapeutic agents[21,22]. Although the intracellular functions of circRNAs have been well studied, their transcellular regulatory networks mediated through EVs have not been systematically elucidated. Research has demonstrated that circRNAs are highly enriched and consistently expressed in EVs. The abundance of circRNAs in EVs is twice that in parental cells and six times greater than that of linear RNAs[23]. This enrichment stems from the unique closed-loop structure of circRNA, which enables its preferential packaging during the sorting of EVs cargo. Furthermore, the lipid bilayer membrane of EVs effectively shields against external nucleases, forming a "dual-stabilization barrier"-the circularized structure of circRNA resists degradation by endogenous RNases, while the EVs membrane prevents attacks by exogenous enzymes. This dual stability endows circRNA of EVs (EVs-circRNA) with exceptional signaling capabilities: They leverage the targeted delivery properties of EVs to accumulate in the tumor microenvironment while retaining circRNA's molecular functions. Consequently, they drive chemoresistance by regulating key signaling pathways or remodeling the immune microenvironment[24-26].

EVs-circRNA has become the focal point of studies on drug resistance mechanisms because of their high abundance, tissue specificity, and transcellular regulatory potential[27-29]. Studies have shown that EVs-circRNA can be used in CRC not only as innovative biomarkers for non-invasive diagnostics and prognosis[30,31] but also as a viable therapeutic strategy for overcoming chemotherapy resistance by reversing the drug-resistant phenotype through targeted intervention. However, research is still in its infancy, and the roles of the majority of EVs-circRNA remain unclear. Their specific mechanisms in CRC drug resistance require further in-depth investigation. In this paper, we first review the intracellular resistance mechanisms of circRNAs and then explore the transcellular delivery of circRNAs via EVs, finally evaluate the potential of EVs-circRNA for clinical applications, providing a theoretical foundation for analyzing CRC resistance mechanisms and developing precise treatment strategies.

INTRINSIC CIRCRNA MECHANISMS UNDERLYING CHEMOTHERAPY RESISTANCE IN CRC

An increasing number of circRNAs are identified as key contributors to chemoresistance development in CRC (Figure 1), including oxaliplatin (OXA), 5-fluorouracil (5-FU), cetuximab, etc. The mechanism primarily involves circRNA acting as a miRNA sponge, influencing miRNA gene expression, regulating related signaling pathways, and affecting cell migration, invasion, and apoptosis. However, the delivery mechanisms of these circRNAs remain incompletely understood. This section details the roles of circRNAs in mediating resistance to key chemotherapeutic agents, focusing on their intracellular regulatory networks (Table 1)[24,32-53].

Figure 1  Circular RNAs in colorectal cancer chemotherapy resistance.

Table 1 Summary of dysregulated circular RNAs in colorectal cancer chemoresistance.

Chemotherapy regimen	CircRNAs	Annotated number	Expression	Samples	Targets/regulators	Function	Ref.
Oxaliplatin	Hsa_circ_0079662	-	Up	Cells	Tumour necrosis factor alpha signaling	Promotes proliferation, migration, invasion, and chemoresistance	Lai et al[24]
	Circ-CD44	Hsa_circ_0000291	Up	Tissues; cells	MiR-330-5p/ABCC1	Promotes proliferation, migration, invasion, and chemoresistance	Zhao et al[32]
	Hsa_circ_0071589	-	Up	Cells	MiR-133b	Aggravates stemness and promotes chemoresistance	Lv et al[33]
	Circ_0000395	-	Up	Cells	MiR-153-5p/myosin VI	Promotes proliferation, migration, and chemoresistance	Xiao et al[34]
	Circ_0082182	Has_circ_0082182	Up	Tissues; cells	MiR-326	Promotes chemoresistance	Wang et al[35]
	Circ-PDIA3	Hsa_circ_0002891	Up	Tissues; cells	MiR-449a	Inhibits pyroptosis and promotes chemoresistance	Lin et al[36]
	Circ-SEC24B	Hsa_circ_0001436	Up	Tissues; cells	Sushi repeat-containing protein X-linked 2	Activates autophagy and promotes chemoresistance	Wang et al[37]
	Circ-PDE4D	Hsa_circ_0072568	Up	Cells	Hsa-miR-569	Inhibits chemoresistance	Li et al[38]
	Circ-ZEB1	Hsa_circ_0000229	Up	Tissues	MiR-200c	Promotes chemoresistance	Chen et al[47]
	Circ_0032833	-	Up	Cells	MiR-125-5p	Promotes chemoresistance	Li et al[48]
	Hsa_circ_0000338	-	Up	Cells	-	Inhibits chemoresistance	Hon et al[49]
5-fluorouracil	Hsa_circ_0002813	-	Up	Cells	MiR-541-3p	Promotes chemoresistance	Cheng et al[39]
	Hsa_circ_0000236	-	Up	Cells	MiR-4769-5p	Promotes chemoresistance
	Circ_0007031	-	Up	Cells	MiR-133b/ABCC5	Promotes chemoresistance	He et al[40]
	Has_circ_0055625	-	Up	Serum	-	Promotes chemoresistance	Liu et al[41]
	Circ_0014130	Hsa_circ_0014130	Up	Tissues; cells	MiR-197-3p/6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3	Promotes chemoresistance	Wang et al[42]
	Circ_0004585	-	Up	Tissues; cells	MiR-874-3p/cyclin D1	Promotes chemoresistance	Wang et al[43]
	Circ-PRKDC	Circ_0136666	Up	Tissues; cells	MiR-375/forkhead box protein M1	Promotes chemoresistance	Chen et al[44]
	Circ-ERBB2	Hsa_circ_0043459	Up	Cells	MiR-181a-5p/phosphatase and tensin homologue deleted on chromosome 10/AKT	Promotes chemoresistance	Zhang et al[45]
	Hsa_circ_0007031	-	Up	Cells	MiR-885-3p/AKT3	Promotes chemoresistance
	Hsa_circ_0000504	-	Up	Cells	MiR-485-5p/signal transducer and activator of transcription 3/AKT3	Promotes chemoresistance	Xiong et al[46]
	Hsa_circ_0048234	-	Down	Cells	MiR-671-5p/epidermal growth factor receptor	Promotes chemoresistance
	Circ-ZEB1	Hsa_circ_0000229	Up	Tissues	MiR-200c	Promotes chemoresistance	Chen et al[47]
	Circ_0032833	-	Up	Cells	MiR-125-5p	Promotes chemoresistance	Li et al[48]
	Hsa_circ_0000338	-	Up	Cells	-	Inhibits chemoresistance	Hon et al[49]
Cetuximab	Circ-IFNGR2	Hsa_circ_001185	Up	Cells	MiR-30b	Promotes chemoresistance	Zhang et al[50]
		Hsa_circ_000339	Up	Cells	MiR-30b	Promotes chemoresistance
	Circular hypoxia inducible factor 1 subunit alpha	Hsa_circ_0007976	Up	Cells	MiR-361-5p	Affects cellular aerobic metabolism, glycolysis levels, and promotes chemoresistance	Geng et al[51]
Irinotecan	Hsa_circ_001680	-	Up	Tissues; cells	MiR-340	Promotes proliferation, migration, and chemoresistance	Jian et al[52]
Cisplatin	CircRNA_101277	-	Up	Tissues; cells	MiR-370/interleukin-6	Promotes chemoresistance	Lv et al[53]

ABCC: Adenosine triphosphate binding cassette subfamily C; AKT: Protein kinase B; CircRNA: Circular RNA; ZEB1: Zinc finger E-box binding homeobox 1.

CircRNA-mediated mechanisms in OXA resistance

Locally advanced or metastatic CRC is commonly treated with OXA chemotherapy[54]. However, patients with advanced or metastatic CRC resistant to OXA have a poor prognosis and few treatment options. Consequently, it is essential to explore the biological mechanisms of OXA resistance and identify novel therapeutic targets to improve chemotherapy sensitivity.

Numerous studies have found that the upregulation of circRNA expression in CRC[24,32] may promote chemotherapy resistance[33-37]. Hsa_circ_0000291 is significantly overexpressed in OXA-resistant CRC tissues and cells, acting as a molecular sponge for miR-330-5p, upregulating adenosine triphosphate binding cassette subfamily C member 1 expression, and promoting OXA chemoresistance in CRC cells[32]. Hsa_circ_0079662 by binding to miR-324-5p, disrupts the inhibition of HOXA9 and activates the tumour necrosis factor alpha pathway, thereby promoting the proliferation, migration, and invasion of CRC cells, leading to OXA resistance in CRC[24]. Hsa_circ_0001436 promotes the deubiquitination of Sushi repeat-containing protein X-linked 2 through the mediation of the deubiquitinating enzyme OTUB1, which activates autophagy and induces chemoresistance in CRC[37]. In contrast, some circRNAs are upregulated in OXA-resistant CRC cells and could contribute to inhibiting chemotherapy resistance in CRC. For example, in the drug-resistant HCT116/L cell line, overexpression of hsa_circ_0072568 reduced hsa-miR-569 levels and increased SPI1 expression, which suppressed drug-resistant cell proliferation and promoted apoptosis, ultimately improving sensitivity to chemotherapy[38].

CircRNA-driven 5-FU resistance

5-FU serves as the primary chemotherapy for CRC, yet approximately half of patients with advanced CRC develop resistance to it[55], highlighting the urgent need to delve deeper into the mechanisms underlying 5-FU resistance. In CRC, 5-FU resistance is closely associated with intracellular regulatory mechanisms[39-41] of multiple circRNAs[42-46]. Cheng et al[39] conducted a comprehensive circRNA expression analysis on two CRC cell lines and their 5-FU-resistant counterparts using high-throughput sequencing techniques to explore the role of circRNAs in 5-FU resistance. Their investigation identified hsa_circ_0002813 and hsa_circ_0000236 as key factors potentially associated with the development of 5-FU resistance. Hsa_circ_0002813 contributes to the modulation of 5-FU resistance by adsorbing miR-541-3p, thus preventing miR-541-3p inhibition of fucosyltransferase 3 (FUT3) and promoting the upregulation of FUT3 expression. On the other hand, hsa_circ_0000236 might regulate 5-FU resistance by adsorbing miR-4769-5p, preventing pleomorphic adenoma gene 1 (PLAG1) inhibition, and indirectly enhancing PLAG1 expression.

In addition, hsa_circ_0000229, circ_0032833, and hsa_circ_0000338 were strongly associated with both OXA and 5-FU chemoresistance[47-49]. Hsa_circ_0000229 Levels in CRC tissues were significantly higher compared to those in adjacent normal tissues. Hsa_circ_0000229 directly binds to miR-200c, acting as a sponge for miR-200c in CRC cells, thereby regulating chemotherapy resistance[47]. Circ_0032833 acted as a sponge for miR-125-5p, inhibiting its activity and indirectly upregulating MSI1 expression, thus promoting chemotherapy resistance[48]. In Hon et al’s investigation[49], hsa_circ_0000338 may act as a tumor suppressor in drug-resistant CRC. Knocking down hsa_circ_0000338 expression resulted in increased sensitivity of drug-resistant CRC cells to chemotherapeutic agents, suggesting that hsa_circ_0000338 may play a role in enhancing chemosensitivity.

CircRNA roles in cetuximab resistance

Cetuximab, combined with chemotherapy, is the standard first-line treatment for patients with RAS-wild-type metastatic CRC; however, most patients eventually develop secondary drug resistance, leading to treatment failure[56,57]. Consequently, understanding the mechanisms underlying cetuximab resistance in CRC patients, particularly at the molecular level, is essential. Moreover, it is necessary to continue searching for molecular markers to facilitate effective treatment with cetuximab.

Overexpression of circIFNGR2 (hsa_circ_001185 and hsa_circ_000339) indirectly upregulates kirsten rat sarcoma 2 viral oncogene homolog expression and activates the downstream signaling pathway via inhibiting miR-30b activity, thereby increasing cetuximab resistance in CRC cells[50]. Moreover, hsa_circ_0007976 was significantly overexpressed in cetuximab-resistant CRC cells. It promoted hypoxia inducible factor 1 subunit alpha upregulation by acting as a molecular sponge for miR-361-5p, disrupting the balance between cellular aerobic metabolism and glycolysis. This metabolic reprogramming ultimately facilitated the development of drug resistance[51]. Therefore, circIFNGR2 and hsa_circ_0007976 may serve as potential therapeutic markers for cetuximab-resistant CRC.

Additionally, circRNAs are crucial in other chemotherapy regimens. Hsa_circ_001680 suppresses miR-340 expression by serving as a competitive endogenous RNA, leading to an upregulation of B lymphoma Mo-MLV insertion region 1. This process boosts CRC cell proliferation, migration, and the cancer stem cell population, which ultimately results in resistance to irinotecan chemotherapy[52]. CircRNA_101277, a newly identified cirRNA, is overexpressed in CRC tumor tissues and strongly correlates with increased CRC cell proliferation. This circRNA exerts its effects by interacting with miR-370, effectively suppressing its activity. As a result, the target gene interleukin (IL)-6 is upregulated, triggering a signaling cascade mediated by IL-6. This pathway ultimately leads to cisplatin resistance in CRC cells, emphasizing the pivotal role of circRNA_101277 in promoting therapeutic resistance in CRC[53].

In summary, circRNAs regulate CRC chemoresistance through cell-autonomous mechanisms, including miRNA sponging, pathway modulation, and interactions with key proteins. However, whether these circRNAs depend on EVs-mediated delivery to transmit drug-resistant phenotypes across cells remains largely uncharacterized. While current studies emphasize the intracellular roles of circRNAs, emerging evidence highlights their potential involvement in intercellular communication. In addition to cell-autonomous mechanisms, EVs-mediated transcellular delivery of circRNAs further extends the dimensions of drug resistance regulation. Notably, EVs have been identified as critical carriers for transferring functional circRNAs between cells, thereby disseminating resistance signals and remodeling the tumor microenvironment. The following section will delve into EVs-mediated circRNA transfer regulating chemotherapy resistance.

EVS-CIRCRNA DELIVERY MEDIATES CHEMORESISTANCE IN CRC

EVs serve as pivotal mediators of intercellular communication by transferring circRNAs between drug-resistant and sensitive CRC cells, thereby regulating chemoresistance through multifaceted mechanisms. The dysregulated expression of EVs-encapsulated circRNAs-whether upregulated or downregulated-exhibits strong correlations with the degree of chemoresistance in CRC, a finding that has propelled EVs-circRNA research into the spotlight. In contrast to the cell-intrinsic circRNA mechanisms detailed earlier, EVs-circRNAs operate via a transcellular regulatory axis: These circRNAs are horizontally transferred within EVs, reprogramming recipient cells to adopt resistant phenotypes. This section systematically examines the dual roles of EVs-circRNAs in either promoting or reversing chemoresistance (Table 2)[25,49,58-66].

Table 2 List of extracellular vesicles-circular RNAs involved in colorectal cancer chemoresistance or chemosensitivity.

	EV content	Expression	EV source	Targets/regulators	Function	Drug	Ref.
Chemosensitivity	Circ-FBXW7	Down	Human fetal colon cell culture	MiR-18b-5p	Increases apoptosis, inhibits epithelial-mesenchymal transition, and improves chemosensitivity	OXA	Xu et al[58]
	Circ_0094343	Down	Cell lines (NCM460 and HCT116)	MiR-766-5p/tripartite motif-containing 67	Inhibits the proliferation, clone formation, glycolysis of colorectal cancer cells, and improves chemosensitivity	5-FU, OXA, and DOX	Li et al[59]
Chemoresistance	Circ_0067557	Up	CAFs	Lin28A, Lin28B	Promotes proliferation, migration, invasion, and chemoresistance	5-FU, OXA	Yang et al[25]
	Hsa_circ_0000338	Up	HCT116-R	-	Promotes chemoresistance	5-FU, OXA	Hon et al[49]
	Hsa_circ_0004085	Up	Fusobacterium nucleatum-infected colon cancer cells	GRP78, ATF6p50	Relieves endoplasmic reticulum stress in recipient cells and promotes chemoresistance	5-FU, OXA	Hui et al[60]
	Circ_0000338	Up	Cell lines (SW480/5-FU and HCT116/5-FU)	MiR-217, miR-485-3p	Promotes chemoresistance	5-FU	Zhao et al[61]
	Cric-N4BP2 L2	Up	CAFs	Eukaryotic translation initiation factor 4A3/phosphatidylinositol 3-kinase/protein kinase B/mammalian target of the rapamycin	Aggravates stemness and promotes chemoresistance	OXA	Qu et al[62]
	Hsa_circ_0005963	Up	Serum and cell lines (SW480, HCT116, and HEK293)	MiR-122/pyruvate kinase muscle isozyme M2	Promotes glycolysis and chemoresistance	OXA	Wang et al[63]
	Circ_0001610	Up	Cell lines (DLD1 and HCT15)	MiR-30e-5p	Increases oxidative phosphorylation activity and stemness phenotype in recipient cells leading to chemoresistance	OXA	Deng et al[64]
	Has_circ_0004771	Up	Serum and cell lines (NCM460, SW620, and HCT116)	MiR-653/ZEB2	Promotes chemoresistance	5-FU	Qiao et al[65]
	Circ_0006174	Up	Cell lines (LoVo/DOX, and HCT116/DOX)	MiR-1205/cyclin D2	Promotes chemoresistance	DOX	Zhang et al[66]

CAFs: Cancer-associated fibroblasts; CircRNA: Circular RNA; DOX: Doxorubicin; EV: Extracellular vesicle; OXA: Oxaliplatin; 5-FU: 5-fluorouracil.

EVs-circRNA can inhibit tumor resistance to chemotherapy

In CRC, specific circRNAs within EVs exhibit reduced expression and potentially inhibit chemotherapeutic resistance. Research findings[58] demonstrated diminished circ_FBXW7 levels among patients with OXA-resistant CRC, similarly observed in CRC cells. Subsequent investigations utilizing cellular and animal experiments revealed EVs-circ_FBXW7 could enter resistant CRC cells, consequently promoting apoptotic processes, suppressing EMT, and reducing OXA outflow, ultimately enhancing the sensitivity of CRC cells to OXA. Li et al[59] discovered that circ_0094343 shows markedly decreased expression throughout CRC tissues, particularly those exhibiting chemoresistance and metastatic properties. EVs carrying circ_0094343 suppressed HCT116 proliferation, colony formation, and glycolysis, enhancing their chemosensitivity to 5-FU, OXA, and doxorubicin. Molecular investigations revealed circ_0094343 functions as a miR-766-5p sponge, modulating tripartite motif-containing 67 expression and consequently inhibiting chemotherapeutic resistance within HCT116 cellular populations.

CircRNA delivery via EVs can enhance tumor resistance to chemotherapy

Several circRNAs found in EVs show elevated expression within CRC[25,49,60-62] and potentially enhance chemotherapeutic resistance[63-66]. Hui et al[60] demonstrated that EVs-delivered hsa_circ_0004085, secreted by fusobacterium nucleatum-infected colon cancer cells, plays a part in the development of chemoresistance in cancer cells, contributes to chemoresistance by reducing endoplasmic reticulum stress, enhancing cell survival under chemotherapeutic agents (e.g., OXA and 5-FU), and diminishing their cytotoxic effects. High expression of hsa_circ_0000338 was observed in drug-resistant CRC cells (HCT116-R) and their EVs, where it exhibited dual roles[49]: Within drug-resistant CRC cells, it may function as a tumor suppressor, but when transferred via EVs from resistant cells to sensitive CRC cells, it displayed oncogenic effects. This suggests that hsa_circ_0000338 may play a crucial role in transmitting chemotherapy resistance through EVs, facilitating the spread of resistance, in line with the findings of Zhao et al[61]. Yang et al[25] demonstrated elevated circ_0067557 levels within cancer-associated fibroblasts-derived EVs, which enhanced CRC cell proliferation, migration, invasion, and chemoresistance while suppressing apoptosis. Their findings indicated that circ_0067557 occupies a central position in resistance development through its interactions with Lin28A and Lin28B targets.

EVs-circRNAs have been shown to have dual regulatory roles in chemoresistance in CRC: (1) They can promote the spread of drug-resistant phenotypes; and (2) They can also reverse drug resistance, highlighting its diagnostic and therapeutic potential. However, there is still a critical gap in the evidence for EVs-mediated transcellular delivery of circRNAs, and some studies have conflicting views with unclear mechanisms (e.g., the tumor-suppressive function of hsa_circ_0000338 in drug-resistant cells vs the oncogenic effect after EVs delivery). In the future, it is necessary to integrate single-cell sequencing and in vivo EVs tracking technologies to resolve the temporal and spatial dynamic delivery network of circRNAs in the tumor microenvironment to resolve the existing controversies. The above mechanistic studies not only reveal the central role of EVs-circRNAs in CRC drug resistance but also lay a theoretical foundation for their clinical translation. Based on this, part IV will systematically explore the clinical prospects of EVs-circRNA.

CLINICAL APPLICATION VALUE OF EVS-CIRCRNA IN CRC

Early screening for CRC is essential for improving therapeutic results and lowering mortality rates. While conventional screening methods including digital rectal examination, fecal occult blood testing, endoscopic colonoscopy, radiological assessment, carcinoembryonic antigen (CEA), and carbohydrate antigen 19-9 (CA19-9) have substantially improved CRC identification rates, superior molecular indicators remain necessary[67]. Notably, EVs-circRNAs have emerged as highly promising non-invasive biomarkers for liquid biopsy, offering superior sensitivity and specificity for CRC diagnosis, treatment, and prognosis compared to conventional biomarkers (Figure 2).

Figure 2 Clinical application of extracellular vesicles-circular RNAs in the diagnosis, treatment, and prognosis of colorectal cancer. EVs: Extracellular vesicles.

Potential markers for CRC diagnosis

The discovery of sensitive biomarkers for the early diagnosis of CRC plays a vital role in enhancing survival outcomes among affected individuals. Evs contain abundant circRNA characterized by remarkable stability, allowing their identification across various biological specimens including serum, urine, and additional physiological fluids. Distinct EVs-circRNA expression profiles observed between cancer patients and healthy subjects establish these circRNAs as compelling options for developing non-invasive screening tools facilitating early CRC diagnosis.

Contemporary research demonstrates that serum EVs-hsa_circ_0004771 expression exhibits significant upregulation across different TNM stages of CRC patients compared to control subjects. Hsa_circ_0004771 displayed remarkable diagnostic performance for colorectal malignancies, achieving an area under the curve (AUC) value of 0.88, with 80.91% sensitivity and 82.86% specificity. Additionally, serum EVs-has_circ_0004771 expression was markedly elevated among early-stage (I-II) CRC patients compared to healthy controls, yielding diagnostic parameters including 0.86 AUC, 81.43% sensitivity, and 80.00% specificity for identifying early-stage disease[68]. Barbagallo et al[69] found elevated levels of circHIPK3 in serum EVs among CRC patients, having an AUC of 0.771, a sensitivity of 71%, and a specificity of 80% for CRC diagnosis. Zheng et al[30] reported that EVs-hsa_circ_0087960 was considered a predictor of CRC diagnosis, which could demonstrate specificity for CRC diagnosis by combining common clinical biomarkers CEA and CA19-9, and improved the diagnostic efficacy of the receiver operating characteristic (ROC) curve to 87.5%. Li et al[70] observed hsa_circ_0003270 upregulation within plasma EVs fractions isolated from individuals with colorectal carcinoma. Statistical evaluation using ROC methodology indicated the potential clinical value of this circRNA as a CRC biomarker. Their analysis revealed diagnostic performance parameters of 75.64% sensitivity and 71.79% specificity for hsa_circ_ 0003270 in distinguishing malignant colorectal pathology. Therefore, hsa_circ_0003270 overexpression represents a promising innovative biomarker candidate for CRC detection. Xie et al[71] explored the potential clinical implications of serum EVs circRNAs within CRC cases. Their investigation determined that hsa_circ_0101802 expression in serum EVs appeared substantially increased among CRC sufferers vs healthy controls. These results indicate that hsa_circ_0101802 potentially serves as a valuable molecular signature facilitating timely CRC recognition. Similarly, Li et al[23] found serum EVs-circKLDHC10 effectively differentiates individuals with CRC from healthy populations, underscoring its utility as a noninvasive screening tool for colorectal malignancies.

Therapeutic targets for CRC

EVs-circRNAs can enter recipient cells, interact with specific miRNAs and RNA-binding proteins, and form functional complexes with therapeutic potential for CRC. Research findings demonstrate that EVs-hsa_circ_0005100 modulates pathological processes in CRC through its interaction with miR-1182, suggesting its potential application as a therapeutic intervention target[72]. Gao et al[73] revealed elevated expression of EVs-hsa_circ_0016866 within CRC samples. Moreover, patients exhibiting heightened hsa_circ_0016866 Levels demonstrated reduced survival compared to their counterparts with diminished expression. Mechanistically, this circRNA promotes cell proliferation, invasion, and migration via the miR-1305/transforming growth factor (TGF)-β2/SMAD family member 3 signaling cascade, consequently influencing clinical outcomes. These findings position EVs-hsa_circ_0016866 as a promising therapeutic target for colorectal neoplasia. In addition, as previously described[25,49,58-60], EVs-circRNAs[61-65] can promote or inhibit CRC chemoresistance through diverse mechanisms, proposing these EVs-circRNAs as biomarkers for assessing the sensitivity of CRC chemotherapeutic agents[66]. Consequently, EVs-circRNAs represent emerging therapeutic opportunities for developing novel strategies against CRC.

Biomarkers for progression and prognostic assessment of CRC

At present, circRNAs are not as well-studied as miRNAs and long non-coding RNAs, and their functions remain largely unknown. Several EVs-circRNAs demonstrate functional significance in CRC development, suggesting their utility as biomarkers for monitoring disease progression and predicting patient outcomes. Shang et al[27] discovered the oncogenic impact of EVs-circPACRGL on CRC proliferation and metastasis. CircPACRGL expression was upregulated and inhibited miR-142-3p and miR-506-3p through sponging, thus deregulating their inhibition of TGF-β1, leading to increased TGF-β1 expression, and promoting the differentiation of N2 neutrophils, which have protumorigenic properties, thereby promoting CRC progression. EVs isolated from CRC blood and cell samples contain abundant circ_001860, which subsequently transmits to recipient cancer cells via EVs delivery. Huang et al[74] discovered that circ_001860 upregulates Zinc finger E-box binding homeobox 1 (ZEB1) by binding to miR-582-5p, thereby facilitating tumor development in vivo and triggering CRC cell proliferation and metastasis in vitro; thus, EVs-circ_001860 accelerated CRC progression via the miR-582-5p/ZEB1 axis. Jiang et al[75] demonstrated that circ_100876 undergoes encapsulation within CRC cellular EVs for extracellular transport, subsequently influencing tumor progression via modulation of the miR-1224-5p/forkhead box protein M1 signaling cascade. Yang et al[26] found that has_circ_0010522 was enriched in plasma EVs from CRC patients. This circRNA exhibited elevated expression, correlating with advancing disease stages and hypoxic cellular conditions. Furthermore, hypoxia-induced EVs-has_circ_0010522 facilitated tumor dissemination through regulation of miR-133a/guanine nucleotide exchange factor-H1/RhoA signaling pathway. Consequently, EVs-hsa_circ_0010522 emerges as a promising molecular indicator for monitoring colorectal malignancy progression. In the progression of CRC liver metastases, the regulatory role of EVs-has_circ_0085159 is crucial. Through enhancing HMGA2 and BMP4/ADAM19 expression, this circRNA emerges as a potential biomarker and therapeutic candidate against metastasis in CRC[76]. Miao et al[77] demonstrated that EVs-hsa_circ_0081069 plays an oncogenic role in CRC development. This circRNA enhances LIM and SH3 protein 1 (LASP1) expression by adsorbing miR-665, thereby influencing the CRC phenotype and promoting progression through the miR-665/LASP1 signaling pathway. Chen et al[78] showed that hsa_circ_0007444, a tumor-suppressive circRNA secreted by EVs, is linked to poor prognosis. Overexpression of hsa_circ_0007444 inhibits CRC cell proliferation, invasion, and migration. Meng et al[79] demonstrated that hsa_circ_0006906 modulates the miR-92a-1-5p/insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) pathway, promoting the interaction between polypyrimidine tract-binding protein 1 and IGF2BP1, thus accelerating CRC progression. Additionally, EVs-hsa_ circ_0006906 has the potential to serve as a biomarker for CRC prognosis, warranting further therapeutic research. Therefore, EVs-circRNA functions as biomarkers for CRC progression and prognosis, demonstrating promising potential for clinical applications.

CONCLUSION

Chemoresistance is a central challenge in the clinical management of CRC, and the dual role exhibited by EVs-circRNAs as key mediators of resistance regulation suggests their potential as therapeutic targets. Despite the promising role of EVs-circRNAs in biomarker development and targeted intervention, current research still faces significant limitations: (1) The gap between basic research and clinical translation needs to be bridged; (2) The inefficiency and lack of standardization of EVs isolation techniques have led to significant heterogeneity of results; and (3) The functional ambivalence and organ-specificity of circRNAs have further limited the generalization of their clinical applications. To break through the bottleneck, we need to focus on multi-dimensional innovations in the future: (1) Technological innovation: Develop high-purity EVs isolation technology, combined with single-cell sequencing and spatial transcriptome to analyze the temporal and spatial dynamic network of circRNAs; (2) Mechanism expansion: Simulate the dynamic delivery of circRNAs with the use of the patient-derived xenograft model or the three-dimensional organoid co-culture system and identify key drug-resistant targets through clustered regularly interspaced short palindromic repeats screening; (3) Extension of research dimension: Breaking through the framework of chemotherapy resistance and exploring the role of EVs-circRNAs in drug resistance to radiotherapy, immunotherapy, and targeted therapy; and (4) Clinical translation advancement: Carrying out multi-center large sample cohort to validate the sensitivity and specificity of markers, with a focus on enrolling CRC patients across diverse disease stages and resistance phenotypes, integrated with multi-omics data analysis, designing engineered EVs drug delivery systems, and promoting interdisciplinary cooperation to optimize precision treatment strategies. EVs-circRNA research is moving from mechanistic analysis to clinical translation, and its dynamic monitoring and targeted intervention potential provide a new perspective for overcoming CRC drug resistance. Despite the challenges, through technological innovation and multidimensional validation, EVs-circRNA is expected to become an important tool for individualized precision medicine, which will ultimately improve patient outcomes and drive a paradigm shift in CRC treatment.