Functions and mechanisms of exosomal lncRNAs in esophageal cancer

Abstract

Esophageal cancer is a prevalent and highly aggressive malignancy with poor clinical outcomes, largely due to late diagnosis and limited effective biomarkers. Improved understanding of its molecular mechanisms and the identification of reliable non-invasive diagnostic indicators are therefore of critical importance. Exosomes are small double-membrane extracellular vesicles present in various biological fluids that mediate intercellular communication through the transfer of bioactive cargo, including proteins and RNAs. Among these cargos, long non-coding RNAs (lncRNAs) packaged within exosomes have emerged as key regulators of tumor initiation, progression, metastasis, and therapeutic resistance. Increasing evidence indicates that exosomal lncRNAs play crucial roles in esophageal cancer by modulating cell proliferation, migration, invasiveness, angiogenesis, and response to treatment, while also exhibiting high stability in circulation, making them promising diagnostic and prognostic biomarkers. This review summarizes recent advances in the biological functions, clinical relevance, and translational potential of exosomal lncRNAs in esophageal cancer, highlighting their emerging value in liquid biopsy and targeted therapeutic strategies.

Introduction

Esophageal cancer (EC) stands out as one of the most aggressive types of gastrointestinal cancers, originating from the epithelial cells of the esophageal. This disease poses a significant threat to global health and life [1]. As one of the most formidable tumors within the gastrointestinal system, EC is associated with high mortality rates and low survival outcomes [2]. Recent data from the International Agency for Research on Cancer indicates that there are around 572,000 new EC cases and approximately 509,000 related deaths, placing it seventh and sixth among the 36 most prevalent cancers, respectively [3].

Histologically, EC is categorized into two primary types: (1) Esophageal squamous cell carcinoma (ESCC) and and (2) esophageal adenocarcinoma (EAC). ESCC recognized as one of the most aggressive human cancers globally and the second most prevalent in China [4], ESCC typically affecting the middle section of the esophageal. EAC which generally impacts the gastroesophageal junction and cardia [5, 6]. Despite significant efforts from medical professionals and researchers to enhance treatment options for EC patients in recent years, the incidence of this cancer continues to rise, and patient prognosis remains poor. Currently, there are no effective treatments available for those with advanced stages of the disease [7, 8]. Early detection and prompt intervention are crucial for improving outcomes in EC; however, many patients are diagnosed at later stages due to the absence of distinct early symptoms.

There is a notable lack of effective screening and diagnostic methods for identifying EC in its initial stages [8], even though early detection can greatly lower mortality rates associated with this cancer. One promising approach for early diagnosis and prognosis prediction is liquid biopsy, which allows for non-invasive, sensitive, and rapid identification of molecular biomarkers linked to tumors [9]. In liquid biopsy procedures, extracellular vesicles (EVs) are a primary focus [10, 11]. These vesicles are categorized into exosomes, microvesicles (MVs), and apoptotic bodies based on their size, production mechanisms, and destinations. Exosomes were first identified in serum in 1983 [12]. They are nano-sized vesicles (ranging from 30 to 150 nm) formed when parts of the plasma membrane invaginate into the cell [13]. Exosomes are secreted by various cell types and carry a diverse array of macromolecules [14–17]. For the study of exosomes in both experimental and clinical settings, reliable isolation and characterization methods are essential. Commonly used approaches for exosome isolation include differential ultracentrifugation, size-based filtration, density gradient centrifugation, polymer-based precipitation kits, and immunoaffinity capture techniques, each with distinct advantages and limitations in terms of yield, purity, and scalability. Following isolation, exosome identification is typically performed using a combination of morphological, molecular, and biophysical techniques. Transmission electron microscopy (TEM) is widely applied to visualize vesicle morphology and size, while nanoparticle tracking analysis (NTA) or dynamic light scattering (DLS) are used to assess size distribution and particle concentration [18, 19]. In addition, western blotting or flow cytometry is commonly employed to detect exosomal surface markers such as CD9, CD63, and CD81, ensuring the authenticity of isolated vesicles [20]. These standardized strategies provide the technical foundation for subsequent analyses of exosomal cargo, including lncRNAs, in esophageal cancer research.

Previous research has established that exosomes serve as crucial mediators for communication between cells, facilitating the exchange of materials [21, 22]. Recent investigations highlight the significant role of exosomes in multiple phases of tumor progression and development, including tumor proliferation, angiogenesis, epithelial-mesenchymal transition (EMT), migration, microenvironment remodeling, and resistance to treatment [23–26]. Numerous studies indicate that the concentration of circulating exosomes (CEs) is markedly elevated in patients with esophageal squamous cell carcinoma (ESCC). Furthermore, these studies reveal that patients exhibiting higher levels of plasma exosomes tend to have a better overall survival rate compared to those with lower levels. Conversely, low exosome levels are associated with poor prognosis [27, 28]. Given their stable nature and the diverse components they carry, exosomes hold promise as valuable biomarkers for diagnosis and prognosis, as well as effective vehicles for targeted drug and biomolecule delivery. Exosomes derived from esophageal cancer can serve as highly efficient biomarkers for early detection, prognosis, and treatment response [25].

Among the various components found in exosomes are long non-coding RNAs (lncRNAs). These lncRNAs are a category of mature RNA molecules exceeding 200 nucleotides in length and lack the capability to encode proteins [29–31]. They play a significant role in the pathogenesis of many cancers, influencing processes such as proliferation [32–34], apoptosis [35], metastasis [36], and metabolism [37]. A study by Tian et al. demonstrated that lncRNAs isolated from exosomes of ESCC cells (specifically PCAT1) were present at elevated levels in the serum of ESCC patients compared to a control group, suggesting that PCAT1 could function as a non-invasive biomarker for ESCC [38]. Other research has also shown that the levels of plasma exosomal lncRNAs are significantly higher in individuals with esophageal cancer than in healthy individuals, with a notable decrease in these lncRNAs following surgical intervention. Consequently, an exosomal lncRNA panel can facilitate accurate diagnosis of esophageal tumors, particularly in the disease’s early stages [39–41]. Timely diagnosis of EC is crucial for initiating prompt treatment in many patients, as delays can lead to disease progression. While non-invasive methods are increasingly used as complementary diagnostic tools for various cancer types [42], a single biomarker with optimal sensitivity and specificity for diagnosing EC is yet to be identified.

This review summarizes findings from recent studies exploring the potential of exosomes found in blood samples from EC patients. The focus is on exosomal lncRNAs as diagnostic, prognostic, and predictive biomarkers in the context of esophageal cancer.

Molecular background: lncRNAs and exosomes in esophageal cancer

Long non-coding RNAs (lncRNAs) are a class of RNA transcripts longer than 200 nucleotides that lack protein-coding capacity but play critical regulatory roles in gene expression at transcriptional, post-transcriptional, and epigenetic levels [43–46]. Initially considered transcriptional noise, lncRNAs are now recognized as key modulators of diverse biological processes, including chromatin remodeling, transcriptional control, cell cycle regulation, differentiation, and apoptosis [47–49] (Fig. 1). Aberrant lncRNA expression has been widely implicated in cancer development and progression, where it contributes to malignant phenotypes such as sustained proliferative signaling, enhanced invasion and metastasis, resistance to apoptosis, and therapeutic failure [50–53]. In esophageal cancer (EC), several dysregulated lncRNAs have been shown to influence tumor behavior by modulating oncogenes, tumor suppressor genes, and microRNA networks, thereby reshaping critical signaling pathways involved in disease progression [54–57].

Fig. 1.

Comprehensive Overview of long non-coding RNAs (lncRNAs) characteristics, functions, and clinical significance. This diagram illustrates the multi-faceted landscape of lncRNAs, highlighting that while only 2% of the genome codes for proteins, lncRNAs represent a significant portion of the non-coding transcriptome. These transcripts lack an open reading frame (ORF) and are categorized based on their genomic location, such as intergenic, intronic, or antisense types. Functionally, lncRNAs regulate gene expression and participate in crucial cellular processes like chromatin remodeling and cell cycle control. In the context of oncology, their dysregulation influences tumor proliferation and drug resistance, acting through mechanisms such as microRNA competition and epigenetic modification. Consequently, specific lncRNAs like SNHG7 and CCAT1 are emerging as stable, high-specificity biomarkers for early cancer detection and clinical applications

Exosomes are nano-sized extracellular vesicles, typically ranging from 30 to 150 nm in diameter, that are released by most cell types and function as essential mediators of intercellular communication [58–62]. By encapsulating proteins, lipids, and nucleic acids, exosomes facilitate the horizontal transfer of molecular information between cells through membrane fusion or endocytic uptake. Importantly, the lipid bilayer of exosomes protects their cargo from enzymatic degradation, conferring exceptional stability in circulation and other biological fluids [63]. In cancer, tumor-derived exosomes actively participate in shaping the tumor microenvironment by promoting angiogenesis, facilitating immune escape, remodeling stromal components, and establishing pre-metastatic niches that support tumor dissemination [23, 64].

A growing body of evidence indicates that lncRNAs are selectively enriched and packaged into exosomes, rather than being passively released, suggesting a regulated sorting mechanism that reflects the biological state of the originating tumor cells [65]. Once delivered to recipient cells, exosomal lncRNAs exert functional effects through multiple mechanisms. These include acting as competing endogenous RNAs (ceRNAs) that sequester microRNAs and derepress downstream targets, forming RNA–RNA duplexes that suppress gene expression, and interacting with transcriptional or epigenetic regulators to alter gene expression programs [66–68]. Through these processes, exosomal lncRNAs extend oncogenic signaling beyond the primary tumor and contribute to systemic disease progression and intercellular crosstalk within the tumor microenvironment [69, 70].

In the context of EC, exosomal lncRNAs have been increasingly recognized as key regulators of tumor growth, invasion, and metastatic potential (Fig. 2). For example Exosomal lncRNA AFAP1-AS1 derived from tumor-associated macrophages enhances the migratory and invasive capacities of EC cells by influencing transcriptional regulators and downstream signaling cascades [71]. Collectively, these findings highlight that exosomal lncRNAs integrate tumor-intrinsic and microenvironmental signals, thereby amplifying malignant phenotypes and facilitating disease progression [72].

Fig. 2.

Mechanism of exosomal lncRNAs in esophageal cancer (EC) progression. Schematic overview of lncRNA trafficking via exosomes in the EC microenvironment. Packaging of nuclear-derived lncRNAs into multi-vesicular bodies (MVBs) and subsequent release as exosomes from EC cells. Exosomal encapsulation provides molecular stability and protection against degradation. Functional delivery to recipient cells, where lncRNAs modulate gene expression through ceRNA activity, epigenetic regulation, and protein interactions. These pathways collectively drive malignant phenotypes such as enhanced invasion and metastasis

Importantly, the combination of cancer-associated expression patterns, functional relevance, and the remarkable stability of exosomal lncRNAs in biological fluids provides a strong biological rationale for exploring them as non-invasive molecular indicators in esophageal cancer. Mechanistically, the biological functions of exosomal lncRNAs in esophageal cancer can be conceptualized within an integrated regulatory framework rather than as isolated pathways. A central mechanism is competing endogenous RNA (ceRNA) activity, whereby exosomal lncRNAs sequester microRNAs and thereby modulate downstream oncogenic and tumor-suppressive gene networks in recipient cells. In parallel, exosomal lncRNAs contribute to immune modulation within the tumor microenvironment by influencing immune cell polarization, cytokine signaling, and immune evasion, particularly through interactions with tumor-associated macrophages and other stromal cells. Beyond these mechanisms, exosomal lncRNAs also participate in epigenetic and transcriptional regulation by interacting with chromatin modifiers and transcription factors. Importantly, these mechanistic axes are highly interconnected and collectively reinforce tumor progression, invasion, and metastatic dissemination in esophageal cancer. Together, these integrated mechanistic insights form the foundation for evaluating exosomal lncRNAs as diagnostic and prognostic biomarkers, which is discussed in detail in the following section.

Exosomal lncRNAs as esophageal cancer biomarkers

Esophageal cancer ranks as the sixth most lethal malignancy globally, contributing significantly to cancer-related mortality. The majority of cases are found in underprivileged regions where patients often experience late diagnoses or face distant metastasis following initial detection [73]. The prognosis for this disease is poor, with a five-year survival rate hovering around 35%. Common symptoms among patients include difficulty swallowing (dysphagia) and weight loss, while less frequent symptoms may involve painful swallowing (odynophagia), hoarseness, cough, chest pain, and melena. Notably, early-stage esophageal cancer typically lacks specific indicators [74, 75].

Currently, various diagnostic methods are employed, including computed tomography (CT), magnetic resonance imaging (MRI), ultrasound imaging, and endoscopy, with endoscopic biopsy being the gold standard for pathological diagnosis. Treatment strategies for esophageal cancer are closely linked to the clinical stage of the disease, which is determined through thorough clinical evaluations. Various detection methods have been utilized to identify potential biomarkers, such as immunohistochemistry, blood-based assays, mRNA profiling, gene expression analysis, and biopsies. Contemporary research efforts are focused on identifying unique molecular tumor biomarkers—such as circulating tumor cells, circulating tumor DNA/RNA, epigenetic markers, and exosomes—to enhance the prognosis, diagnosis, and treatment of esophageal cancer, while also addressing the limitations of current biomarkers [76].

In addition to mRNA-based biomarkers like miRNA, long noncoding RNAs (lncRNAs) are emerging as valuable biomarkers that can be easily extracted from serum and tissue samples [77, 78]. Recent studies have identified several lncRNAs in serum that serve as diagnostic or prognostic indicators, originating from tumor tissue exosomes [79, 80]. This comprehensive review summarizes approximately 20 lncRNAs from both tissue and serum samples of patients with esophageal cancer in Table 1.

Table 1.

Summary of reported exosomal lncRNAs associated with esophageal cancer

lncRNA	Clinical relevance	Sample source	Study model	Patient cohort	Regulation	Diagnostic accuracy	Combination with biomarkers	Key functional role	Exosome isolation & characterization	Detection method	Stability reported	Refs.
PCAT1	Diagnostic/prognostic	Serum/tissue	Human cohort	39 paired ESCC vs. normal tissues	↑	Not reported	miR-326	Promotes ESCC proliferation via miR-326 sponging	Differential centrifugation + TEM	RT-qPCR	Not reported	[85]
FAM225A	Therapeutic	Tissue	Human cohort	30 paired ESCC tissues	↑	Not reported	miR-206/NETO2/FOXP1	Promotes angiogenesis and tumor progression	ExoQuick kit + WB	RT-qPCR	Not reported	[86]
FMR1-AS1	Diagnostic/prognostic (female ESCC)	Tissue	Human cohort	179 ESCC patients	↑	Not reported	Not reported	Activates TLR7–NF-κB signaling in CSCs	Not reported	RT-qPCR/FACS	RNase/Triton-X validated	[87]
ZFAS1	Diagnostic/therapeutic	Tissue	Human cohort	136 ESCC, 64 esophagitis, 69 healthy	↑	Not reported	miR-124/miR-143	Promotes proliferation and invasion via STAT3	Kit-based + TEM + WB	Fluorescent staining	Not reported	[88]
RP11-465B22.8	Therapeutic	Tissue	Human cohort	26 EC patients	↑	Not reported	miR-765	Enhances tumorigenesis via KLK4 axis	Ultracentrifugation + TEM	PKH67 labeling	Not reported	[89]
NR_039819 et al.	Diagnostic	Plasma	Human cohort	317 ESCC, 64 esophagitis, 69 controls	↑	Not reported	Not reported	Associated with ESCC development	Isolation kit + NTA + WB	WB	Not reported	[90]
LINC01711	Diagnostic/monitoring	Tissue	Human cohort	137 paired samples	↑	Not reported	miR-326/FSCN1	Promotes proliferation and invasion	Ultracentrifugation + TEM	RT-qPCR	Not reported	[91]
UCA1	Diagnostic	Plasma/Tissue	Human cohort	30 ESCC plasma, 15 paired tissues	↓	Sens: 86.7%/Spec: 70.2%	miR-613	Inhibits tumor progression	Ultracentrifugation + TEM + NTA	RT-qPCR/WB	Freeze–thaw stable	[92]
PART1	Diagnostic	Plasma	Human cohort	79 ESCC (gefitinib-treated)	↑	Sens: 78.6%/Spec: 86.5%	miR-129	Induces gefitinib resistance	ExoQuick kit + TEM + WB	RT-qPCR	pH/RNase stable	[93]
POU3F3	Diagnostic	Plasma	Human cohort	147 ESCC patients	↑	Sens: 72.8%/Spec: 89.4%	SCCA	Enhances early ESCC detection	Not reported	RT-qPCR	Not reported	[94]
RP5-1092A11	Diagnostic	Tissue	Human cohort	6 ESCC/6 esophagitis/6 controls	↑	Not reported	Not reported	Promotes ESCC proliferation	Kit-based + TEM + WB	RNA-seq/RT-qPCR	Not reported	[38]
MEG3	Therapeutic	Blood/Tissue	Human cohort	28 paired samples	↓	Not reported	miR-149-3p	Suppresses angiogenesis and immune escape	Not reported	RT-PCR/WB	Not reported	[95]
MALAT1	Prognostic	Tissue	Human cohort	132 EC, 50 controls	↑	Not reported	Not reported	Associated with metastasis	Not reported	RT-qPCR	Not reported	[96]
HOTAIR	Diagnostic	Blood	Human cohort	50 ESCC, 20 controls	↑	Not reported	Not reported	Promotes malignant phenotype	Not reported	RT-qPCR	Not reported	[97]

While not all studies detail exosome isolation and identification techniques, many frequently utilize isolation kits. Transmission electron microscopy (TEM) and western blotting are commonly employed for exosome identification, whereas RT-qPCR is often used for detection [81, 82].

The lncRNAs encapsulated in exosomes are outlined in Table 1, which categorizes them into three classes: diagnosis, prognosis, and treatment. Among the 20 lncRNAs listed, FMR1-AS1 is highlighted as a specific marker for diagnosing and prognosing female patients with esophageal squamous cell carcinoma (ESCC).

According to the literature reviewed, the diagnostic accuracy for UCA1, PART1, and Pou3F3 lncRNAs exceeds 70% for both sensitivity and specificity [83]. The primary sources of lncRNAs are generally limited to blood or tissue; however, UCA1 has been identified in both plasma and tissue samples [84]. Data presented in Table 1 indicate that most exosomal lncRNAs associated with esophageal cancer are upregulated in patient tissues or serum, with the exception of UCA1 and Meg3, which are downregulated.

Currently, numerous tumor markers—including SCCA, CEA, CA19-9, IL-6, CYFRA 21 − 1, M-CSF, DKK-1, MMP-9, MiR-18a, MiR-1246, among others—have been assessed for diagnosing ESCC but have proven insufficient for conclusive diagnosis. Notably, POU3F3 has been linked to a specific antigen known as serum squamous cell carcinoma antigen (SCCA), demonstrating enhanced efficacy for early tumor screening and detection of ESCC.

In general, these exosomal lncRNAs have been identified as promising biomarkers for esophageal squamous cell carcinoma (ESCC). However, further research is required to validate their sensitivity and specificity as reliable exosomal biomarkers. Despite the encouraging diagnostic and prognostic potential of exosomal lncRNAs in esophageal cancer, several critical limitations should be considered when interpreting current findings. Substantial heterogeneity exists among studies in terms of patient cohorts, sample sources (plasma versus tissue), exosome isolation techniques, and lncRNA detection platforms, which complicates direct comparison across reports. Moreover, most available studies are retrospective and include relatively small sample sizes, frequently lacking independent validation cohorts. The absence of standardized protocols for exosome purification and lncRNA quantification further limits reproducibility and hinders clinical translation. Collectively, these observations suggest that although individual exosomal lncRNAs demonstrate promising performance, their greatest clinical utility is likely to be achieved through multi-lncRNA panels integrated with conventional biomarkers and clinical parameters, pending confirmation in large-scale, prospective, multicenter studies. It should be noted that several exosomal lncRNAs summarized in Table 1 have been reported based on studies with relatively small patient cohorts. Compared with clinically established biomarkers, exosomal lncRNAs present several conceptual advantages, including improved molecular stability in circulation and closer reflection of tumor-specific alterations. Conventional serum markers often show limited sensitivity and specificity, particularly in early-stage disease, whereas exosomal lncRNAs may offer enhanced discriminatory potential. However, unlike traditional biomarkers that are already standardized and routinely used in clinical practice, most exosomal lncRNAs are still under exploratory investigation, with heterogeneous methodologies and insufficient large-scale validation. Therefore, exosomal lncRNAs are currently better positioned as complementary biomarkers rather than standalone replacements for existing clinical markers. Although these findings provide initial evidence supporting the biomarker potential of exosomal lncRNAs in esophageal cancer, limited sample sizes may reduce statistical power and restrict the generalizability of the results. Therefore, further validation in larger, independent cohorts using standardized methodologies is required before their clinical application.

Conclusion

This review highlights the multifaceted roles of exosomal non-coding RNAs (ncRNAs) in the diagnosis, progression, metastasis, therapeutic resistance, and targeted delivery associated with esophageal cancer (EC). Accumulating evidence underscores the potential of exosomal ncRNAs—particularly long non-coding RNAs (lncRNAs)—as minimally invasive biomarkers and functional mediators of tumor–microenvironment communication, supporting their relevance in both diagnostic and therapeutic contexts.

Despite these promising findings, several critical challenges and knowledge gaps must be addressed to enable clinical translation. Key limitations include the lack of standardized protocols for exosome isolation, characterization, and ncRNA quantification, as well as substantial heterogeneity across study designs, patient cohorts, and analytical platforms. Moreover, diagnostic performance metrics—such as sensitivity, specificity, and ROC-based analyses—are not consistently reported for many candidate exosomal lncRNAs, limiting their clinical interpretability and comparative evaluation. The scarcity of large-scale, prospective clinical studies further restricts the validation of exosomal ncRNAs as reliable biomarkers or therapeutic targets.

Future research should prioritize the development of standardized and reproducible workflows for exosome isolation and analysis, alongside multicenter and longitudinal studies designed to systematically assess diagnostic accuracy. Integrative strategies combining exosomal lncRNA profiling with conventional clinical parameters and other liquid biopsy components—such as circulating tumor DNA and protein biomarkers—may improve diagnostic precision and prognostic stratification. In parallel, emerging technological advances, including single-exosome analysis, high-throughput sequencing, artificial intelligence–assisted data integration, and engineered exosome platforms, are expected to accelerate progress in this field.

Importantly, exosomal ncRNAs also represent promising therapeutic targets and delivery vehicles. The rational design of engineered exosomes for targeted ncRNA modulation or drug delivery holds potential for overcoming therapeutic resistance and enhancing treatment precision in EC. Collectively, advancing from descriptive discovery toward mechanistic understanding, standardized diagnostic evaluation, and rigorous clinical validation will be essential to fully realize the translational potential of exosomal ncRNAs and to integrate these biomarkers into future precision medicine strategies for esophageal cancer.

Author contributions

All authors contributed to the study conception, design, data collection, and written manuscript. All authors read and approved the final manuscript.

Funding

This work was financially supported by Abadan University of Medical Sciences, Abadan, Iran (Grant Number: 1403U-1991).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This study was reviewed and approved by the Biomedical Research Ethics Committee of Abadan University of Medical Sciences (Ethics Code: IR.ABADANUMS.REC.1403.169).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.