Exosomal MicroRNAs in Cancer: Mechanisms, Clinical Applications, and Challenges in Biomarker Discovery

1. Introduction

Cancer remains a major global health challenge, with millions of new cases diagnosed annually. Early detection, accurate diagnosis, and personalized treatment strategies are paramount to improving survival rates and reducing the burden of the disease. In this context, biomarkers play a crucial role in monitoring the disease's onset, progression, metastasis, and therapeutic responses. Conventional biomarkers such as serum proteins, imaging techniques, and tissue biopsies, although useful, present limitations in terms of invasiveness, sensitivity, specificity, and accessibility [1]. Moreover, many of these biomarkers lack the ability to detect cancer at early stages, which is when treatment has the most profound impact on survival [2].

Exosomes are emerging as a potential solution to these challenges. These extracellular vesicles (EVs), typically ranging from 30 to 150 nm in diameter, are secreted by a variety of cells and contain a diverse cargo of proteins, lipids, mRNAs, and microRNAs (miRNAs) [3]. Exosomes facilitate intercellular communication, and their ability to transfer molecular signals between cells has made them a subject of great interest in cancer biology. Of particular importance are the miRNAs carried by exosomes, which are stable and detectable in body fluids like blood, urine, and saliva [4,5].

MiRNAs, small non-coding RNAs of 20–22 nucleotides, regulate gene expression at the post-transcriptional level, and dysregulation of miRNA expression is a hallmark of cancer [6,7]. Exosomal miRNAs are particularly promising as biomarkers because they can reflect the molecular characteristics of their cells of origin, including tumor cells, and thus provide insights into tumor progression and metastasis. Moreover, exosomal miRNAs can be detected using minimally invasive liquid biopsy techniques, providing a novel, non-invasive alternative for cancer diagnosis and prognosis [8]. This review highlights the biogenesis, functional roles, and clinical potential of exosomal miRNAs as cancer biomarkers, emphasizing recent developments, challenges, and future directions in the field.

2. Exosomal microRNA: Characteristics and Biogenesis

Exosomes are secreted by virtually all cell types, and their cargo includes miRNAs, mRNAs, proteins, and lipids, which collectively reflect the cellular state of the donor cell [9]. Exosomal miRNAs are particularly valuable due to their stability in circulation. Unlike free-floating miRNAs, which are prone to degradation by RNases, miRNAs encapsulated in exosomes are protected by the lipid bilayer membrane, ensuring their integrity even in the bloodstream [10].

The biogenesis of exosomes begins with the inward budding of the plasma membrane, forming early endosomes. As these endosomes mature into multivesicular bodies (MVBs), some of the vesicles are trafficked to the cell surface where they fuse with the membrane, releasing exosomes into the extracellular space [11]. The packaging of miRNAs into exosomes is a highly regulated process, involving a complex interplay of RNA-binding proteins and sorting machineries such as the ESCRT (endosomal sorting complex required for transport) pathway and ceramide, which facilitate the sorting of miRNAs into exosomes [12].

Specific RNA-binding proteins such as heterogeneous nuclear ribonucleoproteins (hnRNPs), Tsg101, and YBX1 have been implicated in the selective loading of miRNAs into exosomes [13]. In the case of cancer, tumor-derived exosomes can carry cancer-specific miRNAs, which influence not only the recipient tumor cells but also the surrounding tumor microenvironment (TME) and immune cells. These exosomal miRNAs can act as signaling molecules that modulate key cancer processes, including tumor proliferation, migration, immune escape, and resistance to therapy [14,15].

3. Exosomal microRNAs in Cancer Biology

Exosomal miRNAs play a pivotal role in the pathophysiology of cancer by mediating communication between cancer cells and their microenvironment. Tumor cells can release exosomes that influence both nearby stromal cells and distant organs, contributing to tumor progression, metastasis, and immune modulation [16]. The specific miRNA profiles of exosomes reflect the molecular state of the tumor, enabling exosomal miRNAs to function as molecular signatures of cancer.

• Tumor Progression and Metastasis: Exosomal miRNAs are involved in regulating tumor growth, angiogenesis, and metastasis. For instance, miR-21 is widely recognized as an oncogenic miRNA that is upregulated in many cancers, including breast, colorectal, and lung cancer. MiR-21 promotes tumor growth by targeting tumor suppressor genes like PTEN and PDCD4, and it also plays a role in promoting cancer cell invasion and metastasis [17]. Similarly, miR-10b has been shown to facilitate metastasis by regulating the RhoGTPases and promoting the epithelial-mesenchymal transition (EMT) process, which is crucial for cancer cell dissemination [18].
• Immune Modulation: Exosomes can also influence the immune response to tumors. Tumor-derived exosomal miRNAs can modulate immune cell functions to create an immunosuppressive microenvironment. For example, exosomal miR-155 has been implicated in suppressing dendritic cell activation and promoting regulatory T cell differentiation, thereby inhibiting anti-tumor immune responses [19]. Additionally, exosomes from cancer cells can carry miRNAs that suppress natural killer (NK) cell function, thus enhancing tumor evasion from immune surveillance [20].
• Therapeutic Resistance: Resistance to chemotherapy and targeted therapy is a significant barrier to successful cancer treatment. Exosomal miRNAs have been shown to contribute to therapeutic resistance by modulating drug response pathways. For example, miR-214 in exosomes can promote resistance to chemotherapy by targeting genes involved in apoptosis, such as PTEN and caspase-3 [21]. Similarly, exosomal miRNAs can mediate resistance to targeted therapies, including those aimed at EGFR or HER2, by altering downstream signaling pathways [22].

To illustrate the diversity of exosomal miRNA profiles across various cancers, Table 1 summarizes key miRNAs identified in different cancer types and their functional roles in tumor progression and metastasis.

To further understand how exosomal miRNAs contribute to cancer biology, Table 2 outlines the mechanisms by which specific miRNAs influence critical cancer processes, including tumor growth, metastasis, and immune modulation.

4. Exosomal microRNAs as Cancer Biomarkers

Exosomal miRNAs represent a promising class of biomarkers for early cancer detection, prognosis, and monitoring of therapeutic responses. The ability to detect specific miRNAs in biofluids such as blood, urine, and saliva offers a non-invasive alternative to traditional tissue biopsies, which are often invasive and not feasible for regular monitoring [23].

• Diagnostic Potential: Exosomal miRNAs have shown diagnostic potential in various cancers, including breast, prostate, lung, and pancreatic cancers. In breast cancer, exosomal miR-21 has been proposed as a diagnostic biomarker due to its elevated levels in the plasma of cancer patients [24]. Similarly, miR-155 has been identified as a potential biomarker for early detection of non-small cell lung cancer (NSCLC) [25]. Liquid biopsies based on exosomal miRNA profiling could provide a less invasive and more frequent method for cancer detection, enabling early intervention and improved patient outcomes.
• Prognostic Value: Exosomal miRNAs have been shown to correlate with cancer stage, progression, and metastasis. For example, high levels of miR-210 in exosomes are associated with poor prognosis in colorectal cancer [26]. Additionally, exosomal miRNA signatures may provide insights into cancer recurrence and the likelihood of metastasis, helping to stratify patients into risk groups and guide therapeutic decision-making [27].
• Therapeutic Monitoring: One of the most promising applications of exosomal miRNAs is in monitoring treatment responses. Exosomal miRNAs can serve as real-time biomarkers for assessing how a patient is responding to therapy. For instance, changes in the levels of specific exosomal miRNAs have been used to monitor chemotherapy efficacy and identify emerging resistance [28]. Furthermore, exosomal miRNAs can reflect the molecular changes occurring within the tumor during treatment, offering a window into the dynamics of cancer evolution and therapy-induced alterations [29].

The clinical potential of exosomal miRNAs in cancer detection, prognosis, and therapeutic monitoring is increasingly recognized. Table 3 summarizes their diverse applications, showcasing how exosomal miRNA profiles can aid in early diagnosis and patient management.

5. Technologies for Exosomal miRNA Profiling

A major challenge in utilizing exosomal miRNAs as biomarkers is the efficient and reliable isolation and characterization of exosomes from biological fluids. Several technologies have been developed for the isolation and profiling of exosomal miRNAs, each with its advantages and limitations.

• Exosome Isolation: Techniques such as ultracentrifugation, precipitation methods, and size-exclusion chromatography are commonly used to isolate exosomes. Ultracentrifugation is the most widely used method; however, it is time-consuming and prone to contamination with non-exosomal particles [30]. Recently, microfluidic platforms have been developed to isolate exosomes with high efficiency and reproducibility, enabling faster and more scalable processes [31]. These newer techniques are promising for clinical applications.
• MiRNA Profiling: Once exosomes are isolated, profiling their miRNA content is essential for biomarker discovery. High-throughput sequencing, RT-qPCR, and microarray-based techniques are commonly used to identify and quantify miRNAs. Sequencing offers the most comprehensive profile, but it is also expensive and data-intensive [32]. RT-qPCR, while more cost-effective, requires prior knowledge of the specific miRNAs of interest and may miss novel biomarkers [33]. The development of highly sensitive and accurate profiling technologies is essential for advancing exosomal miRNAs as reliable biomarkers.

As the accurate profiling of exosomal miRNAs is crucial for their potential as cancer biomarkers, Table 4 provides an overview of the most commonly used techniques for exosome isolation and miRNA profiling, highlighting their advantages and limitations.

Efficient exosomal miRNA profiling relies on the development of advanced technologies. Table 5 compares the different methods available for liquid biopsy-based profiling, offering insights into their strengths and limitations for clinical applications."

6. Challenges and Future Directions

Despite their promise, the use of exosomal miRNAs as cancer biomarkers faces several challenges that need to be addressed for their clinical translation.

• Standardization and Reproducibility: One of the major challenges is the lack of standardization in exosome isolation and miRNA profiling protocols. Variability in isolation methods, differences in sample handling, and data processing inconsistencies can lead to discrepancies in results across studies [34]. Standardized methods and quality control measures are essential for ensuring reproducibility and accuracy.
• Biological Variability: Another challenge is the biological variability of exosomal miRNAs. MiRNA expression levels can vary based on tumor type, stage, and even between patients with the same type of cancer [35]. Large-scale clinical studies are needed to validate the potential of exosomal miRNAs in different patient cohorts and cancer types.
• Clinical Validation: Despite the promising preclinical data, clinical validation remains the most significant barrier to the widespread use of exosomal miRNAs in the clinic. Large multicenter trials are necessary to demonstrate the diagnostic, prognostic, and therapeutic utility of exosomal miRNAs across diverse cancer populations [36]. These studies will provide the robust evidence needed to incorporate exosomal miRNA profiling into clinical practice.

Despite the promising clinical applications of exosomal miRNAs, several challenges remain in their widespread use. Table 6 summarizes the key obstacles that need to be addressed for the successful clinical translation of exosomal miRNAs as reliable biomarkers.