Introduction

Oral squamous cell carcinoma (OSCC) is the most common subtype of head and neck squamous cell carcinoma (HNSCC), primarily affecting the mucosa of the tongue, buccal cavity, gingiva, palate, and floor of the mouth.1 According to recent global statistics, approximately 389,485 new cases of lip and oral cavity cancer were diagnosed, and188230related deaths occurred in 2022.2 Despite significant advances in therapeutic interventions, the survival rate and quality of life for OSCC patients remain suboptimal.3,4 This can largely be attributed to the high rates of recurrence and distant metastasis associated with the disease.5,6 Furthermore, the 5-year survival rate for patients with advanced OSCC is typically less than 20%, in stark contrast to the 80% survival rate for those diagnosed at an early stage.7 Consequently, early detection and accurate diagnosis of OSCC are essential for enabling personalized, targeted, and timely treatment strategies that can significantly improve patient outcomes.

The diagnosis of OSCC is commonly based on general clinical examination,8 radiological imaging,9 and surgical biopsy,10 owing to the tumor’s typically superficial location. However, these diagnostic methods primarily identify clinically apparent symptoms, which often leads to a diagnosis at later stages, thereby resulting in a poorer prognosis. Specifically, these techniques struggle to detect subtle biomolecular changes in early-stage tumor tissues, leading to the missed opportunity for early diagnosis and intervention.11,12

Liquid biopsy has emerged as a novel, accurate, and minimally invasive strategy for cancer detection, enabling both early diagnosis and longitudinal monitoring by analyzing tumor-associated biomolecules in body fluids. These analytes include tumor-derived proteins, circulating tumor DNA, circulating tumor cells, and extracellular vesicles (EVs), particularly exosomes.13 While blood remains the most widely utilized medium in liquid biopsy studies,14 other biofluids—such as urine, cerebrospinal fluid, and saliva—are also gaining attention for their diagnostic potential. Saliva, in particular, has been employed in the diagnosis of a range of inflammatory, metabolic, and immune-related conditions.15,16 More recently, its relevance in cancer diagnostics—especially for oral malignancies—has come under increasing focus.17 Notably, for OSCC, saliva may offer diagnostic performance comparable to, or even superior to, blood-based assays.18,19 Due to the close anatomical proximity of OSCC lesions to the oral cavity, tumor-derived components—including exfoliated cells, nucleic acids, cytokines, and EVs—can be directly secreted into saliva.20 Furthermore, saliva collection is entirely non-invasive, painless, and does not induce irritation or damage to tumor tissue, making it an ideal sampling method for repeated assessments and population-level screening.21 Compared to blood, saliva is easier to collect, more economical to store, and does not require anticoagulants or specialized preservation agents, further enhancing its feasibility in clinical and remote settings. However, the diagnostic performance of whole saliva is often hindered by the presence of enzymatic activity and heterogeneous contaminants, which can degrade nucleic acids and proteins, thereby limiting sensitivity and specificity. To circumvent these issues, growing attention has been directed toward salivary exosomes—a more stable and biochemically enriched subpopulation of EVs—as a promising source of OSCC-specific biomarkers.21

Exosomes are nanosized, membrane-bound vesicles (typically 40–160 nm in diameter) secreted by most cell types.22 They function as critical mediators of intercellular communication by transporting nucleic acids, proteins, lipids, and metabolites that reflect the physiological or pathological state of their cells of origin.23–25 Owing to their stability in biological fluids and their capacity to encapsulate disease-relevant cargo, exosomes have emerged as attractive candidates for non-invasive diagnostics and real-time disease monitoring. In recent years, saliva-derived exosomes have been extensively investigated for their utility in OSCC detection. These vesicles carry tumor-specific molecular signatures, and alterations in their concentration or content may serve as early indicators of disease onset and progression.26,27 For example, Nakamichi et al reported increased levels of exosomal marker protein Alix in the saliva of OSCC patients compared to healthy individuals.28 Winck et al employed label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics to demonstrate differential expression patterns in salivary EVs associated with immune responses in OSCC patients.29 Similarly, Langevin et al identified a diagnostic model based on the salivary exosomal miRNAs miR-10b and miR-486-5p, which were significantly altered in OSCC patients.30 Hofmann et al further profiled 119 overlapping miRNAs in plasma- and saliva-derived exosomes from HNSCC patients, some of which were significantly associated with disease-free survival.31 Collectively, these findings underscore the potential of salivary exosomes as a minimally invasive liquid biopsy platform for OSCC. Their molecular profiling can provide real-time insights into tumor biology, aid in early disease detection, and support personalized therapeutic decision-making.

In this review, we provide a comprehensive overview of the current advancements in the application of salivary exosomes for OSCC. We begin by describing the biogenesis and physicochemical properties of exosomes, followed by an evaluation of saliva collection protocols and isolation techniques tailored for diagnostic use. We then discuss recent progress in exosome detection and characterization technologies, encompassing both conventional analytical methods and emerging biosensor-based platforms. Special emphasis is placed on the molecular constituents of salivary exosomes—including nucleic acids, proteins, and lipids—and their potential utility as diagnostic and prognostic biomarkers in OSCC. Finally, we examine the clinical relevance, practical challenges, and future perspectives of salivary exosome-based liquid biopsy for early cancer detection and disease monitoring. We aim to poll and discuss the updated research evidence and perspectives on the collection, separation, and detection technologies of salivary exosomes, and to introduce several components in salivary exosomes with potential to serve as diagnostic biomarkers for OSCC. This work would serve as a timely and comprehensive resource for researchers and clinicians pursuing non-invasive, exosome-driven strategies for the early diagnosis and personalized management of OSCC.

Biogenesis and Sources of Salivary Exosomes Exosomes Biogenesis

Salivary exosomes hold great promise as diagnostic markers for oral squamous cell carcinoma, as they contain a wealth of specific biomarkers from the source cells. This characteristic is closely related to their biological formation mechanism. The biogenesis of exosomes is a dynamic and highly regulated process that remains difficult to observe directly (Figure 1). Exosomes can be formed either through direct outward budding from the plasma membrane (PM) or via a more complex endocytic pathway involving inward budding,32 and they can also be released by intracellular plasma membrane-connected compartments (IPMC).33 In the endocytosis pathway, the PM undergoes invagination to form early endosomes. These early endosomes can either recycle back to the PM—referred to as “recycling endosomes”—or progress through maturation by continuous inward budding, giving rise to numerous intraluminal vesicles (ILVs). Endosomes containing numerous ILVs are termed multivesicular bodies (MVBs).34 While the majority of MVBs fuse with lysosomes, resulting in degradation of their contents, a subset of MVBs enriched with specific molecules—such as the tetraspanin CD63, lysosomal-associated membrane proteins Lysosome-associated membrane glycoprotein 1 (LAMP1) and Lysosome-associated membrane glycoprotein 2 (LAMP2), or the major histocompatibility complex II (MHC-II)—can instead fuse with the PM.35 This fusion event facilitates the release of ILVs into the extracellular space, at which point they are defined as exosomes.

Figure 1 Biogenesis of Exosomes. Exosomes are small extracellular vesicles generated through multiple cellular pathways. They can originate from: (1) direct outward budding of the plasma membrane (PM); (2) inward budding of the PM, followed by maturation through the endocytic pathway; and (3) intracellular compartments (IPMCs) associated with the PM. In the canonical endocytic route, the plasma membrane invaginates to form early endosomes (EEs), which either recycle back to the PM or mature by inward budding to generate numerous intraluminal vesicles (ILVs), resulting in multivesicular bodies (MVBs). While most MVBs fuse with lysosomes for degradation, a subset—enriched in proteins such as CD63, lysosome-associated membrane glycoprotein 1/2 (LAMP1/2), and major histocompatibility complex II (MHC II)—fuses with the PM to release ILVs into the extracellular milieu as exosomes. Exosome formation is tightly regulated by the endosomal-sorting complex required for transport (ESCRT) machinery, although ESCRT-independent mechanisms also exist. For instance, CD63 can mediate protein sorting into ILVs independently of ESCRT, and the melanosome-specific protein Pre-melanosomal protein (Pmel17), along with certain lipids, can contribute to ILV biogenesis in an ESCRT-independent manner. The Rab GTPase family orchestrates the transport, docking, and fusion of MVBs with the PM. Once secreted, exosomes interact with recipient cells through surface receptor binding, direct membrane fusion, or endocytic uptake, thereby modulating downstream intracellular signaling pathways.

The biogenesis of exosomes is orchestrated by multiple protein complexes that regulate distinct steps of ILVs formation and MVBs maturation. Central to this process is the endosomal-sorting complex required for transport (ESCRT), which is involved in sorting cargo into ILVs, membrane remodeling, vesicle budding, and scission.22,36 Additionally, the Rab family of small GTPases regulates MVBs size, intracellular trafficking, and docking at the PM to mediate exosome release.37–40 The syndecan–syntenin–ALIX complex also contributes to ILVs formation by facilitating membrane curvature and cargo recruitment.41 The ESCRT machinery consists of four core subcomplexes—ESCRT-0, -I, -II, and -III—comprising more than 30 proteins, along with several accessory factors such as ALG-2-interacting protein X (ALIX), Vacuolar Protein Sorting 4 (VPS4), and vesicle trafficking 1 (VTA1). These complexes assemble sequentially on the endosomal membrane to drive vesicle budding.42 ESCRT-0 initiates the process by recognizing and clustering ubiquitinated transmembrane proteins; ESCRT-I and -II promote membrane invagination, while ESCRT-III mediates final vesicle scission, releasing ILVs into the MVBs lumen.43 Although ESCRT-dependent pathways are primarily responsible for sorting ubiquitinated cargo into exosomes,44 protein ubiquitination is not an absolute requirement, and alternative mechanisms exist. Exosome biogenesis can also proceed via ESCRT-independent pathways involving specific lipids and tetraspanins. For instance, enzymes such as neutral sphingomyelinase and phospholipase D2 promote ceramide-dependent inward budding of the MVBs membrane.45,46 Tetraspanins, including CD63, facilitate the selective incorporation of proteins—such as melanoma-associated antigens and ceramides—into ILVs independent of the ESCRT machinery.47 Other molecules, such as heat shock cognate protein 70 (HSC70), aid in cargo recruitment (eg, transferrin receptors),48 while pre-melanosomal protein (Pmel17), a melanosome-specific protein with a specialized lumenal domain, can also drive ILVs formation in a lipid-dependent, ESCRT-independent manner.49 Importantly, ESCRT-dependent and ESCRT-independent pathways are not mutually exclusive; both mechanisms may coexist within a single cell or even within distinct MVBs subpopulations.32 Beyond protein-mediated mechanisms, exosome biogenesis is also influenced by the cell of origin, intracellular mechanical forces, growth factors, and various physicochemical conditions.39 Upon release into the extracellular environment, exosomes travel via the circulatory system to distant target tissues, where they interact with recipient cells through surface receptor binding.50 They subsequently initiate downstream signaling cascades or transfer their cargo through membrane fusion, endocytosis, macropinocytosis, or phagocytosis, thereby modulating recipient cell behavior and function.32

Sources of Salivary Exosomes

Saliva is a readily accessible biofluid, secreted by various salivary glands located throughout the oral cavity. Once released into the oral environment, it becomes a complex mixture that includes not only glandular secretions but also serum exudates from the gingival sulcus, and secretions from the nasal and pharyngeal mucosa.51 Saliva is composed predominantly of water, in addition to a diverse array of proteins, enzymes, food debris, shed cells, and microorganisms. It plays a vital role in several physiological processes, including digestion, lubrication, antimicrobial defense, tissue repair, pH buffering, and immune modulation.52 There is a dynamic exchange of substances between the blood and saliva. Biomolecules from the circulatory system can enter saliva through passive ultrafiltration, exudation, or active transport.53 It is estimated that 20–30% of the human salivary proteome overlaps with that of plasma, leading to saliva being referred to as a “plasma ultrafiltrate” or the “mirror of the body”.54 In addition to systemic circulation, salivary components also originate from gingival crevicular fluid and resident oral microbiota.55

Salivary exosomes originate from multiple cellular sources (Figure 2). Exosomes produced by cells located spatially close to the oral cavity can directly enter the saliva. The exosomes produced by the distal cells of the body can transport their cargo through the vascular system to the salivary glands, which are then secreted along with the saliva into the oral cavity.56 Tumors in superficial locations of the oral cavity, such as the tongue, buccal mucosa, and gingiva, come into direct contact with saliva. Exosomes derived from these tumor tissues maintain high and detectable concentrations in saliva.28,57 In addition, various exosomes from distal tumors are also present in saliva. Studies in animals have demonstrated that exosomes from pancreatic cancer cells or exosome-like microcarriers from lung cancer cells reach the oral cavity through circulation, thereby delivering tumor-associated biomarkers into saliva.58,59 Almost all normal cells commonly found in the oral cavity, including epithelial cells and keratinocytes, are capable of producing exosomes.60,61 Salivary gland cells also produce exosomes and enter the oral cavity with saliva via the salivary gland ducts.62 Exosomes may also be produced by the large number of bacteria, and fungi present in the oral cavity.63 While this complexity provides a rich reservoir of physiological and pathological information, it also presents significant analytical challenges, particularly in distinguishing tumor-derived exosomes from background vesicle populations.

Figure 2 Sources of exosomes in the salivary environment. Exosomes in saliva can be directly released into the oral cavity by oral squamous cell carcinoma (OSCC) cells and other epithelial cells of the oral mucosa. Exosomes derived from distal tumor tissues may enter the systemic circulation and be transported to the salivary glands, where they are subsequently secreted into saliva via salivary ducts. The gingival crevicular fluid (GCF), a serous exudate originating from the gingival sulcus, is also enriched with exosomes. Additionally, saliva contains abundant extracellular vesicles of microbial origin, including those secreted by commensal and pathogenic bacteria and fungi. Exosomes are also actively secreted by salivary gland epithelial cells and released into saliva through the glandular ductal system.

Salivary Exosome Components and Potential OSCC Biomarkers

The components of exosomes are sorted from the contents of the origin cell, partially reflecting the characteristics of the origin cell. Exosomes are mainly composed of components such as proteins, nucleic acids, lipids, sugars, and metabolites (Figure 3). Exosomes mediate intercellular communication in different physiological and pathological environments by delivering these components.64 Salivary exosomes also harbor unique biomarkers derived from OSCC cells. Among the most widely used techniques for proteomic analysis of exosomes are Western blotting (WB) and flow cytometry, both of which are effective for detecting surface protein markers. When combined with immunocapture strategies, the sensitivity and specificity of these methods can be significantly enhanced.65,66 For exosomal nucleic acid analysis, commonly employed approaches include polymerase chain reaction (PCR), quantitative reverse transcription PCR (qRT-PCR), and various next-generation sequencing technologies.67 Through continued research efforts, the molecular composition of salivary exosomes has been progressively elucidated, thereby driving the development of novel diagnostic strategies for the early detection of OSCC (Table 1).

Table 1 Potential OSCC Biomarkers in Salivary Exosomes

Figure 3 Molecular composition of salivary exosomes. The ability of exosomes to modulate the immune response is attributed to the presence of molecules such as programmed cell death ligand 1 (PD-L1) and major histocompatibility complex II (MHC-II) on their surface. Proteins associated with the biogenesis of exosomes include tumor susceptibility gene 101 (TSG101), ALG-2-interacting protein X (ALIX), CD63, and Rab proteins. Proteins involved in cell signaling pathways, such as β-catenin, are also present in exosomes. In addition, exosomes contain heat shock proteins (HSPs) and metabolic enzymes. Notably, aquaporin-5 in salivary exosomes highlights the difference between exosomes secreted by salivary glands and those from other sources. Exosomal lipids mainly include cholesterol, sphingomyelin, phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidic acid (PA). The major nucleic acid components in salivary exosomes are DNA, RNA, microRNAs (miRNA), circular RNAs (circRNAs), and long non-coding RNAs (lncRNA).

Nucleic Acid

Exosomes are rich in nucleic acids, including genomic DNA and various RNAs. RNAs are divided into mRNA and non-coding RNA.76,77 Nucleic acids in exosomes reach target cells to enable signaling and ultimately influence their behavior.78 Since tumorigenesis and progression are often associated with abnormal changes in genetic material, the study of exosomal nucleic acids is a focal point for cancer diagnosis using EVs. EVs secreted by tumor cells are enriched with information about these abnormal nucleic acids and can reflect tumor cell status. Researchers can identify potential OSCC diagnostic biomarkers by investigating the association between altered salivary exosome gene expression patterns and OSCC status.

DNA

Back in 2016, F. A. San Lucas et al attempted to analyze the nucleic acid composition of shed exosomes in biofluid samples from patients with pancreaticobiliary cancers. They found that tumor DNA information, including mutations in Notch Receptor 1 (NOTCH1) and BRCA2 DNA repair associated gene (BRCA2), were robustly expressed in shed exosomes. The results suggest that liquid biopsies of shed exosomes may provide a comprehensive analytical assessment of tumors. This approach excludes the necessity of direct tumor sampling for visceral cancers and demonstrates the potential of exosomal DNA in the field of cancer diagnostics.79 Wang et al analyzed DNA in circulating exosomes from pheochromocytoma (PCC) and paraganglioma (PGL) patients and mice. They found that double-stranded DNA (dsDNA) fragments were present in the circulating exosomes of the patients. These fragments were highly consistent with the genomes of the paired tumors. Demonstrated that exosomal dsDNA can be used as a noninvasive biomarker for tumor diagnosis.80 We only retrieved that Mouadh Barbirou et al directly investigated the diagnostic performance of salivary exosomal DNA. They compared their potential as biomarkers for HNSCC by collecting cell-free DNA (cfDNA) and exosomal DNA (exoDNA) from blood and saliva samples of HNSCC patients. In their study, they conducted whole-genome bisulfite sequencing of paired cfDNA and exoDNA in saliva and plasma. There were more methylation differences in saliva exoDNA compared to plasma, demonstrating the potential of salivary exoDNA as a biomarker for HNSCC.68

Overall, current research on salivary exosomal DNA is sparse, and further studies are needed to explore its diagnostic potential in OSCC.

mRNA

Several studies have shown that tumor-derived exosomal mRNA is important for intercellular communication. Specifically, exosomal mRNAs are closely associated with tumor growth, angiogenesis, cell proliferation, immune escape, drug resistance, metastasis, and other behaviors.81 Several studies have investigated the diagnostic potential of salivary mRNA. Li et al used microarray analysis to compare the differences in gene expression in saliva supernatants from OSCC patients and controls. They found that the expression levels of 1679 genes in the saliva supernatants of OSCC patients were significantly different from those of controls. Seven cancer-related mRNA biomarkers (including transcripts of IL8, IL1B, DUSP1, HA3, OAZ1, S100P, and SAT) were upregulated at least 3.5-fold in the salivary supernatants of OSCC patients. ROC curves and classification modeling showed that the combination of these biomarkers had high sensitivity and specificity for the differentiation of OSCC patients from controls.82 The study by Gleber-Netto et al included a large number of clinical samples to investigate the diagnostic potential of multiple salivary transcriptome biomarkers, proteome biomarkers, and risk factor combinations for OSCC. They found that the level of the transcriptome marker DUSP1 in the saliva of OSCC patients was significantly lower than that of healthy controls and the potentially malignant oral disorders group. A multivariate model combining salivary markers and risk factors associated with oral cancer showed good diagnostic efficacy in differentiating OSCC from controls. The results of this study provide clues for the early non-invasive diagnosis of OSCC.83

We have not found studies exploring the potential of salivary exosomal mRNA in OSCC diagnosis. Since mRNA can be stabilized in salivary exosomes, it is feasible to use salivary exosomal mRNA as an entry point for the study of non-invasive methods of disease diagnosis.84

Non-Coding RNA (ncRNA)

The ncRNAs are a class of RNA molecules that do not code for proteins, which are mainly divided into microRNAs (miRNAs), transfer RNAs (tRNAs), small interfering RNAs (siRNAs), piwi-interacting RNA (piRNA), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs).85,86 In OSCC, ncRNAs can be involved in regulating behaviors such as cell proliferation, metastasis, and invasion.87 The study of exosomal ncRNAs will contribute to early cancer diagnosis and the discovery of new targets for effective cancer therapy.

MiRNAs are undoubtedly the stars of tumor diagnostics compared to other ncRNAs. Several of studies have shown that exosomes derived from tumor cells contain a large number of miRNAs, which are considered to be promising diagnostic biomarkers for a variety of cancer types.32 Depending on the cell type from which they originate, these miRNAs can either promote or inhibit cancer growth.81 MiRNAs are among the most abundant small RNAs in human saliva, and this feature is more pronounced in salivary exosomes, attributed to the exosome membrane’s protective effect on miRNAs.88,89 Abnormal expression of exosomal miRNAs may be an excellent diagnostic biomarker in OSCC diagnostic studies. Scott Langevin et al cultured four HNSCC cell lines and normal oral epithelial cells, collected supernatant exosomes, and sequenced miRNAs. All four HNSCC cell lines had highly overlapping exosomal miRNA profiles, and all differed significantly from normal oral epithelial cells. In their work, miRNA demonstrated relatively low sensitivity but high specificity as a non-invasive salivary biomarker for HNSCC, highlighting its strong potential for diagnosis of tumors.30 Chiara Gai et al collected unstimulated saliva from OSCC patients and normal control subjects, obtained exosomes by centrifugation, and finally assessed the expression levels of miRNAs by qRT-PCR. They found a differential expression of miRNAs in salivary exosomes of OSCC patients compared to normal controls. MiR-302b-3p and miR-517b-3p were expressed only in salivary exosomes of OSCC patients, whereas miR-512-3p and miR-412-3p were upregulated. They also discussed the potential of saliva as a diagnostic biofluid, noting that saliva collection technology offers superior cost-effectiveness and operational feasibility compared to blood samples.69 He et al used miRNA microarray technology to obtain differentially expressed miRNAs from salivary exosomes of healthy controls and OSCC patients. The qRT-PCR validation revealed a significant increase of miR-24-3p in the salivary exosomes of pre-surgical OSCC patients compared to controls. ROC analysis showed miR-24-3p to have extremely high diagnostic accuracy for OSCC ([AUC] = 0.738, P = 0.02), suggesting that salivary exosomal miR-24-3p has the potential as a novel diagnostic biomarker for OSCC.57 Amina Fouad Farag et al utilised qRT-PCR to analyse the changes in microRNA expression in saliva from healthy individuals, smokers, and patients with OSCC. They found that OSCC patients had significantly higher expression of miRNA-200a and miRNA-134 than the other two groups.70 Shanaya Patel et al constructed a combinatorial miRNA model consisting of salivary exosomes miR-30a, miR-140, miR-143, and miR-145 for oral cancer diagnosis. ROC analysis showed that these miRNAs had high sensitivity and specificity for OSCC, and their diagnostic results were consistent with the characteristics of the tissue samples. Subsequent miRNA-mRNA regulatory network analyses also revealed that these miRNAs correlate with oral cancer behaviors such as disease progression, recurrence, and chemotherapy resistance. This multiple miRNA signature has higher efficacy in early detection and is clinically relevant to the disease progression and overall survival rate of OSCC patients, which is of great significance for the research on the early diagnosis of cancer.71 To establish a diagnostic tool for HNSCC in synergy with plasma exosomes, Linda Hofmann et al analyzed the characterization of saliva-derived exosomes from patients with HNSCC and healthy individuals. In their miRNA profiling results, eight miRNAs had significantly lower expression ratios in the HNSCC group compared to the healthy individuals group, with miR-203 a-3p showing the largest fold change and miR-133a-5p showing the highest significance.66 Subsequent study performed a comprehensive and exploratory comparison of exosomal miRNA profiles from plasma and salivary in HNSCC patients. Their proposed two tumor-specific exosomal miRNA panels from plasma and saliva showed great potential in diagnosing HNSCC patients, Union for International Cancer Control (UICC) staging, and human papillomavirus (HPV) status. These results advance the diagnostic applications of salivary exosomal miRNAs.31 In a study conducted by Aditi Patel et al, saliva, tumor tissues, and adjacent non-cancerous tissues were collected from patients with oral cancer and subjected to miRNA sequencing analysis. The results revealed that the expression level of miR-1307-5p was significantly elevated in both salivary exosome samples and cancerous tissues compared to non-cancerous controls. This enrichment has been hypothesized to serve as a potential indicator of poor prognosis in affected patients.72 Similarly, Cosmin Ioan Faur et al employed qRT-PCR to assess the differential expression of salivary exosomal miR-10b-5p and miR-486-5p in patients diagnosed with oral cavity and oropharyngeal squamous cell carcinoma (OPC), in comparison to healthy volunteers. Their findings demonstrated that miR-486-5p was upregulated in the patient group, whereas miR-10b-5p was downregulated, suggesting their potential utility as diagnostic biomarkers for oral and oropharyngeal malignancies.73 qPCR-based detection kits hold potential for transformation into cost-controlled, high-throughput, and relatively straightforward tumor screening or clinical testing programs.

The role of ncRNAs such as lncRNAs, circRNAs, tRNAs, siRNAs, and piRNAs in the progression of OSCC has been extensively investigated. The lncRNA TIRY can facilitate oral cancer proliferation, invasion, and metastasis by promoting epithelial-mesenchymal transition.90,91 CircRNA induces tumor immune escape in OSCC92. These ncRNAs can be used as biomarkers for cancer diagnosis and prognosis, as their expression is associated with various clinical variables in cancer, and investigating them will contribute to the discovery of new diagnostic and therapeutic strategies for OSCC.93–95 However, studies on ncRNAs other than miRNAs in the field of salivary exosome biopsy have not been conducted.

Protein

Research on the exosomal proteome facilitates the search for unique disease biomarkers. Until 2024, the Vesiclepedia database contains 566 911protein entries. Some of the common proteins are HSP and cytoskeleton proteins. Many conserved proteins are shared between different EVs, and many more are different because of the way EVs are synthesized and the way proteins are sorted in the cell of origin, reflecting specific characteristics of the cell of origin.96 Changes in these proteins can be informative for tumor diagnosis. The ability of exosomes to regulate the immune response is due to the presence of molecules such as PD-L1 and MHC-II on the surface. According to electron microscopy, salivary exosomes are also abundant in tetraspanins.84 Tetraspanins are widely involved in biological functions such as protein transport and signaling. For example, CD63 is involved in ESCRT-independent exosome biogenesis.47 The proteins associated with the ESCRT-dependent pathway in salivary exosomes are Hrs, flotillin, tumor susceptibility gene 101 (TSG101), and ALIX. Rab proteins, membrane transport proteins, and annexins are responsible for the regulation of membrane binding during exosome biogenesis and remain in the exosome after its formation. Several proteins that play important roles in cell signaling pathways, such as β-catenin, Wnt5B, or Notch ligand Delta-like 4, have also been identified in exosomes.53 In addition, exosomes contain heat shock proteins, cytoskeletal proteins (actin and myosin), integrins, and metabolic enzymes. Finally, water channel protein-5 and others in salivary exosomes highlight the difference between exosomes secreted by the salivary glands and exosomes from other sources.97 Numerous studies have shown that variations in the protein fractions of salivary exosomes are closely associated with OSCC status. To investigate the differences in exosomes in the oral fluid of oral cancer patients and healthy individuals, Ayelet Zlotogorski-Hurvitz et al conducted a clinical study. They obtained exosomes by ultracentrifugation and then analyzed these exosome samples for morphological and molecular differences using AFM, NTA, ELISA, and WB. The results showed that exosomes in the oral fluid of oral cancer patients not only had increased particle concentrations and particle sizes but also had changes in protein expression levels on their surfaces. CD81 and CD9 expression were reduced, while CD63 expression was higher.74 Fatima Qadir et al suggested that exosomes derived from cancer cells can express surrogate oncogenic markers such as the membrane protein CEP55. That exosomal CEP55 in saliva or blood can be used as a cancer biomarker for the non-invasive diagnosis and prognosis of HNSCC.98 Linda Hofmann et al found that exosomes isolated from the saliva of HNSCC patients contained high levels of CD44v3, PD-L1, and CD39. Changes in the expression of these proteins partly reflect the immunosuppressive microenvironment induced by HNSCC and are expected to be diagnostic biomarkers. Their research not only explores the molecular composition of salivary exosomes but also delves into their immunosuppressive functions, integrating both aspects. By combining the most discriminative surface protein panel with a streamlined miRNA panel and pairing them with efficient magnetic bead capture technology, a “multiparametric liquid biopsy tool” can be developed for head and neck cancer auxiliary diagnosis, prognosis assessment, and prediction of immunotherapy efficacy.66 Natalie Bozyk et al also focused on the effect of OSCC on salivary exosome protein fractions, quantifying salivary exosome protein content from OSCC patients, oral potentially malignant disease (OPMD) patients, and healthy controls using mass spectrometry. They found that a screened panel of three proteins highly expressed in OSCC salivary exosomes, protonated schiff base of Ret7 (PSB7), APC membrane recruitment protein 3 (AMER3), and lysyl oxidase-like protein 2 (LOXL2), could serve as highly sensitive biomarkers for differentiation between healthy controls and patients with OSCC. This study prospectively enrolled patients with OPMD and successfully developed a proteomics detection panel capable of distinguishing healthy individuals from OPMD patients. This achievement directly aligns with core clinical needs for early detection and risk stratification, demonstrating significant translational medical value.75

Lipids

Similar to the cell membrane, the surface of exosomes is composed of lipid bilayers. Compared to the plasma membrane of the origin cell, the membrane of exosomes is more robust and resistant to degradation, which may be attributed to their higher cholesterol, desaturated lipids, and sphingomyelin content.99,100 Therefore, researchers often describe lipids on the surface of exosomes as “natural barriers” or “transporters” for proteins and nucleic acids. Exosomal lipids mainly include cholesterol, sphingomyelin, phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidic acid (PA), with cholesterol being the most abundant.101 In exosomes from different sources, the abundance of these lipids varies. We did not retrieve studies on lipid components in salivary exosomes and tumor diagnosis. Research in this area would be beneficial to deepen the understanding of exosome formation mechanisms and promote early diagnosis of tumors.

Technologies for Saliva Sampling and Salivary Exosome Characterization and Detection Saliva Sample Collection

To obtain sufficient diagnostic information from saliva for OSCC, standardized and appropriate saliva collection methods are essential (Figure 4). To minimize contamination and variability, current protocols generally recommend collecting saliva samples from fasting individuals in the morning. Participants should avoid alcohol consumption for at least 24 hours prior to collection to prevent mucosal irritation, and refrain from eating, drinking, smoking, and performing oral hygiene procedures for at least 8 hours before sampling.51,102 It is advised that subjects stand during collection, as salivary flow rates have been reported to be higher in the standing position compared to sitting or lying down.103 Saliva can be either whole saliva or glandular saliva. Additionally, based on whether external stimulation is applied during the collection process, samples can be classified as stimulated or unstimulated saliva.104

Figure 4 Saliva collection techniques and salivary exosome isolation methods. Whole saliva is collected by four main methods: the spitting method, the draining method, the suction method, and the cotton swab method. Salivary gland saliva is collected by a cannula, Lashley cup, or modified Carlson Crittenden device. Saliva sample preservation requires the addition of enzyme inhibitors and transfer under frozen conditions. Common exosome isolation methods include centrifugation, ultrafiltration, sedimentation, size exclusion chromatography (SEC), and immunoaffinity capture. In addition to these, there are emerging technologies such as microfluidic chips.

Whole saliva, also referred to as mixed saliva, is the most commonly collected form. Four principal techniques are used for whole saliva collection: Spitting method, which is simple, suitable for all age groups, and associated with relatively low inter-individual variability.102 Draining method, which involves allowing saliva to drip passively from the lower lip into a container, thereby minimizing irritation and preserving the native state of the sample. Suction method, which utilizes pipettes or syringes to aspirate saliva directly from the oral cavity floor.105 Cotton swab method, in which a cotton swab is placed in the oral cavity until saturated and then transferred to a collection container.51 In contrast, glandular saliva collection requires specialized devices such as cannulas, the Lashley cup, or the modified Carlson–Crittenden device to isolate saliva from specific salivary glands.104,106 In recent years, companies such as DNA Genotek, QIAGEN, and Malvern Medical Developments have developed customized alternative kits or devices for saliva collection. These kits often include preservatives or employ specially designed containers and materials to ensure sample stability and prevent contamination. They are widely used in clinical and molecular diagnostic settings, including for the detection of viral diseases such as mumps and rubella.107,108 If not for immediate use, saliva samples need to be frozen as soon as possible after initial centrifugation. Saliva samples can be stored at either −20 °C or −80 °C, and repeated freezing and thawing should be avoided.109,110 In addition, protease inhibitors should be added to minimize degradation of protein content during storage and analysis.

Unstimulated whole saliva is generally considered to have greater diagnostic potential than stimulated glandular saliva in the context of OSCC diagnosis. Unlike glandular saliva, whole saliva is in direct contact with OSCC lesions and is therefore more likely to contain exosomes derived from OSCC. Although stimulated saliva yields a larger volume over the same collection period, it is often more dilute, resulting in lower exosome concentrations and reduced sensitivity for diagnostic applications.103 Further clinical studies are warranted to validate the diagnostic value of different saliva types and collection methodologies in OSCC. Optimizing sampling strategies is crucial, as poor-quality or improperly collected samples can compromise the accuracy of diagnostic results. As research advances, the development of more user-friendly, cost-effective, and high-sensitivity saliva collection technologies is expected to further enhance the clinical utility of salivary diagnostics for OSCC.

Isolation of Salivary Exosomes

Exosome isolation is one of the key steps in exosome research. There are a range of well-developed techniques for exosome extraction from saliva and other samples. Common exosome isolation methods include centrifugation, ultrafiltration, precipitation, size exclusion chromatography, and immunoaffinity capture. In addition, innovative technologies such as microfluidic chips are also emerging (Figure 4).

The centrifugation method is the most common isolation method and is currently the gold standard for exosome isolation.111 By generating centrifugal force through the rotation of rotors, the centrifugation method allows different particles in a liquid to settle at various speeds. The centrifugation methods commonly applied to exosomes include differential ultracentrifugation and density gradient centrifugation. The former gradually removes cell debris, apoptotic vesicles, and other components from the sample by adjusting the centrifugal force.112 The latter utilizes sucrose, nycodenz, and iodoexanol media to form density gradients, leading to an orderly distribution of particles with different densities in different density compartments of the media.113 The centrifugation method is suitable for large-volume samples but also suffers from drawbacks including time-consuming and low purity.114 Moreover, the centrifugation process could disrupt the morphology and biological activity of exosomes.115

The ultrafiltration method isolates EVs by forcing the sample through a membrane with specific pore sizes, which can be adjusted to screen for different sizes of EVs. The ultrafiltration method is easily operated and does not require expensive equipment, but the membrane life and separation efficiency are often significantly reduced due to clogging of the membrane pores.116 Moreover, the size consistency and purity of ultrafiltered vesicles are inferior, requiring additional processing to separate exosomes from contaminating proteins.117

The precipitation method utilizes materials such as the hydrophilic polymer polyethylene glycol (PEG) to trap water molecules in the sample, continually decreasing the solubility of the exosome to facilitate its settling. The precipitation method is convenient to employ, with high exosome yield but low purity and high cost, and there are already many mature kits on the market utilizing the precipitation method to achieve exosome isolation.100,118

The size exclusion chromatography (SEC) method separates based on particle size. When the sample flows through SEC fillers (such as SEC columns) with specific pores, the fillers adsorb proteins and lipids with smaller sizes. In comparison, larger-sized exosomes flow faster and are preferentially eluted into the sample collection tubes at specific stages. The advantages of the SEC method are high product purity and minimal influence on exosome characteristics. However, the implementation of the SEC method requires special equipment and is hardly applied to large-volume samples.114,119

The immunoaffinity capture method utilizes ligands or indicators with specific immunoaffinity to recognize protein markers present on exosomes, such as transmembrane proteins and heat shock proteins (HSP). The immunoaffinity capture method allows simultaneous exosome isolation, quantification, and subpopulation identification, which has great potential for disease diagnosis, especially liquid biopsy.114,120,121

Microfluidics technology has continued to develop in recent years, leading to the development of microfluidic chips capable of rapid, automated isolation of exosomes.122 Microfluidic chips can be equipped with microfilters, nano-arrays, and nanowires while utilizing acoustic nanofiltration, dielectrophoretic segregation, or tangential flow filtration to achieve efficient and high-purity exosome isolation.123 Microfluidic chips enable high throughput in situ exosome isolation and analysis, and as it continues to develop, the cost will decrease.

These technologies perform differently when applied to different types of samples and combining them may help to maximize the benefits and avoid the drawbacks.124 For example, the samples after centrifugation or ultrafiltration followed by size exclusion chromatography can provide better exosome samples for proteomics and functional studies.125 Conjugation of specific antibodies on microfluidic chips enables efficient exosome isolation.126

In recent years, exosome isolation technologies have been under constant improvement and innovation, with some lower-cost, more efficient, and more personalized technologies emerging. For example, Suchi Gupta et al simplified the centrifugation step by introducing a sucrose cushion into the centrifugation process. Because of the simplification of the centrifugation step, the time and cost spent as well as the destruction of exosomes are reduced.127 Gu et al have developed an acoustic fluid centrifugation technology that combines acoustic wave actuation and spin of a fluidic droplet to achieve rapid concentration and size-based separation of nanoparticles. Researchers have developed a variety of latex and magnetic beads encapsulating specific receptors that target exosomal membrane markers such as CD63 and Alix. The beads adsorbed exosomes were then placed in special buffers to release the target exosomes.128 Novel magnetic beads coated with polycationic polymers can also utilize the negatively charged phosphatidylserine on the exosome surface to isolate exosomes in large quantities from sample.129 Feng et al coated an amphiphile-dendrimer supramolecular probe (ADSP) on nitrocellulose membranes for exosome capture. The ADSP is composed of highly branched globular dendrimers functionalized with amphotericin B (AMB) molecules. The AMB molecule on ADSP can interact with EVs and promote EVs segregation. They also combined ADSP with an automated printing workstation to enable high throughput capture and analysis of EVs. In addition, this workstation can also be used for the exploration of glycosylation modification on the surface of EVs, which provides a new tool for the functional research of EVs.130 Recently, Zhang et al developed a multivalent cholesterol-modified paranemic crossover DNA construct by DNA nanotechnology. As an effective synthetic nano glue, this structure promotes the rapid merging of nanoscale vesicles into micrometer-sized clusters, followed by low-speed centrifugation to enable rapid enrichment of EVs within minutes. This method effectively avoids the destruction of the structure of EVs by complex ultracentrifugation and provides an efficient and reliable tool for EVs research.131 In conclusion, the exosome isolation method continues to be optimized and innovated and will certainly provide a more robust foundation for the research and application of exosomes in the future.

The isolation of exosomes from saliva poses unique technical challenges compared to other biological fluids such as blood. Saliva collection methods—such as spitting, draining, or suction—typically yield limited sample volumes, rendering volume-dependent techniques like differential ultracentrifugation less practical. As a result, isolation protocols for salivary exosomes must prioritize high recovery efficiency and purity from minimal input volumes. In this context, precipitation-based commercial kits and immunomagnetic bead capture methods have shown favorable compatibility with saliva samples, as they require smaller volumes while maintaining effective isolation performance.132,133 However, the heterogeneous composition of saliva introduces additional complications. Salivary exosomes are often contaminated with food debris, digestive enzymes, and microbial components, all of which can interfere with downstream analysis and compromise diagnostic accuracy. To address this, enzyme inhibitors are commonly added during sample processing, and preliminary purification steps—such as low-speed centrifugation or filtration—are employed to remove large particulate matter. These pre-treatment steps, however, may also risk compromising the structural integrity or biological activity of exosomes. Moreover, the inherently higher viscosity of saliva compared to other biofluids necessitates dilution with suitable buffers prior to isolation, which can introduce variability in exosome yield and concentration.113 Currently, there is no standardized, universally accepted protocol for the isolation of salivary exosomes. Future efforts should focus on the development and validation of reproducible workflows tailored to the physicochemical properties of saliva. Importantly, such protocols must also account for their impact on the fidelity of downstream molecular profiling and the clinical applicability of exosome-based diagnostic assays.

Salivary Exosomes Characterization and Observation

The characterization of salivary exosomes is essential for assessing sample quality, optimizing diagnostic workflows, and enabling the identification of robust and reproducible biomarkers. Accurate characterization provides critical information on exosome size distribution, concentration, morphology, and surface marker expression, thereby enhancing the reliability of subsequent molecular analyses. A range of analytical techniques—such as nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and various electron microscopy methods—have been widely employed in salivary exosome research. These tools not only facilitate quality control of isolated exosomes but also support the validation of their diagnostic potential in the context of OSCC and other pathologies.

Saliva Exosome Characterization

Extensive physicochemical heterogeneity exists between exosomes. Characterization of exosomes is essential for quality assessment of clinical saliva samples, disease diagnosis and diagnostic markers studies (Table 2).

Table 2 Characterization Technologies for Exosomes

As the preferred technology for characterization of exosomes, NTA utilizes the Brownian motion of exosomes in solution. The NTA captures the variation of scattered light from exosomes in the field of view under laser irradiation by the camera and then calculates the sample concentration and particle size.151 The principle and operation of NTA are simple and suitable for analyzing multiple sets of samples. However, the concentration of vesicles is often underestimated due to limitations of the instrumental detection of particle size.134

The principle of DLS is similar to the NTA. Particle size and concentration are inferred by using a fast photon detector to monitor the temporal intensity changes in the scattered light produced by the laser irradiation of particles.147 The DLS offers a broader detection range, however, its measurements are susceptible to the physicochemical properties of the particles. In samples containing a small number of micrometer-sized exosomes, this sensitivity may lead to significant deviations in particle size estimation.135

Fluorescence correlation spectroscopy (FCS) has also been commonly used for exosome characterization. It uses the fluctuation of fluorescent signals generated by fluorescently labeled molecules as they move through a small volume of sample to analyze the characteristics of the labeled molecules. In addition, it can also be used to characterize proteins on the surface of exosomes.136 The primary advantage of FCS over DLS is its ability to detect individual fluorescently labeled molecules. This feature makes it possible to have a detection limit below 50 nm while being less prone to erroneous results when detecting larger particles.137

TRPS technology is also capable of determining exosome particle concentration and size.138 TRPS measures the transient change in electrical resistance produced by individual nanoscale particles as they pass through a tunably sized pore embedded in an elastic membrane and determines the number and size of particles passing through the pore by monitoring and analyzing the change in electrical current. TRPS features high-resolution, real-time analysis. For exosome identification, TRPS suffers from certain important drawbacks, such as the possibility of membrane clogging with repeated use, followed by a decrease in assay stability. At the same time, the background noise of the instrument system could obscure the signals of tiny vesicles, resulting in a decrease in sensitivity.139

Flow cytometry is also frequently applied to exosome characterization.140,141 However, the scattering intensity of exosomes typically drops below the detection limit of most flow cytometers.142 To detect exosomes by normal flow cytometry, it is necessary to first increase the surface area of the exosome sample by binding it with fluorescein-conjugated antibodies or silica beads.143,144 Another serious obstacle is the clustering of small exosomes, where conventional flow cytometers have difficulty in resolving the scattered signals generated by multiple exosomes that are clustered and only record them as a single object.145 In recent years, with the development of nanoscale flow cytometry (nFCM), the analysis of vesicles has become more precise, which will expand the detection limit of exosomes.146

In addition to the above common exosome characterization technology, microfluidics and single particle interferometric reflectance imaging sensor (SP-IRIS) are also rapidly developing, and their application in exosome characterization is also worth expecting.123,152

Salivary Exosome Observation

Exosomes are nanoscale membrane vesicle structures, for direct observation of their natural morphological structure and assessment of the purity of the sample, the primary tool is the microscope. The most common microscopes used for observing exosomes include transmission