Exosomes: Mediators of cellular communication in potentially malignant oral lesions and head and neck cancers

Exosomes are a unique type of extracellular vesicles that contain a plethora of biological cargo such as miRNA, mRNA, long non-coding RNA, DNA, proteins and lipids. Exosomes serve as very effective means of intercellular communication. Due the presence of a lipid bilayer membrane, exosomes are resistant to degradation and are highly stable. This makes them easily identifiable in blood and other bodily fluids such as saliva. The exosomes that are secreted from a parent cell directly release their contents into the cytoplasm of a recipient cell and influence their cellular activity and function. Exosomes can also transfer their content between cancer cells and normal cells and regulate the tumor microenvironment. Exosomes play a vital role in tumor growth, tumor invasion and metastasis. Exosomes provide a multitude of molecular and genetic information and have become valuable indicators of disease activity at the cellular level. This review explores the molecular characteristics of exosomes and the role that exosomes play in the tumorigenesis pathway of potentially malignant oral lesions and head and neck cancers The application of exosomes in the treatment of oral cancers is also envisioned. Exosomes are very small and can easily pass through various biological barriers, making them very good delivery vectors for therapeutic drugs as well as to selectively induce DNA’s mRNA and miRNAs into targeted cancer cells.


Introduction
Exosomes are a form of extracellular vesicles (EVs) that were first described by Pan and Johnstone in 1980.They were first identified as endocytic vesicles released by maturing reticulocytes. 1,2osomes are formed intracellularly, and the size of exosomes ranges from 30nm-150nm.The exosomes contain a plethora of biological entities such as proteins, lipids, DNA, microRNA, messenger RNA and long noncoding RNA enclosed in a lipid bilayer. 3,4osomes originally form as intraluminal vesicles within the endosome and are released to the environment by fusion with the plasma membrane. 5Exosomes are formed by the invagination of the endosomal plasma membrane during the transformation of an early endosome into a late endosome.The late endosomes, also known as microvesicular bodies, fuse with the cell membrane and release their contents into the extracellular environment and are now called exosomes 6 (Figure 1).The formation of exosomes depends on two different pathways; the first an endosomal sorting complex required for transport (ESCRT)-dependent mechanism, and an ESCRT-independent mechanism. 7Several factors influence the release of exosomes into the extracellular environment, such as oxidative stress and hypoxia.In addition, drugs such as sitafloxacin, pentatrezole and fenoterol activate the production of exosomes. 8osomes are an unique mode of intercellular communication for transferring bioactive cargo to recipient cells. 9s exosomes are released into the extracellular compartment, they are richly available in most of the body's fluids

REVISED Amendments from Version 1
The revised version elaborates on the morphological and molecular characteristics of exosomes.The role of salivary exosomes in dysplastic lesions and oral cancer is described.The different methods by which exosomes can be employed for chemotherapeutic drug delivery is explained.The mechanism by which exosomes bring about drug resistance in tumor cells is explained.The challenges related to the clinical application of exosomes is described.Two figures have also been added.
including saliva.The lipid bilayer membrane that forms around the exosome capsule protects their cargo from degradation and RNAse damage, thus allowing exosomes to be highly stable in circulation. 10osomes play a vital role in transferring molecular mediators and promote cell-to-cell communication both locally and to distant sites. 11When exosomes release the protein, signaling molecules, mRNA and miRNA into the cytoplasm of the target cells, this modifies the cell biology of that target cell. 12e mRNA and the miRNA that are present in exosomes can be translated into proteins in the target cells that they enter, thereby transferring genetic information from one cell to another cell. 13is review elucidates the molecular characteristic of exosomes and their implication in the biology of potentially malignant oral disorders and head and neck cancers.

Characteristic features of exosomes
Zlotogorski et al. 14 elucidated the molecular characteristics of exosomes through several molecular techniques such as transmission electron microscopy, nanoparticle tracking, atomic force microscopy, ELISA and Western blot.In his experimental analysis he compared the morphological features and molecular characteristic of exosomes in oral fluids of healthy individual and oral cancer patients.The details are as follows.

Transmission electron microscopy
Oral fluids collected from 36 Oral Cancer patients and from 25 Healthy individuals was centrifuged to remove the cell fragments and residual organelles and the supernatant was removed.The pellet that remained after the removal of the supernatant was washed and purified These pellets were dehydrated and fixed with epoxy resins and ultrathin sections were cut and the ultrastructural features were evaluated under a TEM.The ultrastructural features of the pellets that were obtained from the saliva of both healthy individuals and oral cancer patients showed nanoparticles that resembled exosomes.They appear as round-shaped vesicles surrounded by bilayered membranes.

Nanoparticle tracking analysis
The number of exosomes in saliva as determined by nanoparticle tracking analysis was 17.9AE12.45E8particles/ml in healthy individuals, while that in oral cancer patients was 36.0AE7.5E8particles/ml.The average size of the nanoparticles in healthy individuals was 49.05AE32.87nm and that of exosomes in oral cancer patients was 95.36AE36.76nm, which is much larger than that of healthy Individuals.

Atomic force microscopy
The 3D topographical images of the nanoparticle in the Oral fluids of both healthy individuals and oral cancer patients appeared as circular bulging structures, but the height of the particles in oral cancer patients was larger than the height of those in healthy individuals.

Enzyme linked immunosorbent analysis (ELISA)
Oral fluids collected from oral cancer patients (n=36) and health individuals (n=25) was evaluated for amount of exosomal proteins CD63, CD9 and CD81.The concentration of CD63 in the exosomes in the oral fluids was higher in oral cancer patients (234AE79 pg/ml) compared with that of health individuals (176AE42.3pg/ml) however there was no significant difference (p=0.2).
The concentration of CD81 in exosomes in the oral fluids of patients with oral squamous cell carcinoma was 61.1AE37 pg/ml, and that concentration of CD81 in the exosomes of oral fluids of healthy individuals was 201AE79.5 pg/ml.This difference is statistically significant (p=0.032).The concentration of CD9 in the exosomes of oral fluids of oral cancer patients was 104.1AE18 pg/ml, while that in the exosomes in the oral fluids of healthy individuals was 152.9AE24.4pg /ml.This difference is not statistically significant.

Western blot
In oral cancer patients the exosomal pellets obtained from the oral fluids showed the presence of the glycosylated form of CD63 as a prominent band at 53Kd.With regard to CD81, the exosomes obtained from the oral fluids of oral cancer patients and healthy individuals showed a band at the expected 26 kDa area of the protein.The exosomes obtained from the oral fluid of both oral cancer patients and healthy individuals showed a specific band at 28kDa area to represent CD9.However, in oral cancer patients the intensity of the band was lower than that in healthy individuals.
Mathivanan et al also found a significant increase in the expression of CD63 and a decrease in the expression of CD9 and CD81 in exosomes present in the saliva of oral cancer patients compared to that of the exosomes in the saliva of healthy individuals. 6Exosomes are recognized by the molecular biomarkers that they express; namely, CD63, CD9, CD81, Alix, TSG101 and hsp70. 15oteins that are encapsulated and transported by exosomes are responsible for regulating the fusion, migration, and adhesion to the target cells.7][18][19][20] The miRNAs in the exosomes are of higher concentration and greater stability while they are in circulation as they are within encapsulated vesicles. 11,21,22The proteases and RNAases in circulation cannot act on the exosomal proteins; hence they have a longer half-life than the free molecules. 19Exosomes are enriched in cholesterol, diglycerides, glycerophospholipids, phospholipids and sphinolipids or glyceromides. 23osomes in oral potentially malignant disorders Li et al. isolated mesenchymal stem cells from the clinical tissue samples of normal oral mucosal tissues, dysplastic oral lesions and oral squamous cell carcinoma tissue.The exosomes were isolated from the Mesenchymal stem cells and a microarray analysis of the exosomes showed that miR 8485 was differentially expressed in the three groups.The miRNA-8485, when transfected into dysplastic oral mucosal cell lines (DOK) as well as tongue squamous cell carcinoma cell line SCC15, caused rapid growth, promoted migration and invasion of the cells. 24ng et al. found that when MSC-EV-miR-185 was pasted onto buccal lesions in dimethylbenzanthracene (DMBA) induced Oral Potentially Malignant disorders (OPMD) model it remarkably attenuated the severity of inflammation and significantly decreased the incidence and the number of dysplastic characteristics in the OPMD tissue.Also, these cells showed a low immunohistochemical expression of PCNA and CD 31.By activating caspase 3 and 9 of the apoptotic pathway, the miR-185 targeted the Akt pathway. 25osomes in oral submucous fibrosis Oral submucous fibrosis is a multifactorial precancer disorder that is caused by chewing areca nuts.Liu et al. isolated adipose-derived mesenchymal stem cell exosomes from the fibroblasts of oral submucous fibrosis patients and normal individuals.These ADSC-Exos were found to be positive for CD63.The mRNA expression levels of COLIA 1 and COLIA III were down regulated in fibroblasts that were stimulated with both TGF-Beta and ADSC-Exos in the culture media.Similarly, the expression of Collagen I and Collagen III was downregulated in fibroblasts that were treated with both TGF Beta and ADSC-exosomes.The ADSC-exosomes inhibited the p38 MAPK signalling pathway and reduced the expression of collagen I and Collagen III.
However, the expression levels of matrix metalloproteinase (MMP)1 and MMP3 were significantly upregulated in fibroblasts when they were stimulated with both TGF beta and ADSC-Exosomes in the culture media.With the ability of ADSC-Exos to inhibit the P38 MAPK signaling pathway, this biomarker can serve as remarkable treatment option for Oral submucous fibrosis. 26 another study, Zhou et al. isolated exosomal long non-coding RNA ADAMTS9-AS2 from tissue samples of oral submucous fibrosis and oral squamous cell carcinomas.miRNAs regulated by lncRNA ADAMTS9-AS2 enriched the metabolic pathway, epithelial mesenchymal transition, p13K-Akt pathway and pathways of cancer and enhanced the malignant potential of OSF.ADAMTS9-AS2 plays a crucial role in altering the cell microenvironment during the carcinogenesis process of oral submucous fibrosis and, thus is an ideal marker for early diagnosis of OSCC in oral submucous fibrosis. 27osomes in oral lichen planus Oral lichen planus is a chronic inflammatory disease of the oral mucosa with an unknown etiology which is characterized by abnormal activity in the T-cell mediated immune response and is also regarded as a "potentially precancerous disorder " . 28,29un et al. 30 isolated exosomes from the lesions of oral lichen planus and from saliva of 16 patients and eight agematched normal individuals.The miRNA microarray analysis showed that there were 21 miRNAs that showed a 2-fold increase in the saliva samples of oral lichen planus compared to that in normal individuals.Among all the miRNAs that were identified, hsa-miR4484, hsa-miR1246 and hsa-miR1290 were found to be upregulated in the salivary exosomes of oral lichen planus patients.The miRNA 4484 can target a multitude of genes and initiate their translation into proteins that alter the cellular mechanisms.

Exosomes in oral cancer
Exosomes derived from oral cancer are saucer-like in shape with a membranous structure.The size of the exosomes in the saliva of oral cancer patients can be as large as 400 nm in diameter. 14Western blot can detect proteins CD63, Rab 5, CD9 and Alix in the exosomes derived from oral cancer patients.Exosomes derived from oral cancer cell lines contain close to 267 proteins. 31osomes are involved in several cellular mechanisms of oral cancers such as tumor growth, invasion, metastasis and chemoresistance 9,32 (Figure 2).Exosomes secreted by neoplastic cells into the tumor microenvironment (TME) play a vital role in tumor growth, invasion and metastasis. 33Exosomes are also found to promote epithelial-mesenchymal transition during the progression of squamous cell carcinomas of the tongue. 34,35In the study done by Veread et al., the exosomes isolated from HSC 3 cancer cell extracts when tested with ELISA also showed the presence of CAV-1 along with CD 63, CD 9 and CD 81. 34Dayan et al., in their study identified cancer derived exosomes by the presence of TSGO1, with EMT epithelial-mesenchymal transition process and the trans differentiation of fibroblast-to-CAF-like cell in the TME and suggested that molecular cross talk can be exploited to design therapeutic strategies. 35ng et al. found that in oral cancer patients with lymphatic metastasis, exosomal laminin 332 was highly expressed. 36heodoraki et al. 37 found that exosomal PD-L 1 was associated with the clinical stage of oral cancer.Rabinowitz also found exosomes enriched with miRNA in the tissue samples of tongue oral squamous cell carcinomas. 38The exosomes in saliva from oral cancer patients are larger compared to that of normal exosomes.The density of CD63 in the exosomes of oral cancer patients is markedly increased when compared with normal exosomes. 39Yet the density of the other surface markers of exosomes CD9 and CD81 is significantly reduced in saliva of oral cancer patients when compared with that of normal individuals. 14osomes from hypoxic oral squamous cell carcinoma cells are found to deliver the miR-21 to tumor OSCC cells.This promotes a prometastatic behavior among tumor cells. 40Exosomes can also spread the invasive potential to noninvasive cells by transferring oncogenic miRNAs.Highly invasive tongue cancer cells can release exosomes containing miR-200-3p that will prevent the expression of CHD 9 and WRN in non-invasive cells and confer an invasive potential to these cells as well. 41Qadir et al. found that CEP55 (a centrosomal protein) was present in all the exosomes released from cell lines of Head and Neck Carcinoma cells and absent in the exosomes released from normal oral keratinocytes. 42u et al. found that oral squamous cell carcinoma cells released chemo resistant exosomes which induce cisplatin resistance in OSCC.These exosomes can upregulate miR-21 and downregulate the expression of phosphate and tensin homolog and programmed cell death. 43angevi et al. found that salivary exosomes expressed elevated levels of miRNA 486-5p in oropharyngeal squamous cell carcinomas compared to controls. 44He et al. isolated salivary exosomes from oral squamous cell carcinoma patients and quantified them by NTA and characterized them by TEM and found that salivary exosomes had higher level of miR24-3p in oral squamous cell carcinoma patients. 45rly-stage tumors release exosomes that contain several tumor markers into saliva and hence they can serve as noninvasive, efficient diagnostic tools. 46Faur et al. found that miR-10b-5p, miR-486-5p, miR-24-3p and miR-200a in the exosomes of saliva are the most useful salivary biomarkers of head and neck cancer. 47In addition other biomarkers such as A2M, HPa, MUC5B, LGALS3BP, IGHA1, PIP, PKM1/M2, GAPDH, have also be identified in the salivary exosomes derived from oral cancer patients. 48Moreover, Human Papilloma virus has been detected in the salivary exosomes of patients with oro-pharyngeal carcinomas. 49osomal miR-29a-3p derived from OSCC cells can promote proliferation and invasion of OSCC cells by enhancing the polarization of M2-subtype of macrophages, the tumor associated macrophages (TAMs). 50When OSCC cells release exosomes that contain THBS1, they communicate with M1 subtype of macrophages and transform them into TAMs.These TAMs can now promote migration of OSCC cells. 51u et al. found that oral cancer cells released exosomes that contained TGF-beta.these exosomes are internalized by natural killer cells in the microenvironment.During the early stages, the proteins in the exosomes enhanced the function of the NK cells.Yet, with a longer incubation time, the TGF-beta gradually inhibited the cellular cytotoxicity of NK cells.The killer function of natural killer cells also decreased.A PANTHER protein class analysis showed that there is salient enrichment of proteins related to localization and adhesion of exosomes to their recipient cells.Then the exosomes fuse with the cell membrane of the recipient cell and transfer their contents in the recipient cell. 52osomes derived from oral cancer cell contain NAP1 that enhanced the cytotoxicity of NK cells. 53Exosomes derived from oral squamous cell carcinomas also contain the biomarker EGFR. 54Most often EGFR and CD 9 are in the same exosome. 55The EGFR secreted by OSCC cells play a vital role in the EMT of epithelial cells.Cetuximab is not able to inhibit the EGFR mediated EMT transition of the transformed OSCC cells. 56Metastatic OSCC cells actively secrete chaperone-rich exosomes that are rich in stress re resistant protein HSP. 57ncer associated fibroblasts Cancer associated fibroblasts promote tumor progression mainly through actively communicating with cancer cells.CAFs-associated exosomes mediate migration and invasion of OSCC cells.CAF's derived exosomes exert a stronger effect on upregulation of MMP-3, MMP-9, N-Cadherin and Beta catenin.miR-382-5p are transferred from CAFs to OSCC cells through exosomes.The expression of miR-382-5p in CAFs is elevated by ~3.83 fold compared to that of normal fibroblasts.58 Languino et al. showed that exosomes released from cancer-associated fibroblasts transferred TBRII cells to malignant keratinocytes and activates them to be responsive to the TGF B ligand.59 Exosomes in salivary gland malignancies Yang et al. in their study found that exosomes loaded with epiregulin from salivary adenoid cystic carcinoma induced epithelial-mesenchymal transition by down regulating the expression of E cadherin.Epilegrin-enriched exosomes derived from salivary adenoid cystic carcinoma can also enhance invasion and metastasis of this tumor.60 Hou et al. found that exosomes-derived salivary adenoid cystic carcinoma 833 cells target the tight junction proteins claudin-1, Zo-1 and beta catenin and enhance migration and invasion of the tumor cells.61 Exosomes are an unique biological entity that play an important role in pathogenesis of potentially malignant oral disorders and head and neck cancers (Tables 1 and 2).
Exosomes in oral cancer treatmentdrug delivery Precise targeted delivery of chemotherapeutic drugs is one of the best methods to reduce the toxic side effects of chemotherapy.Exosomes have superior drug delivery properties, such as good stability, allowing them to travel to distant target organs.The hydrophilic core encapsulates water soluble drug molecules.Exosomes are highly biosafe and do not induce an immune response in the body. 62,63Exosomes can track and monitor tumor progression and drug resistance in real-time giving information on drug heterogeneity. 64A proteome map of human parotid exosomes has also been developed using multi-dimensional protein identification technology, which helps in discovering exosome protein markers related to oral cancer. 65able 1.Exosomal components in the biology of potentially malignant oral disorders.
Increased the severity of inflammation.Decreased the number of dysplastic features.

Reversed the upregulation of Collagen I and
Collagen III that is induced by TGF-β1.
Downregulation of COLIA 1 and COL3A I mRNA.
Reversed the upregulation of phosphorylation of p38 that is induced by TGF-.
Acts on the Epithelial -Mesenchymal transition pathway.
Act on the metabolic pathways.

Suppresses the malignant transformation of oral submucous fibrosis
Can also inhibit the cell growth, migration and invasion of oral squamous cell carcinoma cells.
Immune reaction or represents a protective mechanism against a pathological stimulus.
Table 2. Exosomal components in the biology of head and neck Cancers.

Exosomal component Target/Tissue Biological function
Exosomal miRNA 8485 from mesenchymal stem cells of dysplastic oral leukoplakia. 24splastic oral mucosal cell line and human tongue squamous cell carcinoma cell line.
Promotes proliferation, migration, and invasion of dysplastic oral keratinocytes.
Programmed death ligand 1 and cytotoxic T lymphocyte associated protein 4 present in the exosomes from head and neck cancer cells. 37duce apoptosis of T cells.Inhibit T cell proliferation.
Promotes tumor cell evasion from the immune system.
Prometastatic behaviour of tumor cells.
Transfers cisplatin-resistance to nonresistant OSCC cell lines.
Enhances the invasive potential of non-invasive tumor cells.
Screening and diagnosis of oral cancers.

Proliferation of Oral Squamous cell carcinoma cells
Exosomes can be used as natural drug delivery vehicles for oral cancer.Exosomes can deliver their contents to the target cell by binding to the cell membranes and the receptors present on the surface membrane and then by cytocytosis. 66[69][70][71][72][73] One of the passive forms of exosomal drug loading is to transfect the drugs to be loaded into the donor cells and then encapsulate the drugs into the exosomes inside the donor cells.Although the drugs might have a toxic effect on the donor cell, this method is safe and is used in immunotherapeutic treatment for cancer. 68Another method of passive drug loading is to separate and purify the exosomes from the donor cell and mix them with the drug to be loaded.Although the drug loading is slow, the integrity of the exosomal membrane is maintained. 67e of the active methods of loading exosomes with drugs is to prepare a pore in the exosomal membrane by an electrical field and then to allow the therapeutic drug to penetrate the exosomes.By this method, hydrophilic molecules are transported into the hydrophilic core of the exosomes. 70In another active method the exosomes and the drugs are first frozen and then thawed. 73osomes can also serve as carriers of therapeutic small molecules, proteins and nuclei acids for therapeutic application in diseases.Exosomes have an amazing advantage of good biocompatibility, almost non-toxic side effects and can be used as a sound drug delivery system.The lipid bilayer membrane of exosomes protects their contents from degradation and  50 Promotes M2 subtype macrophage polarization.These macrophages produce VEGF, PDGF, cytokines and MMPs.

Promotes proliferation and invasion of Oral Squamous cell carcinoma
Promotes angiogenesis, cancer growth and metastasis.

Transform macrophages into tumor associated macrophages
Regulates the migration of tumor cells.
Exosomal TGF B. 52 Reduces the expression of surface receptors NKp30 and NKG2D on natural killer lymphocytes.
Inhibits the cellular cytotoxicity of natural killer lymphocytes.
Enhanced the cytotoxicity of natural killer cells.
Migration and invasion of CAL 27 lines.
Migration and invasion of tumor cells.

Exosomal component Target/Tissue Biological function
Epiregulin-enriched exosomes Derived from adenoid cystic carcinomas. 60wn regulates the expression of E-Cadherin.Enhance the expression of vascular endothelial growth factor receptor 1 in lung endothelial cells.

Enhances invasion and metastasis
Generates a premetastatic niche at the site of future metastasis.
Promotes tumor cell migration and invasion.
destruction, and they are highly stable in circulation.The small size of exosomes gives them the ability to cross various biological barriers such as the bloodbrain barrier and reach the target cell or organ. 66osomes carry hydrophilic molecules such as miRNAs.An effective way of cancer treatment would be load the exosomes with miRNAs that can inhibit OSCC progress such as miR-1294, miR-6887-5p or miR-101-3p.It is revealed that Fe3O4 nanoparticles and a constant magnetic field can induce exosomal miR-21-5p upregulation.This method of a combination of nanomaterials and a magnetic field improves the precise localization of drug, drug retention and the drug half-life and reduces the drug dose that is needed and improves drug efficacy. 74,75acha et al. in their review on the role of extracellular vesicles in the tumor microenvironment and in anti-cancer drug resistance deduced that by exploiting their molecular cargo the extracellular vesicles can be developed into efficient drug vehicles for cancer treatment.Bioengineered EV's loaded with chemotherapeutic agents or ligands which target malignant cells can be used for cancer treatment. 76The presence of CD47 on exosomes produces a signal that protects them from phagocytosis. 77Delivering specific DNA, RNA or proteins via exosomes can be an interesting method of treating oral cancers.
Modifications of the cargo of exosomes to express CD3 antibodies reprograms exosomes to activate T cells, and thereby, exosomes can be engineered to generate allogenic therapeutics with defined immune targeting properties. 78osomes have also been recognized as modulators of drug resistance.They can mediate drug resistance by directly exporting of sequestrated cytotoxic drugs and this reduces the effective concentration of the drugs at the target sites.Exosomes can also serve as a sink for immunotherapies, as their surface contain cellular antigens that are a target for monoclonal antibody-based drugs.The bioavailability of these drugs at the tumor site is diminished. 76 addition, exosomes can also communicate drug resistance to cancer cells that are drug sensitive.The presence of some microRNA's-21 in exosomes was found to promote anticancer drug resistance to cisplatin in oral cancer cell lines. 43o mitigate exosomes that promote drug resistance, these exosomes are to be specifically removed and the secretion of the beneficial exosomes is to be maintained. 76allenges in the clinical application of exosomes One of the challenges application of exosomes in oral cancer therapeutics is that the isolation of exosomes is time consuming, laborious, costly and at times inefficient.The purity and the quality of the exosomes is not sufficient. 75The translation of Exosome based therapeutic vesicles requires large-scale good manufacturing practice production. 79bstantial amount of clinical grade exosomes has been obtained through differential ultra centrifugation and tangential flow filtration.By Tangential flow filtration and size exclusion chromatography large volumes of exosomes can be isolated from cell culture media. 80 is challenging to maintain the stability of the exosomes in saliva for a long period of time.The action of exosomes is also affected by several pre-analysis parameters, that will reduce the efficacy of exosomes in the diagnosis of oral cancers. 81r the application of exosomes for oral cancer treatment, the route of exosome delivery, the optimal dose to be employed and the frequency of the treatment needs to be determined to realise the maximum clinical efficiency with minimal side effects.Extensive clinical research with high quality exosomes is necessary to accomplish this. 82ere are strategies to develop exosome-based vaccine for treating oral cancer.Yet, because of their heterogenous origin and the differential effect that the oral cancer derived exosomes exhibit, it is challenging to develop these vaccines.Also, derived exosomes may inhibit anti-tumor mechanisms and promote metastasis and hence will be risky to employ them in vaccine preparation. 83

Conclusion
Exosomes are extracellular messengers that transport and exchange valuable molecules across cells and this has a huge influence on the cellular activity of the recipient cell as well as their microenvironment.An astute evaluation of the components of exosomes will provide a greater insight into the underlying molecular mechanism of the disease process.Exosomes are found in abundance in biofluids such as saliva, which can be readily and easily obtained through noninvasive methods.Salivary exosomes are more stable and can be widely used for the diagnosis and early detection of oral cancers.miRNAs in the exosomes provide molecular and genetic information that can assist in the prognosis and for disease monitoring of oral cancer patients.Further investigation in the area of salivary exosomes, will unravel the biological mechanism of exosomes in the tumorigenesis pathway of oral precancers and cancers.Exosomes are very small and can easily pass through various biological barriers, making them very good delivery vectors for therapeutic drugs as well as to selectively induce DNA's mRNA and miRNAs into targeted cancer cells.
Strength: The authors have analyzed data from relevant scientific literature and have systematically presented the way in which exosomes are mediators of intercellular communications in premalignant and malignant oral lesions.In addition, the limitations and challenges encountered in the clinical application of exosomes is also described.
I feel this manuscript can be approved in the present format.

Are all factual statements correct and adequately supported by citations? Yes
Is the review written in accessible language?Yes

Are the conclusions drawn appropriate in the context of the current research literature? Yes
Competing Interests: No competing interests were disclosed.

Are all factual statements correct and adequately supported by citations? Yes
Is the review written in accessible language?Yes

Are the conclusions drawn appropriate in the context of the current research literature? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Cancer Biology, Immunology, Obesity I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Version 1
Reviewer Report 29 June 2023 https://doi.org/10.5256/f1000research.139868.r177886The review by Solomon et al., focuses on the role of exosomes, extracellular vesicles that mediate intercellular communication, in the pathogenesis of potentially malignant oral lesions and head and neck cancers.The authors discuss the molecular characteristics of exosomes, highlighting their resilience due to their lipid bilayer membrane and their capacity to transfer biological material, such as miRNA, mRNA, long non-coding RNA, DNA, proteins, and lipids, between cells.They emphasize the importance of exosomes in tumor growth, invasion, and metastasis, and their potential use in diagnosing disease activity.The review also explores the potential of using exosomes as therapeutic vectors due to their size and ability to traverse biological barriers.

Strengths:
The review provides a comprehensive discussion on the emerging role of exosomes in the context of potentially malignant oral lesions and head and neck cancers, a relatively novel area of research. 1.
It delves into the molecular and genetic information carried by exosomes, thereby providing deep insights into their functioning and influence on cancer cells.

2.
The review brings to light the potential of exosomes as biomarkers, which is crucial for early diagnosis and personalized treatment strategies.

3.
It also explores the potential use of exosomes as therapeutic delivery vectors, which opens a new avenue in the treatment of these types of cancers.

Limitations:
The title might benefit from minor rephrasing to improve readability.Important literature is missing 1,2 .Authors should cite these and many other important recent references.Readers might benefit from references to key studies that have shown the importance of exosomes in potentially malignant oral lesions and head and neck cancers.

2.
It does not address potential challenges or limitations in utilizing exosomes for diagnosis or therapy, which would provide a more balanced perspective.

3.
Authors should have at least one or two figures in the paper.4.
Although the conclusion acknowledges the need for further investigation in the area of salivary exosomes, it could also discuss potential challenges in this line of research or any practical barriers to using exosomes as diagnostic markers or therapeutic vectors.

5.
Rather than simply stating the need for more research, the authors could suggest specific areas for future study or the kinds of experiments that could be valuable in this field.

English Language:
The language is generally clear and well-written.However, there are some phrases that could be reworded for clarity and flow.For instance, "Exosomes provide a multitude of molecular and genetic information and have become valuable indicators of disease activity at the cellular level" could be reworded to "Exosomes, rich in molecular and genetic information, have emerged as valuable indicators of cellular disease activity."There are many such instances.
1.The title of the manuscript has been corrected as per the suggestion.
Important literature is missing 1,2 .Authors should cite these and many other important recent references.Readers might benefit from references to key studies that have shown the importance of exosomes in potentially malignant oral lesions and head and neck cancers.
The recommended references have been included in the manuscript.

2.
It does not address potential challenges or limitations in utilizing exosomes for diagnosis or therapy, which would provide a more balanced perspective.
The potential limitation of utilizing exosomes for diagnosis and therapy has been addressed in the manuscript.

3.
Authors should have at least one or two figures in the paper.
Figures have been included in the manuscript.

4.
Although the conclusion acknowledges the need for further investigation in the area of salivary exosomes, it could also discuss potential challenges in this line of research or any practical barriers to using exosomes as diagnostic markers or therapeutic vectors.
The practical barriers has been included in the manuscript.

5.
Rather than simply stating the need for more research, the authors could suggest specific areas for future study or the kinds of experiments that could be valuable in this field.
This has been included in the manuscript.

Monica Charlotte Solomon
The authors thank the reviewer for their suggestion and recommendations The first sentence of the Introduction is clearly untrue.I am not sure what the authors meant to say but to state that exosomes were first described in 2014 is incorrect by about 40 years.Exosomes were first described in the 1970s.
Thank you, this has been corrected in the manuscript.
○ Page 3 TEM section.This section contains a single sentence, and it requires a reference.And in the same section what are the pellets that the authors are referring to?
The details of the same has been included in the manuscript.

○
Page 3 ELISA section.The authors report the levels of CD63 observed in exosomes from the oral fluids from oral cancer patients compared to normal individuals; however, there is no mention as to whether the differences are statistically significant or how many patients were sampled.Looking at the numbers I would predict that the observed levels overlap and are unlikely to be significantly different.But this may just reflect a small sampling number.Neither is reported but the authors should address The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com

Figure 2 .
Figure 2. Role of exosomes in oral cancer.

©
2023 Bhat A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Ajaz A Bhat Department of Human Genetics-Precision Medicine in Diabetes, obesity and Cancer Research Program, Obesity and Cancer Research Program, Sidra Medicine, Doha, Qatar Main Findings: For example: "Exosomes: Mediators of Cellular Communication in Potentially Malignant Oral Lesions and Head and Neck Cancers" or "The Role of Exosomes in Cellular Communication: Implications for Potentially Malignant Oral Lesions and Head and Neck Cancers." 1.

Is the topic of the review discussed comprehensively in the context of the current literature? Partly Are all factual statements correct and adequately supported by citations? Yes Is the review written in accessible language? Yes Are the conclusions drawn appropriate in the context of the current research literature? Partly Competing Interests:
1. Kalluri R, McAndrews KM: The role of extracellular vesicles in cancer.Cell.2023; 186 (8): 1610-1626 PubMed Abstract | Publisher Full Text 2. Maacha S, Bhat AA, Jimenez L, Raza A, et al.: Extracellular vesicles-mediated intercellular communication: roles in the tumor microenvironment and anti-cancer drug resistance.Mol Cancer.2019; 18 (1): 55 PubMed Abstract | Publisher Full Text No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
The authors thank the reviewer's for their suggestions and recommendation.Thank you for your time and patience.The title might benefit from minor rephrasing to improve readability.For example: "Exosomes: Mediators of Cellular Communication in Potentially Malignant Oral Lesions and Head and Neck Cancers" or "The Role of Exosomes in Cellular Communication: Implications for Potentially Malignant Oral Lesions and Head and Neck Cancers."

the topic of the review discussed comprehensively in the context of the current literature? Partly Are all factual statements correct and adequately supported by citations? Yes Is the review written in accessible language? Yes Are the conclusions drawn appropriate in the context of the current research literature? Yes Competing Interests:
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The authors review the literature surrounding exosomes generally and more specifically the role of exosomes in oral cancer.Although this manuscript is presented as a Review it reads more like a list of observations or facts.The authors provide two organized tables outlining the exosomal components in the biology of malignant disorders (table1) or head and neck cancers (table 2) but there is overlap.For example, the first example in Table1(Oral Disorders) is dysplastic oral mucosal cell line and human tongue squamous cell carcinoma cell line which should properly be in Table2(head and neck cancers).The first sentence of the Introduction is clearly untrue.I am not sure what the authors meant to say but to state that exosomes were first described in 2014 is incorrect by about 40 years.Exosomes were first described in the 1970s.The authors report the levels of CD63 observed in exosomes from the oral fluids from oral cancer patients compared to normal individuals; however, there is no mention as to whether the differences are statistically significant or how many patients were sampled.Looking at the numbers I would predict that the observed levels overlap and are unlikely to be significantly different.But this may just reflect a small sampling number.Neither is reported but the authors should address this deficiency.The authors state that "The size of the exosomes in the saliva of oral cancer patients are also larger compared to that of healthy individuals and range from 20-400nm in diameter".So what are the sizes of healthy individuals for comparison?They must be extremely small if they are less than 20nm.Page 5, last sentence of paragraph 3. "Early-stage tumors release exosomes that contain several tumor markers into saliva and hence they can serve as non-invasive, efficient diagnostic tools."Ok,that is fine but what are these "several tumor markers"?andhowcan they serve as diagnostic tools?On page 5, 5 th paragraph the authors state that "hypoxic oral squamous cell carcinoma cells are found to deliver the miR-21 to normal OSCC cells."How is this possible?By definition "normal cells" can't be OSCC (oral squamous cell carcinoma).Please correct and clarify what you mean.The authors list several studies that identified numerous miRNAs (9 different miRNAs) in the exosomes of oral cancer patients and only one is shared amongst the reports.A review article should be synthesizing these reports and sifting through the observations to present the reader with the best evidence.That is not clear here.No competing interests were disclosed.
Interests: Nil Reviewer Report 26 June 2023 https://doi.org/10.5256/f1000research.139868.r177869© 2023 Barrett J. ○ Page 3 TEM section.This section contains a single sentence, and it requires a reference.And in the same section what are the pellets that the authors are referring to? ○ Page 3 ELISA section.○ Page 5 paragraph 2. ○ ○ ○ Reviewer Expertise: Translational head and neck cancer biology I confirm that I