Salivary Chemical Barrier Proteins in Oral Squamous Cell Carcinoma—Alterations in the Defense Mechanism of the Oral Cavity

Oral squamous cell carcinoma (OSCC) is one of the most frequent types of head and neck cancer. Despite the genetic and environmental risk factors, OSCC is also associated with microbial infections and/or dysbiosis. The secreted saliva serves as the chemical barrier of the oral cavity and, since OSCC can alter the protein composition of saliva, our aim was to analyze the effect of OSCC on the salivary chemical barrier proteins. Publicly available datasets regarding the analysis of salivary proteins from patients with OSCC and controls were collected and examined in order to identify differentially expressed chemical barrier proteins. Network analysis and gene ontology (GO) classification of the differentially expressed chemical barrier proteins were performed as well. One hundred and twenty-seven proteins showing different expression pattern between the OSCC and control groups were found. Protein–protein interaction networks of up- and down-regulated proteins were constructed and analyzed. The main hub proteins (IL-6, IL-1B, IL-8, TNF, APOA1, APOA2, APOB, APOC3, APOE, and HP) were identified and the enriched GO terms were examined. Our study highlighted the importance of the chemical barrier of saliva in the development of OSCC.


Introduction
The oral cavity is one of the most frequent sites of head and neck cancers, developing predominantly as oral squamous cell carcinoma (OSCC) in the upper aerodigestive epithelium [1,2].OSCC accounts for nearly 95% of all cancer types in the oral cavity, 2% of all malignant lesions, and more than 30,000 new cases per year worldwide [3].OSCC mainly affects the elderly population since the average age of diagnosis is around 60 years with an approximately 2:1 male:female ratio [4].In Europe, the age-standardized rates for both the incidence and mortality of oral cavity and pharyngeal cancers are high, without substantial improvements in the last decades [5].Among the European countries, Hungary has the highest rate of incidence and mortality of OSCC [5].OSCC is frequently being diagnosed in advanced stages and, despite considerable progress in surgical methods, radio-, chemo-, and immunotherapy, the long-term survival rate is around 50%.In contrast, the recovery rates for patients with early stage lesions may be up to 80% [6].Unfortunately, with the exception of biopsy, considered as the gold standard procedure, there are no evidence-based, reliable, non-invasive methods for large-scale screening and early detection of OSCC [7].Besides genetic risk factors, such as the overexpression of NPM, CDK1, and NDRG1 genes and underexpression of CHES1 [8], tobacco and alcohol consumption and poor oral hygiene are also risk factors in the carcinogenesis of OSCC [9][10][11].Microbial infections and/or dysbiosis are also associated with the development of OSCC, and the impact of HPV infection was highlighted as well [12].Studies have shown that bacteria, such as Porphyromonas gingivalis, Fusobacterium nucleatum, Peptostreptococcus, Filifactor, Parvimonas, Pseudomonas, Campylobacter, and Staphylococcus species, can participate in the development of OSCC [13].Moreover, Robayo et al. highlighted the importance of the co-infection of HPV and Streptococcus anginosus in the development of OSCC [14].
Saliva is a complex mixture of organic and inorganic compounds continuously secreted from major and minor salivary glands and the gingival crevice [15].It is a very dilute body fluid composed of approximately 99% water with varying (0.7-2.4 µg/µL) protein concentrations [16], showing high variability depending on the age, sex, sample collection time, and health status of the oral cavity.Saliva contains more than 2700 proteins, and the most abundant proteins belong to the antimicrobial and immunomodulatory protein (AMP) family [17].AMPs are elements of the innate immune system [18] and constitute the first line of defense in protecting the host from invading pathogens by creating a chemical barrier [19].In the human body, the chemical barriers contain several prototypic AMPs, such as defensins, dermcidin, and LL-37 cathelicidin [20], and there are several proteins with much higher concentrations compared with prototypic AMPs.These proteins, e.g., lactotransferrin, lipocalins, lysozyme-C, lacritin, etc., belong to the highly abundant body fluid proteins, with various defense functions [20].The non-invasive collection and continuous availability of saliva make it an excellent target for omics and biomarker studies.Unsurprisingly, the protein composition of saliva has been analyzed by several workgroups aiming to identify new biomarkers, indicating its relevance to medical applications [21].Since OSCC is one of the most frequent tumor types in the oral cavity and the analysis of salivary proteins has a high impact, many studies have been carried out in order to identify the alterations in the saliva of patients with OSCC compared with controls [22][23][24][25].Our workgroup also demonstrated changes in the proteome and transcriptome of saliva from patients with OSCC [26][27][28][29].
Considering that saliva contains many chemical barrier proteins and the fact that OSCC can alter the protein composition secreted into saliva, our aim was to analyze the effect of OSCC on the salivary chemical barrier proteins.We collected publicly available datasets regarding the analysis of salivary proteins from patients with OSCC and controls and we searched for differentially expressed chemical barrier proteins.Network and GO analyses of the differentially expressed chemical barrier proteins were performed as well.

Results
In our study, we reutilized publicly available datasets in order to analyze salivary proteins from patients with OSCC and matched controls originating from the ProteomeXchange [30] and PubMed [31] repositories.We searched for chemical barrier proteins in the downloaded datasets in the UDAMP database previously created by our workgroup [32] and we searched for chemical barrier proteins with significantly different expression between OSCC and control groups.Our evaluation relied on the results of the statistical analyses performed by the authors.

Differential Expression of Chemical Barrier Proteins in Saliva of Patients with OSCC
Our analysis revealed that 94 chemical barrier proteins showed significantly elevated amounts in the saliva of patients with OSCC compared with controls.From the 94 proteins that showed elevated expression, 77 AMPs (Table 1), 10 complement system proteins (Table 2), and 7 cytokines (Table 3) were identified.Tumor necrosis factor Immunomodulatory effect [96] Besides the upregulated salivary chemical barrier proteins, we identified 28 AMPs with significantly lower amounts in the saliva of patients with OSCC compared with the controls (Table 4).All downregulated proteins belonged to the AMP family.Transmembrane protease serine 11D Protease activity [114] Considering the number of chemical barrier proteins that showed altered expression between patients with OSCC and controls, the presence of the tumor in the oral cavity could alter the expression profile and/or secretion of AMPs and other barrier components, and could also alter the defense function of the barrier.
From the set of differentially expressed chemical barrier proteins identified by our meta-analysis, we found five salivary proteins where the data were contradictory.In some cases, the authors reported significantly elevated amounts in patients with OSCC, while other studies suggested decreased expression of the proteins compared with controls (Table 5).We examined the clinical data presented by the authors of the studies, and one of the possible reasons for the contradictory data could be the different grades and stages of the tumors.The clinical data suggest that the localization of the tumor in the oral cavity has a slight effect on the secreted chemical barrier proteins.Moreover, the difference between the studied cohorts was also a possible reason for the contradictory data, since the secreted components of saliva can vary between different populations.

Network Analysis of Chemical Barrier Proteins Affected by OSCC
In order to gain more insight into the biological consequences of the changes in the chemical barrier composition, the up-and down-regulated chemical barrier proteins were further subjected to network analysis.We used the STRING-DB (version 11.5) [117] and Cytoscape software (version 3.9.1)[118] to map the interactions between the proteins.Furthermore, to gain more insights into the biological functions of the revealed AMPs, we performed gene ontology (GO) analyses using ClueGO (version 2.5.9)[119] Cytoscape plug-in.
The protein-protein interaction networks of the up-regulated salivary chemical barrier proteins in OSCC with their 50 first shell interactors are presented in Figure 1 and Figure S1.
possible reasons for the contradictory data could be the different grades and stages of the tumors.The clinical data suggest that the localization of the tumor in the oral cavity has a slight effect on the secreted chemical barrier proteins.Moreover, the difference between the studied cohorts was also a possible reason for the contradictory data, since the secreted components of saliva can vary between different populations.

Network Analysis of Chemical Barrier Proteins Affected by OSCC
In order to gain more insight into the biological consequences of the changes in the chemical barrier composition, the up-and down-regulated chemical barrier proteins were further subjected to network analysis.We used the STRING-DB (version 11.5) [117] and Cytoscape software (version 3.9.1)[118] to map the interactions between the proteins.Furthermore, to gain more insights into the biological functions of the revealed AMPs, we performed gene ontology (GO) analyses using ClueGO (version 2.5.9)[119] Cytoscape plug-in.
The protein-protein interaction networks of the up-regulated salivary chemical barrier proteins in OSCC with their 50 first shell interactors are presented in Figures 1 and  S1.The analysis of the chemical barrier proteins with their first shell interactors detected 116 proteins with 334 connections.The network analysis revealed that most of the chemical barrier proteins were part of two core clusters that interacted with each other.Many different proteins with high numbers of interactions are present, such as apolipoprotein A1, antithrombin III, IL-6, and complement C3.On the other hand, we identified two additional clusters with a small number of proteins (FABP4 and LIPE; MUC7, MUC16, LGALS3, and LGALS3BP) and without interactions with the core network.
In order to identify the top hub proteins in the network of the chemical barrier proteins altered by OSCC, the datasets were analyzed by CluePedia [120] and CytoHubba [121].The top 10 identified hub proteins in the network of up-regulated proteins in OSCC are represented in Figure 2.
different proteins with high numbers of interactions are present, such as apolipoprotein A1, antithrombin III, IL-6, and complement C3.On the other hand, we identified two additional clusters with a small number of proteins (FABP4 and LIPE; MUC7, MUC16, LGALS3, and LGALS3BP) and without interactions with the core network.
In order to identify the top hub proteins in the network of the chemical barrier proteins altered by OSCC, the datasets were analyzed by CluePedia [120] and CytoHubba [121].The top 10 identified hub proteins in the network of up-regulated proteins in OSCC are represented in Figure 2. The hub proteins observed in the network of the up-regulated proteins were mainly apolipoproteins, cytokines, and haptoglobin (Figure 2), indicating their important function in the chemical barrier of the oral cavity.
To obtain functional information, the enriched GO terms were examined using ClueGO (Table S1), and the top 10 enriched GO terms were visualized in Figure 3.The hub proteins observed in the network of the up-regulated proteins were mainly apolipoproteins, cytokines, and haptoglobin (Figure 2), indicating their important function in the chemical barrier of the oral cavity.
To obtain functional information, the enriched GO terms were examined using ClueGO (Table S1), and the top 10 enriched GO terms were visualized in Figure 3.The GO functions enriched in the network of up-regulated proteins (Figure 3) w mainly related to defense mechanisms, such as defense, inflammatory, and humoral i mune responses.However, the cellular immune response, cytokine signaling pathwa wounding, and the regulation of protease activity were observed as well.
The data support that, during tumor development, the humoral immune respon are activated, creating an inflammatory environment that has already been linked to t development of OSCC [122][123][124].
We also examined the network of the chemical barrier proteins down-regulated The GO functions enriched in the network of up-regulated proteins (Figure 3) were mainly related to defense mechanisms, such as defense, inflammatory, and humoral immune responses.However, the cellular immune response, cytokine signaling pathways, wounding, and the regulation of protease activity were observed as well.
The data support that, during tumor development, the humoral immune responses are activated, creating an inflammatory environment that has already been linked to the development of OSCC [122][123][124].
We also examined the network of the chemical barrier proteins down-regulated in the saliva of patients with OSCC (Figure 4 and Figure S2).The network analysis identified 61 proteins with 158 connections and revealed one complex cluster that included most of the chemical barrier proteins in connection with most of the remaining AMPs.We also identified three additional small clusters (LACRT and SDC1; KNG1, KLKB1, and PRCP; GM2A, GLB1, NAGA, HEXA, HEXB, and CHIT1) without interactions with the core network.Compared with the network obtained from the up-regulated proteins, the network of the down-regulated chemical barrier proteins in OSCC showed a lower number of protein clusters.
The analysis of the top 10 hub proteins was also performed on the down-regulated proteins, but only the first shell interactor proteins were present as hubs.
The enriched GO terms in the network of down-regulated proteins were also examined (Table S2), and the top 10 enriched GO terms were visualized, as shown in Figure 5.The network analysis identified 61 proteins with 158 connections and revealed one complex cluster that included most of the chemical barrier proteins in connection with most of the remaining AMPs.We also identified three additional small clusters (LACRT and SDC1; KNG1, KLKB1, and PRCP; GM2A, GLB1, NAGA, HEXA, HEXB, and CHIT1) without interactions with the core network.Compared with the network obtained from the up-regulated proteins, the network of the down-regulated chemical barrier proteins in OSCC showed a lower number of protein clusters.
The analysis of the top 10 hub proteins was also performed on the down-regulated proteins, but only the first shell interactor proteins were present as hubs.
The enriched GO terms in the network of down-regulated proteins were also examined (Table S2), and the top 10 enriched GO terms were visualized, as shown in Figure 5.In the case of the network of down-regulated proteins, the enriched GO terms were mainly related to the adaptive and cellular immune responses, suggesting alterations in the immune response during tumor development.

Discussion
OSCC is a common type of head and neck carcinoma with high incidence and prevalence, creating a socio-economical burden.The mortality rate of the disease is high, mainly due to late diagnosis.Since the survival rate of the disease is low, it is extremely important to better understand the molecular mechanisms behind the progression and development of OSCC [125].In this study, we aimed to examine the chemical barrier proteins in the saliva of patients with OSCC and controls in order to investigate the effect of the tumor on the barrier of the oral cavity.Therefore, the scientific literature was reviewed and searched for datasets regarding the analysis of the protein content of saliva from the above-mentioned groups.
Altogether, we collected 30 datasets from the PubMed and ProteomeXchange repository that examined the differences in the salivary proteins of patients with OSCC and healthy controls.We collected the differentially expressed proteins based on the statistical analyses applied by the authors, and we identified the components of the salivary chemical barrier by searching in the UDAMP database.After evaluation, we found 127 proteins that showed different expression patterns between the OSCC and control groups.Of the 127 proteins, 94 were up-regulated (Tables 1-3) and 28 proteins were down-regulated in OSCC compared with controls (Table 4).We also found five proteins with contradictory expression profiles in the two groups; several studies indicated up-regulation and other studies indicated down-regulation of these five proteins (Table 5).Most of the up-regu- In the case of the network of down-regulated proteins, the enriched GO terms were mainly related to the adaptive and cellular immune responses, suggesting alterations in the immune response during tumor development.

Discussion
OSCC is a common type of head and neck carcinoma with high incidence and prevalence, creating a socio-economical burden.The mortality rate of the disease is high, mainly due to late diagnosis.Since the survival rate of the disease is low, it is extremely important to better understand the molecular mechanisms behind the progression and development of OSCC [125].In this study, we aimed to examine the chemical barrier proteins in the saliva of patients with OSCC and controls in order to investigate the effect of the tumor on the barrier of the oral cavity.Therefore, the scientific literature was reviewed and searched for datasets regarding the analysis of the protein content of saliva from the above-mentioned groups.
Altogether, we collected 30 datasets from the PubMed and ProteomeXchange repository that examined the differences in the salivary proteins of patients with OSCC and healthy controls.We collected the differentially expressed proteins based on the statistical analyses applied by the authors, and we identified the components of the salivary chemical barrier by searching in the UDAMP database.After evaluation, we found 127 proteins that showed different expression patterns between the OSCC and control groups.Of the 127 proteins, 94 were up-regulated (Tables 1-3) and 28 proteins were down-regulated in OSCC compared with controls (Table 4).We also found five proteins with contradictory expression profiles in the two groups; several studies indicated up-regulation and other studies indicated down-regulation of these five proteins (Table 5).Most of the up-regulated proteins belonged to the AMP family, but cytokines and complement system proteins were identified as well.In the case of the down-regulated proteins, only the members of the AMP family were identified.

Amylases and Mucins in the Chemical Barrier of Patients with OSCC
Amylases and mucins are the most abundant proteins in the saliva [17], maintaining the homeostatic functions, but also parts of the chemical barrier.Owing to their hydrolytic activity, amylases can alter the biofilm formation of bacteria by cleaving the polysaccharide backbone of extracellular polymeric structures [97].However, evidence shows that amylase can bind to the amylase-binding protein of Streptococcus species and can induce biofilm formation [97]; therefore, the effect of amylases in the formation of bacterial biofilms is still not clear.Mucins are high-molecular-weight glycoproteins also acting in the chemical barrier.MUC5B and MUC7 can interact with a variety of bacteria, such as Streptococcus species and Pseudomonas aeruginosa, and pathogenic fungi, such as Candida albicans, to prevent the activity and further invasion of these microorganisms [77].Our analyses revealed elevated levels of MUC7 and MUC16 in the saliva of patients with OSCC compared with controls (Table 1), indicating their role in tumor progression.We also identified that the level of amylases was down-regulated in patients with OSCC compared with controls (Table 4).Since the major salivary proteins are affected by OSCC, further study would be necessary to gain more insight into the role of these proteins in cancer development.

Proteases and Protease Inhibitors in the Salivary Chemical Barrier of Patients with OSCC
Besides amylases, other hydrolases, such as proteases, are also affected by OSCC.Proteases and protease inhibitors are constitutive parts of each chemical barrier of the human body.A variety of cells express and secrete a wide range of proteolytic enzymes in order to defend the host against potential pathogens by the degradation of proteins involved in the life cycle of pathogens.Saliva contains a broad spectrum of proteases and, since salivary proteases were recognized as potential biomarkers for oral cancer [126], they have high relevance in the homeostatic and pathological processes of the oral cavity.Since proteases are double-edged swords capable of degrading host proteins as well, protease inhibitors are crucial for the host.While the protease inhibitors of the host provide a defense against their own proteases, they can also inhibit the proteases secreted by pathogenic microorganisms [127], indicating their important role in chemical barriers.The list of the differentially expressed chemical barrier proteins in OSCC contained many proteases.The levels of MMP1, MMP9, myeloblastin, prostasin, and stromelysin-1 were elevated in the saliva of patients with OSCC, while lower amounts of ER aminopeptidase 2, kallikrein 11, and lysosomal Pro-X carboxypeptidase were identified compared with controls (Tables 1 and 4).Many protease inhibitors, such as SERPINs and inter-alphatrypsin inhibitor heavy chains, showed elevated expression profiles in OSCC (Table 1), while other protease inhibitors, like cystatins and SPINK5, showed reduced expression profiles in OSCC compared with controls (Table 4).Our results indicate that the proteolytic and anti-proteolytic activity of the saliva is altered in patients with OSCC; however, the alteration may not shift the balance between the proteases and protease inhibitors, since the effect of up-regulated proteins can be balanced with the down-regulation of other proteases and protease inhibitors.

Contribution of Cytokines in the Salivary Chemical Barrier of Patients with OSCC
The intention to maintain the balance in homeostatic functions can also be observed in the case of up-regulated pro-and anti-inflammatory cytokines.Cytokines are small proteins secreted by a variety of cells and have specific effects on the interactions and communications between cells.Cytokines can fulfill autocrine, paracrine, or endocrine actions and have pleiotropic effects on target cells [96].The fact that cytokines are part of the top 10 hub proteins in the network of chemical barrier proteins up-regulated in OSCC (Figure 2) highlights the importance of these proteins in tumor development.Proinflammatory cytokines, such as IL-1α, IL-1β, IL-6, and TNF, showed higher amounts in the saliva of patients with OSCC, indicating the involvement of the inflammatory environment in tumor progression [128].Additionally, our workgroup demonstrated that IL-6 is a robust biomarker for OSCC in saliva [28].However, along with the pro-inflammatory cytokines, the levels of Il-10 and IL-13 anti-inflammatory cytokines were upregulated in OSCC as well (Table 3).This suggests that the body tries to fight against the inflammatory pathways activated by the pro-inflammatory cytokines and tries to keep the balance between the proand anti-inflammatory processes.

Members of S100 Protein Family Are Affected by OSCC
Besides cytokines, other proteins, such as S100 proteins, can also participate in the regulation of cellular responses.Members of the S100 family are Ca 2+ -binding proteins with potent antimicrobial activity against pathogens [129].The secreted forms of S100 proteins also have paracrine effects by regulating different cell types, such as immune cells, endothelial cells, and muscle cells [129].Our analysis revealed that eight S100 proteins (S100A2, S100A7, S100A7A, S100A8, S100A9, S100A11, S100A12, and S100P) were upregulated in the saliva of patients with OSCC compared with controls (Table 1), while a lower amount of S100A4 was identified in OSCC compared with controls (Table 4).S100A7, or psoriasin, is a well-known potent AMP that mainly can be found on the surface of the skin [20].S100A9 was also identified by our workgroup as a potential salivary biomarker of OSCC [26].Our examination revealed that many S100 proteins are affected by OSCC, indicating their important role in the pathomechanism of this type of cancer.

OSCC Can Enhance the Complement System in the Salivary Chemical Barrier
While the different mediators can activate a variety of cellular responses, the humoral immune response can be activated as well, such as antibody production by plasma cells or the activation of the complement system.The complement system is part of the immune system that enhances the clearance of microorganisms and damaged cells, promotes inflammatory responses, and disrupts the cell membrane of pathogens [95].The complement system is composed of several small proteins mainly synthesized by the liver in precursor forms [95].The activation of the complement proteins follows a cascade model; after activation, proteases in the system cleave their targets and start the cascade system.Our analysis revealed 10 complement system proteins that were up-regulated in the saliva of patients with OSCC compared with controls (Table 2).Complement proteins are already associated with oral cancer [130], and evidence suggests that they have important roles in the tumor microenvironment as well [131].Therefore, complement proteins are possible targets for anticancer therapies [131].Our network analysis highlighted that some complement proteins, such as complement C3 and complement C9, are hub proteins that are important in communication with the other subnetworks (Figure 1).

Elevated Levels of Apolipoproteins in the Saliva of Patients with OSCC
The levels of many apolipoprotein forms, such as Apo AI, Apo B-100, Apo D, and Apo E, were elevated in the saliva of patients with OSCC (Table 1).The main function of these proteins is the construction of lipoproteins, such as chylomicron, VLDL, LDL, or HDL, that carry triglycerides, cholesterol, cholesterol esters, and other types of lipids in the circulation system [132].Besides their important role in lipid transport, apoproteins also take part in host defense mechanisms.The antimicrobial activity of Apo A1 against Staphylococcus epidermidis has been described [42] and antimicrobial peptides derived from the further processing of Apo B acting against Salmonella strains have also been identified [133].Our results indicate that apolipoproteins are important hub proteins in the protein-protein interaction network (Figure 2), highlighting the importance of apoproteins in the chemical barrier of the oral cavity.

Salivary Chemical Barrier Proteins with Contradictory Expression Profile in OSCC
Among proteins that were clearly up-or down-regulated in the saliva of patients with OSCC, five AMPs were found to be differentially expressed between OSCC and control groups, but the way that they changed was contradictory (Table 5).There is evidence in the scientific literature of either the up-and down-regulation of these salivary proteins in OSCC.The possible reason for this contradiction may be the different stages of the tumors and the difference between the studied cohorts.One of our previous studies highlighted the importance of population-tailored studies [26].Depending on the different sex, age, and ethnicity of the studied cohorts, the expression profiles of the salivary chemical barrier proteins may be different from each other.

Alterations in the Defense Mechanism of the Oral Cavity
Oral microorganisms, including bacteria, archaea, fungi, viruses, and protozoa, are closely associated with the physiological/pathological state of the oral cavity.Currently, more than 1000 bacterial species are known in the oral cavity [134], including Actinobacteria, Bacteroidetes, Chlamydia, Euryarchaeota, Fusobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Tenericutes species [135].Along with the classical proteomics studies [136], metaproteomics approaches have emerged for examining the connections between the proteome of the oral cavity and the proteome of the oral microbiota [137][138][139].Studies described that oral microbiome dysbiosis can lead to the development of pathological changes in the oral cavity, such as caries and periodontal diseases [140], and is also associated with systemic diseases, such as obesity, diabetes [134], lung cancer [137], and oral cancer [134].Our analysis revealed that OSCC can alter the secretion of many chemical barrier proteins in the saliva and, as the tumor alters the protein content of the chemical barrier, the defense mechanism of the oral cavity can change, allowing uncontrolled proliferation of distinct microbial species.Data in the literature highlight that HPV, Porphyromonas gingivalis, Fusobacterium nucleatum, Peptostreptococcus, Filifactor, Parvimonas, Pseudomonas, Campylobacter, and Staphylococcus species are known pathogens that participate in the development of OSCC [13,14].One of the possible reasons for the dysbiosis could be the change in the defense function of the chemical barrier caused by the altered expression of the defense and regulatory proteins.As OSCC is still an emerging problem in our society, it is extremely important to better understand the biological events leading to tumor progression.The analysis of the chemical barrier proteins and their interactions with the oral microbiome can highlight additional layers of the host-tumor interactions and can be important for the design of new possible therapies.Therefore, further studies are needed to investigate the defense mechanism of the saliva against the above-mentioned pathogens involved in tumor development.

Limitations of the Study
In this study, we revealed that the differential expression and/or secretion of chemical barrier proteins into saliva could be linked to OSCC and we highlighted the importance of this protein family in the progression of OSCC.However, the majority of the data related to differentially expressed proteins originated from survey studies, and most of the changes have not been validated yet.Therefore, the validation of the changes in these proteins will be an important task in further studies.While the available databases contain a huge amount of information, our knowledge of protein-protein interactions is constantly improving, revealing more and more interaction partners of specific proteins.Thus, our interaction networks represent our current knowledge of the interactions between the proteins making up the chemical barrier and may change when databases are further updated.Our study focused only on the composition of the salivary chemical barriers; however, analyses of cellular responses [141] and small molecules secreted into saliva [142] can add additional layers to our knowledge of the development and progression of OSCC.

Examination of Chemical Barrier Proteins in OSCC Datasets
Datasets involving human saliva samples from patients with OSCC and healthy controls were retrieved from PubMed [31] and ProteomeXchange [30] until May 2023.Datasets were selected for examination if samples from patients with OSCC and matched controls were examined and their comparison was carried out.Altogether, 30 datasets were selected for examination (Table 6).The chemical barrier proteins were searched in the downloaded datasets by using the UDAMP database [32].Those chemical barrier proteins whose level showed a statistically significant change in OSCC samples compared with controls were listed and assigned for further analyses.Our evaluation relied on the results of the statistical analysis performed by the authors of the datasets.
In order to create and analyze the interaction networks of the differentially expressed chemical barrier proteins, the STRING-DB and Cytoscape software programs were used, as described by Kumar et al. [32].The differentially expressed proteins originated from the statistical analyses performed by the authors.Briefly, interaction networks of the differentially expressed proteins and up to 50 of their first shell interactors were retrieved.The top 10 genes based on their network degree were imported into ClueGO in order to examine the GO terms enriched in the networks of chemical barrier proteins.The gene names were uploaded and, after species selection (Homo sapiens [9606]), "Functional Analysis" in the "Analysis Mode" menu was used and pathways were searched by setting the "Significance" in the "Visual Style" menu and to "Show only Pathways with pV ≤ 0.05000".All the other settings remained as default.The top 10 enriched GO terms were selected based on the number of detected genes.The gene visualization threshold for the CluePedia analysis was set to 1000 and the other analysis parameters were set to default.The identification of hub proteins was performed using the CytoHubba plug-in and the top 10 nodes ranked by degree were selected.

Conclusions
In this study, we highlighted the importance of the chemical barrier of saliva in the development of OSCC.As pathogenic microorganisms can participate in the development of OSCC, the alteration in the defense function of the saliva may contribute to tumor progression.Our study can serve as a starting point for further examinations regarding the possible link between the altered defense mechanism and tumor progression.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ijms241713657/s1, Figure S1: Network view of the interaction network of the upregulated chemical barrier proteins in saliva from patients with OSCC; Figure S2: Network view of the interaction network of the downregulated chemical barrier proteins in saliva from patients

Figure 1 .
Figure 1.The interaction network of the upregulated chemical barrier proteins in saliva from patients with OSCC.Each circle represents a protein and the lines indicate interactions.The proteins are labeled with their gene name.The high-resolution image of this network is presented in Figure S1.

Figure 2 .
Figure 2. Top 10 hub proteins in the network of salivary chemical barrier proteins upregulated in OSCC.The proteins are labeled with their gene name.A darker color means a higher number of connections.

Figure 2 .
Figure 2. Top 10 hub proteins in the network of salivary chemical barrier proteins upregulated in OSCC.The proteins are labeled with their gene name.A darker color means a higher number of connections.

Figure 3 .
Figure 3. Top 10 enriched GO terms for chemical barrier proteins up-regulated in the saliva of tients with OSCC.The enriched GO terms were ordered according to the gene count.

Figure 3 .
Figure 3. Top 10 enriched GO terms for chemical barrier proteins up-regulated in the saliva of patients with OSCC.The enriched GO terms were ordered according to the gene count.

22 Figure 4 .
Figure 4. Network view of the interaction network of the down-regulated chemical barrier proteins in saliva from patients with OSCC.Each circle represents a protein, and the lines indicate interactions.The proteins are labeled with their gene name.The high-resolution image of this network is presented in Figure S2.

Figure 4 .
Figure 4. Network view of the interaction network of the down-regulated chemical barrier proteins in saliva from patients with OSCC.Each circle represents a protein, and the lines indicate interactions.The proteins are labeled with their gene name.The high-resolution image of this network is presented in Figure S2.

Figure 5 .
Figure 5. Top 10 enriched GO terms for chemical barrier proteins down-regulated in the saliva of patients with OSCC.The enriched GO terms were ordered according to the gene count.

Figure 5 .
Figure 5. Top 10 enriched GO terms for chemical barrier proteins down-regulated in the saliva of patients with OSCC.The enriched GO terms were ordered according to the gene count.

Table 1 .
AMPs with increased amount in saliva of patients with OSCC compared with controls.The functions of proteins in the chemical barrier are indicated as well.

Table 2 .
Complement system components with increased amounts in saliva of patients with OSCC compared with controls.The functions of the proteins in the chemical barrier are indicated as well.

Table 3 .
Cytokines with increased amount in saliva of patients with OSCC compared with controls.The functions of the proteins in the chemical barrier are indicated as well.

Table 4 .
AMPs with decreased amounts in saliva of patients with OSCC compared with controls.The functions of the proteins in the chemical barrier are indicated as well.

Table 5 .
AMPs with contradictory expression profiles in the saliva of patients with OSCC compared with controls.Different studies showed elevated or decreased expression in saliva.The functions of the proteins in the chemical barrier are indicated as well.

Table 6 .
List of datasets used in this study.The dataset identifier and the name of the source database along with references are listed for each processed dataset.