Identification of Host-Immune Response Protein Candidates in the Sera of Human Oral Squamous Cell Carcinoma Patients

One of the most common cancers worldwide is oral squamous cell carcinoma (OSCC), which is associated with a significant death rate and has been linked to several risk factors. Notably, failure to detect these neoplasms at an early stage represents a fundamental barrier to improving the survival and quality of life of OSCC patients. In the present study, serum samples from OSCC patients (n = 25) and healthy controls (n = 25) were subjected to two-dimensional gel electrophoresis (2-DE) and silver staining in order to identify biomarkers that might allow early diagnosis. In this regard, 2-DE spots corresponding to various up- and down-regulated proteins were sequenced via high-resolution MALDI-TOF mass spectrometry and analyzed using the MASCOT database. We identified the following differentially expressed host-specific proteins within sera from OSCC patients: leucine-rich α2-glycoprotein (LRG), alpha-1-B-glycoprotein (ABG), clusterin (CLU), PRO2044, haptoglobin (HAP), complement C3c (C3), proapolipoprotein A1 (proapo-A1), and retinol-binding protein 4 precursor (RBP4). Moreover, five non-host factors were detected, including bacterial antigens from Acinetobacter lwoffii, Burkholderia multivorans, Myxococcus xanthus, Laribacter hongkongensis, and Streptococcus salivarius. Subsequently, we analyzed the immunogenicity of these proteins using pooled sera from OSCC patients. In this regard, five of these candidate biomarkers were found to be immunoreactive: CLU, HAP, C3, proapo-A1 and RBP4. Taken together, our immunoproteomics approach has identified various serum biomarkers that could facilitate the development of early diagnostic tools for OSCC.     

Evaluation of salivary endothelin-1 levels in oral squamous cell carcinoma and oral leukoplakia

Oral squamous cell carcinoma (OSCC) is the most frequent malignant neoplasia of the oral cavity, which largely compromises the patient's life quality. Therefore, the identification of biomarkers for this kind of cancer is essential to provide a better diagnosis and prognosis for patients. Endothelin-1 is a peptide produced mainly by endothelial cells, and might be found in several body fluids, such as saliva, milk, urine, cerebrospinal fluid and plasma. It has been demonstrated that expression of this peptide is increased in a great number of neoplasias, including oral carcinoma. The identification of salivary biomarkers would be a useful tool for scanning and monitoring patients with risk of developing OSCC, as well to early detect recurrence, or the formation of a new primary tumor. In the present study, we have analyzed the levels of endothelin-1 in saliva obtained from patients with OSCC or oral leukoplakia, in comparison to healthy control patients. This study also evaluated the salivary ET-1 levels in patients with complete remission of OSCC. The results revealed no statistical difference in salivary endothelin-1 levels, neither in OSCC nor in oral leukoplakia, even when conditions such as elderly, sex and hypertension were taken into consideration. Although, ET-1 might display an important role in OSCC, its levels in saliva do not seem to be a good marker of neoplasias grade or malignant transformation.     

Saliva proteome profiling reveals potential salivary biomarkers for detection of oral cavity squamous cell carcinoma

Oral cavity squamous cell carcinoma (OSCC), which is frequently associated with poor prognosis and mortality, is a leading cause of cancer‐related death worldwide. Discovery of body fluid accessible biomarkers is needed to improve OSCC screening. To this end, we profiled proteomes of saliva from the healthy volunteers, the individuals with oral potentially malignant disorders (OPMD), and the OSCC patients by means of SDS‐PAGE coupled with LC‐MS/MS. In the control, the OPMD, and the OSCC groups, 958, 845, and 1030 salivary proteins were detected, respectively. With spectral counting‐based label‐free quantification, 22 overexpressed salivary proteins were identified in the OSCC group compared with the healthy controls and the OPMD individuals. Among them, resistin (RETN) was subjected to further validation with an independent cohort using ELISA. The data confirmed that the salivary RETN levels in the OSCC patients were significantly higher than that in the healthy or in the OPMD group. Moreover, the elevated levels of salivary RETN were highly correlated with late‐stage primary tumors, advanced overall stage, and lymph‐node metastasis. Our results not only reveal that profiling of saliva proteome is feasible for discovery of OSCC biomarkers, but also identify RETN as a potential salivary biomarker for OSCC detection.     

Screening for potential serum biomarkers in rat mesangial proliferative nephritis

Mesangial proliferative nephritis (MesPGN) is a common kidney disease worldwide. The main feature of the disease is mesangial cell proliferation‐induced injury to kidney function. In this study, we explored serum biomarkers for kidney function injury in anti‐Thy1 nephritis. We found that mesangial proliferation were increased on days 5 and 7, and recovered by day 14 in anti‐Thy1 nephritis. 24‐h urine protein, the ratio of urine protein to urine creatine, serum creatine, and blood urea nitrogen, were increased at days 5 and 7 in the model. We found that TXN, BET1, PrRP, VGF, and NPS differed strongly from controls on days 5 and, associated with kidney injury when detected by SELDI‐TOF MS. Moreover, we applied LC‐MS to detect differential protein expression and found A2M, C3, ITIH4, ITIH3, VDBP, AFM, and SERPINF2 to be upregulated, and ES1, HPX, SERPINC1, SERPINA1F, SERPINA4, SERPINA3K, SPI, TF, VNN3, SERPINF1, and PON1 to be downregulated, on days 5 and 7, associated with kidney injury. The levels of VNN3 and VDBP were validated by Western blotting. Overall, this study explored a group of candidate biomarkers of mesangial proliferation inducing kidney injury, to provide the basis of an assessment model for MesPGN in the future.     

Meta-omics analysis indicates the saliva microbiome and its proteins associated with the prognosis of oral cancer patients

Saliva is a biofluid that maintains the health of oral tissues and the homeostasis of oral microbiota. Studies have demonstrated that Oral squamous cell carcinoma (OSCC) patients have different salivary microbiota than healthy individuals. However, the relationship between these microbial differences and clinicopathological outcomes is still far from conclusive. Herein, we investigate the capability of using metagenomic and metaproteomic saliva profiles to distinguish between Control (C), OSCC without active lesion (L0), and OSCC with active lesion (L1) patients. The results show that there are significantly distinct taxonomies and functional changes in L1 patients compared to C and L0 patients, suggesting compositional modulation of the oral microbiome, as the relative abundances of Centipeda, Veillonella, and Gemella suggested by metagenomics are correlated with tumor size, clinical stage, and active lesion. Metagenomics results also demonstrated that poor overall patient survival is associated with a higher relative abundance of Stenophotromonas, Staphylococcus, Centipeda, Selenomonas, Alloscordovia, and Acitenobacter. Finally, compositional and functional differences in the saliva content by metaproteomics analysis can distinguish healthy individuals from OSCC patients. In summary, our study suggests that oral microbiota and their protein abundance have potential diagnosis and prognosis value for oral cancer patients. Further studies are necessary to understand the role of uniquely detected metaproteins in the microbiota of healthy and OSCC patients as well as the crosstalk between saliva host proteins and the oral microbiome present in OSCC.     

Downregulation of Microrna‐148a in Cancer‐Associated Fibroblasts from Oral Cancer Promotes Cancer Cell Migration and Invasion by Targeting Wnt10b

It is well established that crosstalk between cancer‐associated fibroblasts (CAFs) and cancer cells plays a critical role in the occurrence and development of oral squamous cell carcinoma (OSCC). The molecular mechanisms underlying such interaction, however, remain far from clear. Accumulating data have indicated that microRNAs involved in tumor microenvironment, particularly in CAFs, contribute to the activation of fibroblasts and metastasis of cancer cells. Here, we showed that miR‐148a was downregulated in CAFs compared with normal fibroblasts isolated from clinical OSCC tissue. Investigation of miR‐148a function in fibroblasts demonstrated that overexpression of miR‐148a in CAFs significantly impaired the migration and invasion of oral carcinoma cells (SCC‐25) by directly targeting WNT10B. Taken together, these data suggested that miR‐148a might be a novel candidate target for the treatment of OSCC.     

Analysis of tissue and salivary nicotinamide N-methyltransferase in oral squamous cell carcinoma: basis for the development of a noninvasive diagnostic test for early-stage disease

Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer worldwide. In recent decades, the 5-year mortality rate is approximately 50% around the world. As reliable biomarkers of oral cancer are still lacking, it is necessary to identify new target molecules for early diagnosis, effective therapy, and monitoring of the disease. In the present work, we focused on the expression of the enzyme nicotinamide N-methyltransferase (NNMT). We analyzed enzyme activity in 37 paired tumor and non-tumor tissues and found that activity levels are significantly higher in tumor compared with adjacent normal oral mucosa. Interestingly, oral epithelium surrounding tumor of unfavorable cases (N+) seems to display higher activity levels compared with that of favorable ones (N0). Western blot analyses were performed to evaluate protein levels in saliva samples from patients with OSCC and healthy subjects. Preliminary results indicated an up-regulation of salivary NNMT in tumor. This study shows a marked increase in enzyme activity in oral cancer and suggests that adjacent normal tissue of unfavorable cases seems to change toward cancer. Moreover, it is conceivable to hypothesize that NNMT could represent a potential biomarker for early and non-invasive diagnosis of oral cancer.     

Screening and identification of autophagy-related biomarkers for oral squamous cell carcinoma (OSCC) via integrated bioinformatics analysis

Increasing evidences have showed that autophagy played a significant role in oral squamous cell carcinoma (OSCC). Purpose of our study was to explore the prognostic value of autophagy-related genes (ATGs) and screen autophagy-related biomarkers for OSCC. RNA-seq and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database following extracting ATG expression profiles. Then, differentially expressed analysis was performed in R software and a risk score model according to ATGs was established. Moreover, comprehensive bioinformatics analyses were used to screen autophagy-related biomarkers which were later verified in OSCC tissues and cell lines. A total of 232 ATGs were extracted, and 37 genes were differentially expressed in OSCC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that these genes were mainly located in autophagosome membrane and associated with autophagy. Furthermore, the risk score on basis of ATGs was identified as potential independent prognostic biomarker. Moreover, ATG12 and BID were identified as potential autophagy-related biomarkers of OSCC. This study successfully constructed a risk model, and the risk score could predict the prognosis of OSCC patients accurately. Moreover, ATG12 and BID were identified as two potential independent prognostic autophagy-related biomarkers and might provide new OSCC therapeutic targets.     

Diagnostic model of saliva peptide finger print analysis of oral squamous cell carcinoma patients using weak cation exchange magnetic beads

Saliva diagnostics utilizing nanotechnology and molecular technologies to detect oral squamous cell carcinoma (OSCC) has become an attractive field of study. However, no specific methods have been established. To refine the diagnostic power of saliva peptide fingerprints for the early detection of OSCC, we screened the expression spectrum of salivary peptides in 40 T1 stage OSCC patients (and healthy controls) using MALDI-TOF-MS combined with magnetic beads. Fifty proteins showed significantly different expression levels in the OSCC samples (P<0.05). Potential biomarkers were also predicted. The novel diagnostic proteomic model with m/z peaks of 1285.6 Da and 1432.2 Da are of certain value for early diagnosis of OSCC.     

S100A8 is identified as a biomarker of HPV18-infected oral squamous cell carcinomas by suppression subtraction hybridization, clinical proteomics analysis, and immunohistochemistry staining

The purpose of this work is to differentiate between the Human papillomaviruses 18 positive (HPV18+) and negative (HPV18-) oral squamous cell carcinomas (OSCC) in oral cancer patients with cancer-associated oral habits (betel quid chewing, cigarette smoking, and alcohol drinking). Both gene and protein expression profiles of HPV18+ and HPV18- OSCC were compared: we then further explored the biological effect of HPV in oral cancer. Suppression subtraction hybridization (SSH), clinical proteomics analysis, and immunohistochemistry (IHC) staining were carried out in the HPV18+ and HPV18- OSCC groups. HPV typing detection revealed that 11 OSCC tissues from 82 patients were positive for HPV18. The SSH experiment showed that 4 cancer-associated genes were highly transcribed within 11 cDNA libraries of HPV18+ OSCC, including poly(ADP-ribose)polymerase I (PARP1), replication protein A2 (RPA2), S100A8, and S100A2. Clinical proteomics analysis indicated that there was over 10-fold overexpression of Stratifin, F-actin capping protein alpha-1 subunit (CapZ alpha-1), Apolipoprotein A-1 (ApoA-1), Heat-shock protein 27 (HSP27), Arginase-1, p16INK4A, and S100 calcium-binding protein A8 (S100A8) in HPV18+ OSCC. Interestingly, the results from SSH and protemics analysis showed that S100A8 was overexpressed in HPV18+ OSCC. Moreover, IHC staining demonstrated that S100A8 was up-regulated in HPV18+ OSCC tissues. Our results suggest that S100A8 plays an important role in oral carcinogenesis following HPV18 infection; therefore, S100A8 may be a powerful biomarker of HPV18 as well as a potential therapeutic target for HPV18+ OSCC patients. The study is the first to identify S100A8 as a biomarker in HPV-associated cancer. Furthermore, this is also the first study to discover a biomarker by combining SSH, clinical proteomics, and IHC stain analysis in oral cancer-associated research.     

IL‐1β Promotes Malignant Transformation and Tumor Aggressiveness in Oral Cancer

Chronic inflammation, coupled with alcohol, betel quid, and cigarette consumption, is associated with oral squamous cell carcinoma (OSCC). Interleukin‐1 beta (IL‐1β) is a critical mediator of chronic inflammation and implicated in many cancers. In this study, we showed that increased pro‐IL‐1β expression was associated with the severity of oral malignant transformation in a mouse OSCC model induced by 4‐Nitroquinolin‐1‐oxide (4‐NQO) and arecoline, two carcinogens related to tobacco and betel quid, respectively. Using microarray and quantitative PCR assay, we showed that pro‐IL‐1β was upregulated in human OSCC tumors associated with tobacco and betel quid consumption. In a human OSCC cell line TW2.6, we demonstrated nicotine‐derived nitrosamine ketone (NNK) and arecoline stimulated IL‐1β secretion in an inflammasome‐dependent manner. IL‐1β treatment significantly increased the proliferation and dysregulated the Akt signaling pathways of dysplastic oral keratinocytes (DOKs). Using cytokine antibodies and inflammation cytometric bead arrays, we found that DOK and OSCC cells secreted high levels of IL‐6, IL‐8, and growth‐regulated oncogene‐α following IL‐1β stimulation. The conditioned medium of IL‐1β‐treated OSCC cells exerted significant proangiogenic effects. Crucially, IL‐1β increased the invasiveness of OSCC cells through the epithelial‐mesenchymal transition (EMT), characterized by downregulation of E‐cadherin, upregulation of Snail, Slug, and Vimentin, and alterations in morphology. These findings provide novel insights into the mechanism underlying OSCC tumorigenesis. Our study suggested that IL‐1β can be induced by tobacco and betel quid‐related carcinogens, and participates in the early and late stages of oral carcinogenesis by increasing the proliferation of dysplasia oral cells, stimulating oncogenic cytokines, and promoting aggressiveness of OSCC. J. Cell. Physiol. 230: 875–884, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Metabolic changes during malignant transformation in primary cells of oral lichen planus: Succinate accumulation and tumour suppression

Oral squamous cell carcinoma (OSCC) is usually diagnosed at late stages, which leads to high morbidity. There are evidence that chronic inflammation (eg oral lichen planus [OLP]) was a risk factor of OSCC, but often misdiagnosed or ignored until invasion and metastasis. By applying precision medicine, the molecular microenvironment variations and relevant biomarkers for the malignant transformation from OLP to OSCC can be fully investigated. Several studies pointed out that the metabolic pathway were suppressed in OSCC. However, it remains unclear how the systemic profile of the metabolites change during the malignant transformation. In this study, we examined and compared the mucosa samples from 11 healthy individuals, 10 OLP patients and 21 OSCC patients. Based on the results, succinate, a key metabolite of the tricarboxylic acid cycle pathway, was accumulated in the primary cultured precancerous OLP keratinocytes and OSCC cells. Then, we found that succinate activated the hypoxia‐inducible factor‐1 alpha (HIF‐1α) pathway and induced apoptosis, which could also be up‐regulated by the tumour suppressor lncRNA MEG3. These results suggested the critical roles of succinate and MEG3 in the metabolic changes during malignant transformation from OLP to OSCC, which indicated that succinate, HIF1α and downstream proteins might serve as new biomarkers of precancerous OLP for early diagnosis and therapeutic monitoring. In addition, succinate or its prodrugs might become a potential therapy for the prevention or treatment of OSCC.     

MicroRNA alterations and associated aberrant DNA methylation patterns across multiple sample types in oral squamous cell carcinoma

Background

MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.

Methods

TaqMan® qRT-PCR arrays and individual assays were used to profile miRNA expression in a panel of 25 tumors with matched adjacent tissues from patients with OSCC, and 8 control paired oral stroma and epithelium from healthy volunteers. Associated DNA methylation changes of candidate epigenetically deregulated miRNA genes were measured in the same samples using the MassArray® mass spectrometry platform. MiRNA expression and DNA methylation changes were also investigated in FACS sorted CD44^high oral cancer stem cells from primary tumor samples (CSCs), and in oral rinse and saliva from 15 OSCC patients and 7 healthy volunteers.

Results

MiRNA expression patterns were consistent in healthy oral epithelium and stroma, but broadly altered in both tumor and adjacent tissue from OSCC patients. MiR-375 is repressed and miR-127 activated in OSCC, and we confirm previous reports of miR-137 hypermethylation in oral cancer. The miR-200 s/miR-205 were epigenetically activated in tumors vs normal tissues, but repressed in the absence of DNA hypermethylation specifically in CD44^high oral CSCs. Aberrant miR-375 and miR-200a expression and miR-200c-141 methylation could be detected in and distinguish OSCC patient oral rinse and saliva from healthy volunteers, suggesting a potential clinical application for OSCC specific miRNA signatures in oral fluids.

Conclusions

MiRNA expression and DNA methylation changes are a common event in OSCC, and we suggest miR-375, miR-127, miR-137, the miR-200 family and miR-205 as promising candidates for future investigations. Although overall activated in OSCC, miR-200/miR-205 suppression in oral CSCs indicate that cell specific silencing of these miRNAs may drive tumor expansion and progression.     

Circular RNA circMDM2 accelerates the glycolysis of oral squamous cell carcinoma by targeting miR‐532‐3p/HK2

Circular RNAs (circRNAs) function as an essential regulator in the progression of oral squamous cell carcinoma (OSCC). However, the potential roles and mechanism of circRNAs in OSCC are still elusive. Here, this research investigates the roles and molecular mechanism of novel circRNA (circMDM2) in OSCC progression. Clinically, circMDM2 was overexpressed in OSCC tissue and cells, and the overexpression served as a poor prognostic factor for OSCC patients. Functionally, cellular experiments confirmed that circMDM2 accelerated OSCC cell proliferation and glycolysis in vitro and circMDM2 knockdown repressed the tumour growth in vivo. Mechanistically, circMDM2 sponged miR‐532‐3p to promote the hexokinase 2 (HK2), forming the circMDM2/miR‐532‐3p/HK2 axis. In conclusion, these findings demonstrated that circMDM2/miR‐532‐3p/HK2 axis promotes the proliferation and glycolysis of OSCC, rendering a potential diagnostic biomarker and prospective therapeutic target for OSCC.     

Comprehensive bioinformatics analysis combined with experimental validation to screen biomarkers for malignant transformation of oral leukoplakia

Oral leukoplakia (OLK) is the most common potentially malignant disorders in the oral cavity. This study aimed to screen the key genes of OLK malignant transformation using the Gene Expression Omnibus (GEO) database and experiments. In this study, the GEO database was employed to screen OLK malignant transformation-related genes, which were subsequently identified with a series of bioinformatic analyses. External validation showed that the model based on LAPTM4B, NR3C1, and COX6A1 had high accuracy in diagnosing OLK malignant transformation. Furthermore, the DMBA-induced potentially malignant disorders and OSCC models in vivo and real-time PCR experiment in vitro further verified the database analysis results. In conclusion, three key genes (LAPTM4B, NR3C1, and COX6A1) were screened as potential biomarkers for the diagnosis and treatment of OLK malignant transformation.     

Proteomic and phosphoproteomic landscape of salivary extracellular vesicles to assess OSCC therapeutical outcomes

Circulating extracellular vesicles (EVs) have emerged as an appealing source for surrogates to evaluate the disease status. Herein, we present a novel proteomic strategy to identify proteins and phosphoproteins from salivary EVs to distinguish oral squamous cell carcinoma (OSCC) patients from healthy individuals and explore the feasibility to evaluate therapeutical outcomes. Bi-functionalized magnetic beads (BiMBs) with Ti (IV) ions and a lipid analog, 1,2-Distearoyl-3-sn-glycerophosphoethanolamine (DSPE) are developed to efficiently isolate EVs from small volume of saliva. In the discovery stage, label-free proteomics and phosphoproteomics quantification showed 315 upregulated proteins and 132 upregulated phosphoproteins in OSCC patients among more than 2500 EV proteins and 1000 EV phosphoproteins, respectively. We further applied targeted proteomics by coupling parallel reaction monitoring with parallel accumulation-serial fragmentation (prm-PASEF) to measure panels of proteins and phosphoproteins from salivary EVs collected before and after surgical resection. A panel of three total proteins and three phosphoproteins, most of which have previously been associated with OSCC and other cancer types, show sensitive response to the therapy in individual patients. Our study presents a novel strategy to the discovery of effective biomarkers for non-invasive assessment of OSCC surgical outcomes with small amount of saliva.     

Serum biomarker discovery related to pathogenesis in acute coronary syndrome by proteomic approach

Acute coronary syndrome (ACS) results from inadequate supply of blood flow from the coronary arteries to the heart or ischemia. ACS has an extremely high morbidity and mortality. The levels of biomarkers currently used for detection of ACS also increase in response to myocardial necrosis and other diseases and are not elevated immediately after symptoms appear, thus limiting their diagnostic capacity. Therefore, we aimed to discover new ACS diagnostic biomarkers with high sensitivity and specificity that are specifically related to ACS pathogenesis. Sera from 50 patients with ACS and healthy controls (discovery cohort) each were analyzed using mass spectrometry (MS) to identify differentially expressed proteins, and protein candidates were evaluated as ACS biomarkers in 120 people in each group (validation cohort). α-1-acid glycoprotein 1 (AGP1), complement C5 (C5), leucine-rich α-2-glycoprotein (LRG), and vitronectin (VN) were identified as biomarkers whose levels increase and gelsolin (GSN) as a biomarker whose levels decrease in patients with ACS. We concluded that these biomarkers are associated with the pathogenesis of ACS and can predict the onset of ACS prior to the appearance of necrotic biomarkers.     

Quantitative Proteomic Analysis of Oral Brush Biopsies Identifies Secretory Leukocyte Protease Inhibitor as a Promising,Mechanism-Based Oral Cancer Biomarker

A decrease in the almost fifty percent mortality rate from oral cancer is needed urgently. Improvements in early diagnosis and more effective preventive treatments could affect such a decrease. Towards this end, we undertook for the first time an in-depth mass spectrometry-based quantitative shotgun proteomics study of non-invasively collected oral brush biopsies. Proteins isolated from brush biopsies from healthy normal tissue, oral premalignant lesion tissue (OPMLs), oral squamous cell carcinoma (OSCC) and matched control tissue were compared. In replicated proteomic datasets, the secretory leukocyte protease inhibitor (SLPI) protein stood out based on its decrease in abundance in both OPML and OSCC lesion tissues compared to healthy normal tissue. Western blotting in additional brushed biopsy samples confirmed a trend of gradual decreasing SLPI abundance between healthy normal and OPML tissue, with a larger decrease in OSCC lesion tissue. A similar SLPI decrease was observed in-vitro comparing model OPML and OSCC cell lines. In addition, exfoliated oral cells in patients’ whole saliva showed a loss of SLPI correlated with oral cancer progression. These results, combined with proteomics data indicating a decrease in SLPI in matched healthy control tissue from OSCC patients compared to tissue from healthy normal tissue, suggested a systemic decrease of SLPI in oral cells correlated with oral cancer development. Finally, in-vitro experiments showed that treatment with SLPI significantly decreased NF-kB activity in an OPML cell line. The findings indicate anti-inflammatory activity in OPML, supporting a mechanistic role of SLPI in OSCC progression and suggesting its potential for preventative treatment of at-risk oral lesions. Collectively, our results show for the first time the potential for SLPI as a mechanism-based, non-invasive biomarker of oral cancer progression with potential in preventive treatment.