Patients and tissue specimens
All research involving human participants had been approved by the authors’ Institutional Review Board (IRB) and all clinical investigations had been conducted according to the principles expressed in the Declaration of Helsinki. A written informed consent was obtained from all the participants and the content of the informed consent was approved by the respective Institutional Research Board namely, Cancer Institute WIA; Protocol 1 HNCOG (Cancer Institute, Women India Association; Protocol 1 Head and Neck Co- operative Oncology Group) SBDCECM105/13/58 (Sree Balaji Dental College and hospital Ethical Committee Meeting reference number 105/13/158) and the Department of Oral medicine and Radiology, Sree Ramachandra Dental College and hospital from June 2018 till January 2019. Institutional Ethics Committee approval (IEC No.CSP/17/AUG/60/239) and Ethics approval from Department of Oral and maxillofacial pathology, Saveetha Dental College and hospital from December 2017 to August 2019 (SRB/SDMDS11/170MP/01) was obtained before the commencement of the study.
Patient tissue samples
Histologically apparently normal adjacent tongue tissues along with paired early staged OTSCC tumour tissues (n=10) were obtained from patients presenting with OTSCC and undergoing surgery. Formalin fixed paraffin embedded samples from buccal leukoplakia (n=50), oral cancers (n=71) were obtained from Sree Balaji Dental College and hospital and formalin fixed tissues from oral submucous fibrosis samples (n=32) and normal buccal mucosa tissues (n=42) were obtained during third molar extraction were collected from Sree Ramachandra Dental College and Hospital. This was an independent cohort (n=195) for validation studies. Saliva samples (n=80) were collected from oral cancer patients (n=45), patients with oral potentially malignant lesions (n=15) and normal (n=20) obtained from Saveetha Dental College and Hospital and Sree Ramachandra Dental College and Hospital. Additionally, formalin fixed paraffin embedded sections from retrospective series of exclusively early staged tongue cancer patients [T1 and T2] (n=150) were obtained from Cancer Institute WIA who had been treated between 1995 to 200 for validation studies of the findings with the complete treatment follow up. All the FFPE sections (n=345) used for the study were histologically examined by oral pathologist VS and PR.
Patient Saliva samples
ELISA was done for saliva samples (n=80) collected from Oral cancer patients, patients presenting with oral potentially malignant lesions and absolute normal volunteers. The study participants were requested to refrain from drinking, eating, chewing tobacco or smoking 1 hour prior to the collection of saliva. After obtaining the informed consent of the patient, 0.5 to 1 ml of whole unstimulated saliva was collected by passive expectoration and patients were asked to spit into a 50-mL sterile tube containing 10µL of proteinase inhibitor (Proteinase inhibitor cocktail P2714 Sigma Aldrich). The saliva samples were transferred to 1.5-mL sterile microtubes and centrifuged for 3 minutes at 13,000 rpm. Supernatants, separated from the cellular phase, were immediately aliquoted and stored at -80oC within 60 minutes after saliva collection.
Proteomics - Proteins Labelling with CyDyes
Pooled OTSCC tumour and pooled adjacent uninvolved tissues were used for the proteomic profiling. Lysine labelling protocol, (minimal labelling) used in this study is described before. The processed tissue proteins were labelled individually with dyes Cy3 and Cy5 while the pooled tissue proteins prepared by mixing equal aliquot of protein from all samples in an experimental set up were labelled with Cy2. The final volume for all preparations was adjusted to a total of 340 uL with rehydration buffer (7 M urea, 2 M thiourea, 1% IPG buffer, 50 mM DTT, 4% CHAPS, and a trace amount of bromophenol blue). A reciprocal labelling experiment was also performed.
Proteomics – 2D gel electrophoresis
Two-dimensional gel electrophoresis of CyDye labeled proteins was done as described before[10, 11] with the following modifications. Eighteen cm IPG strips of pH 4–7 (GE Healthcare, Uppsala, Sweden) was employed in the first dimension. Labelled proteins were focused for a total of 80,000 Vhs at a constant temperature (20oC) under linear voltage ramp after an active IPG rehydration at 30 V in a IPGPhor III (GE Healthcare, Uppsala, Sweden) apparatus. Following IEF, each IPG strip was placed in the equilibration buffer containing 2% DTT first followed by incubation in another buffer in which the DTT was replaced by 2.5% iodoacetamide. The second dimension PAGE (12.5%) was carried out in an EttanDaltSix systems (GE Healthcare, Uppsala, Sweden) at 1 W/gel for 1 hr and 13 W/ gel for 5 hr. All experimental procedures were performed in dim light or in the dark.
Protein Visualisation and DeCyder Image analysis
The protocols for the protein visualization and image analysis using DeCyder has been mentioned previously. Briefly, after second dimension electrophoresis, the gels were scanned with Typhoon FLA 9500 Variable Mode Imager (GE Healthcare, Uppsala, Sweden). Cy2, Cy3 & Cy5 images were captured using the settings recommended by the manufacturer. A DeCyder differential in-gel analysis (DIA) module was used for image analysis between samples within the same gel while a DeCyder biological variation analysis (BVA) module was performed for pairwise image analysis among multiple gels. Student’s t-test and ANOVA were used to compare the average spot volume and differences of protein abundance for all detectable spots between the tumor and normal groups. Reciprocal dye labelling was performed to normalize bias in labelling.
Protein Identification and Mass Spectrometry (MS)
Pooled tongue tissue proteins (250 µg) were separated on 18 cm IPG strips of pH 4-7 in the first dimension. First and second dimension electrophoresis were done as given under 2D DIGE method. The second-dimension gels were stained with colloidal coomassie blue G-250 and gel spots from this preparative gel were excised manually for in-gel trypsin digestion and LC-MS/MS was performed. Extracted peptides were dried under vacuum for 90 min and stored at 4 °C. Zip tip purified peptides were analysed using nano-RPLC (Thermo Scientific, USA) coupled with an Orbitrap Elite Mass spectrometer (Thermo Scientific, USA). Peptides were ionized by positive mode electrospray with an ion spray voltage of 1.9 kV. The MS data were acquired in positive ion mode over mass range m/z 350–4000 Da using Xcalibur software (version 2.2.SP1.48) (Thermo Scientific USA). MS data were analysed using Proteome Discoverer software v.1.4 (Thermo Scientific) using Sequest algorithm with database downloaded from Uniprot as described earlier. The combined list of official gene symbols corresponding to the identified proteins was used for input. We used STRING (www.string.db.org) network construction.
The IHC detection methods was as mentioned previously. Briefly, IHC for vimentin was performed on 5 μm sections of FFPE tissues. The sections were deparaffinized in xylene and rehydrated in absolute ethanol. Antigen retrieval was done with 0.05M citrate Buffer (pH 9) in pressure cooker for 20 minutes. Endogenous peroxidase activity was blocked by incubation in 0.03% hydrogen peroxide in distilled water for 10 minutes and then washed with phosphate buffered saline (PBS). Sections were counterstained with hematoxylin, dehydrated, and mounted in DPX. Positive controls and negative controls were included appropriately where primary anti-body was replaced with 2% BSA in negative control. Immunostaining of the sections was reviewed with the corresponding haematoxylin and eosin-stained sections.
Immunohistochemical scoring for the target was done as described earlier. Briefly, the percentage grade of stained tumor cells was scored as 0, negative; 1, <10%; 2, 11–50%; 3, 51–80%; or 4, >80% positive cells and the intensity of stain was scored as 0, negative; 1, weak; 2, moderate; or 3, strong. The immunoreactive (IR) score was calculated as a product of the percentage grade and intensity score with the IR score ranged from 0 to 12. The immunoreactivity was divided into three groups on the basis of the final score: negative immunoreactivity was defined as a total score of 0, low immunoreactivity was defined as a total score of 1–4, and high immunoreactivity was defined as a total score >4. The immunostaining of the tumor invasive front was evaluated using the same method as mentioned for tumor areas.
ELISA in Saliva Samples
The RayBio® Human Vimentin ELISA (Enzyme-Linked Immunosorbent Assay) kit is used to quantify the expression of vimentin in saliva samples (n=80), of which saliva samples from patients with oral cancer (n=45), patients with oral potential pre-malignant lesions (n=20) and healthy volunteers (n=15) were used. This is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human Vimentin in saliva samples. This assay employs an antibody specific for human Vimentin coated on a 96-well plate. Standards and samples are pipetted into the wells and Vimentin present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human Vimentin antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Vimentin bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. The standard graph was plotted with the vimentin standard protein provided in the kit. Using the standard graph the protein concentration were extrapolated for the unknown OD values obtained from saliva samples obtained from patients and healthy volunteers.
The relative levels of stained protein spots compared with the internal standard spots were analyzed by DeCyder Difference In-gel Analysis (DIA) and DeCyder Biological Variation Analysis (BVA) software modules (GE Healthcare). Student’s t-test was used to calculate statistically significant differences between 2 groups in relative abundance of individual protein spots among the groups in 2D-DIGE. P<0.05 was considered statistically significant. Other statistical analysis was done using SPSS (IBM Corporation version 16).