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ORIGINAL ARTICLE
Year : 2022 | Volume
: 18
| Issue : 9 | Page : 191-196
Expression of syndecan-1 in different grades of oral squamous cell carcinoma: An immunohistochemical study
Priyanka K Shetty1, Nitin Gonsalves1, Dinkar Desai1, Siddharth Pandit1, Chethan Aradhya2, Mohammed Shahid1, Shubhalakshmi1, Sharan J Shetty1
1 Department of Oral Pathology and Microbiology, A.J. Institute of Dental Sciences, Mangaluru, Karnataka, India
2 Department of Oral Pathology and Microbiology, Century Dental college, Kasargod, Kerala, India
Correspondence Address:
Priyanka K Shetty
Oral Pathology and Microbiology, A.J. Institute of Dental Sciences, NH 66, Kuntikana, Mangaluru - 575 004, Karnataka
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/jcrt.JCRT_1715_20
Background: Syndecans are a family of transmembrane proteins, belonging to heparin sulphate proteoglycan family and are localized entirely to the epithelial cells with the stratified squamous epithelia. They are involved in cell–cell adhesion and interaction with the extracellular matrix and play a critical role in cell growth, differentiation, cell morphology, and migration. The down regulation of syndecan-1 indicates loss of cellular adhesion and possibility of invasion. The present study is aimed to evaluate the difference in immunohistochemical expression of syndecan-1 in different grades of oral squamous cell carcinoma (OSCC) patients and control group.
Methods: The present study consists of 42 cases of paraffin-embedded tissue sections of OSCC; 14 well differentiated, 14 moderately differentiated, and 14 poorly differentiated. As a control, 10 paraffin-embedded tissue sections of unaffected oral mucosa were used. The sections were stained for immunohistochemical expression of syndecan -1. The intensity of staining was scored. The immunohistochemistry scores for each sample were obtained by Tissue Quant software.
Results: Statistical analysis revealed that there was a significant decrease in intensity of staining between normal and different grades of OSCC.
Conclusion: This study shows that as cellular differentiation was lost, syndecan-1 expression was less. This provides an insight and understanding of the pathophysiology of the invasive process of OSCC and helps in establishing the prognostic link.
Keywords: Cell adhesion molecules, immunohistochemistry, oral squamous cell carcinoma, syndecan-1
Oral squamous cell carcinoma (OSCC) is one of the common malignancies in worldwide with an annual incidence of 300,000 cases and ranks sixth most common neoplasm in the world.[1],[2] OSCC is epithelial in origin and representing up to 80–90% of all malignant neoplasms of the oral cavity.[3] OSCC is categorized as an invasive growth and usually associated with regional metastases at diagnosis.[4] About 98% of OSCC in western population are found in patients more than 40 years of age, and the male to female ratio was found to be 2:1.[5] The 5-year survival rate of OSCC patients is about 50%.[1] OSCC has been usually diagnosed in advanced stages and, therefore, having a high morbidity and mortality rate.[6] The etiology of OSCC appears as multifactorial and includes intrinsic and extrinsic factors. Tobacco, dietary factors, and high alcohol consumption are regarded as important extrinsic factors; however, cumulative genetic alterations, nutritional deficiency, and immunity of patients are main intrinsic factor.[7],[8]
The syndecans are a family of transmembrane cell surface heparan sulfate (HS) proteoglycans, which regulate cell–cell and cell–extracellular matrix adhesion, migration, and growth factor activity of cells.[9],[10] The syndecan family consists of four members (syndecan 1–4) encoded by four different genes.[11] Among them, syndecan-1 is known to bind with cell-to-cell and cells-to-interstitial matrix.[11] Unstable expression or function of syndecan molecules has been implicated in tumor development, tumor cell differentiation, invasion, and metastasis.[12] Syndecan-1 is mainly expressed in epithelium and mesenchymal tissue.[13] In addition, loss of epithelial syndecan-1 has been associated with poor prognosis in some forms of cancer.[14] However, in mature tissues, syndecan-1 expression is localized entirely to the epithelial cells with the stratified squamous epithelia showing the most expression. Decreased epithelial expression of syndecan-1 has been reported in OSCC.[15] As the differentiation of cancer decreases, the expression of syndecan also decreases; hence, its expression may be high in well-differentiated tumor, whereas it may suppress in poorly differentiated squamous cell carcinoma. The purpose of this study was to analyze syndecan-1 expression in different histological grades of OSCC from the oral cavity.
> MethodsStudy consists of 42 cases of paraffin-embedded tissue sections of OSCC; 14 well differentiated, 14 moderately differentiated, and 14 poorly differentiated. Ten paraffin-embedded tissue sections of unaffected oral mucosa were taken as control from a tertiary care hospital, Mangalore (India). Institutional ethical committee has approved the study, and written informed consent was obtained from each participating individual.
Immunohistochemistry for syndecan-1
Formalin-fixed paraffin-embedded wax blocks were used to obtain 4-μm sections for immune staining. The cut sections were then transferred on to a coated slide. Incubation was done at a temperature range of 55–60 °C. The tissue sections were deparaffinised in fresh xylene for 10 min and rehydrated through decreasing concentrations of absolute alcohol (three series).
Antigen retrieval used for syndecan-1 was done by enzymatic treatment. Antigen retrieval solution for syndecan-1 was EDTA buffer. The solution was prepared by diluting one part of 50X Tris EDTA epitope retrieval buffer (PathnSitu, CA, USA) in 49 parts of diluted water. The slides were then removed from the microwave and rinsed in distilled water followed by 5 min immersion in wash buffer. Immuno wash buffer (25X), Tris-buffered saline with Tween 20 (PathnSitu, CA, USA), one part of 25X wash buffer was diluted with 24 parts of distilled water. After rinsing in wash buffer, the slides were drained and blotted gently. Endogenous peroxidase activity was quenched by treating the sections with PolyExcel H2O2 (PathnSitu, CA, USA) for 10 min. The slides were then rinsed in distilled water for 2 min continued by buffer wash (two changes) for 3 min each and then blotted.
The sections were incubated with rabbit monoclonal anti-human syndecan-1 antibody for 45 min at room temperature. The procedure was carried out in a moist chamber and care was taken not to let the section dry. The sections were rinsed in wash buffer (two changes) for 5 min each. The slides were then treated with PolyExcel Target Binder (PathnSitu, CA. USA) at room temperature for 10 min and blotted gently. The slides were incubated with PolyExcel Poly Horseradish peroxidase (PathnSitu, CA, USA) for 30 min. The solution comprises secondary antibody conjugated to non-biotinylated polymer HRP reagent. Again slides were rinsed in wash buffer (two changes) for 5 min each and blotted gently. The slides were then treated with PolyExcelStunn (3,3'-diaminobenzidine) (PathnSitu, CA, USA) solution. It was prepared by adding 1 drop of Stun DAB chromogen to 1 mL of Stunn DAB Buffer. The slides were incubated in the solution for 5 min at room temperature.
The slides were counterstained with Harri's haematoxylin for 2 min, washed in tap water, dehydrated, and mounted with a glass coverslip and xylene-based medium. In the present study, normal mucosa was used for the positive control. Endothelial staining of blood vessels within the connective tissue served as an internal control.
The slides were observed in a bright field microscope at a magnification of 10× for analyzing intensity of staining. Good quality of images was obtained [Figure 1], [Figure 2], [Figure 3], [Figure 4]. All the images were analyzed using “TissueQuant” software, Karnataka, India which works on the principle of staining intensity quantification. TissueQuant has been used for color-based quantification and segmentation-based analysis in many studies.[16],[17],[18],[19],[20] The positively stained area and the average intensity of the positively stained specimen can be obtained using “TissueQuant.” TissueQuant software works on the principle of scoring pixels of an image based on a reference color representing a specific biological component. The software provides facility to adjust color settings to choose various color shades of a particular color to be considered for analysis as positively stained area in the image[21] In this study, the reference color was selected to quantify brown staining indicating positivity for syndecan-1. The same analysis was repeated for all the images.
Figure 1: Syndecan-1 expression in normal mucosa with intense staining (10×)
Figure 2: Syndecan-1 expression in well-differentiated oral squamous cell carcinoma (10×)
Figure 3: Syndecan-1 expression in moderately differentiated oral squamous cell carcinoma (10×)
Figure 4: Syndecan-1 expression in poorly differentiated oral squamous cell carcinoma (10×)The immunohistochemical scores were subjected to statistical analysis using Anova and Tukey's test to validate the statistical comparison and correlation between the groups.
> ResultsOn applying the TissueQuant principle, the pixel scoring showed a marked increase in scoring in normal tissue (mean value 15.3669). In comparison with the normal tissue, the pixel scoring of well-differentiated squamous cell carcinoma (mean value 11.1246) is more than in poorly differentiated squamous cell carcinoma (mean value 3.1740).
This quantifies that the intensity of syndecan-1 expression decreases as OSCC differentiates further, and the result shows the association between the different groups of cancerous tissue with regard to intensity of staining [Table 1]. The intensity of the OSCC samples ranged from 5.501 to 8.232.
In normal control, the mean value is found to be 15.367, whereas in well-differentiated squamous cell carcinoma, it is 11.125; in moderately differentiated squamous cell carcinoma, it is 7.833; and in poorly differentiated squamous cell carcinoma, the mean is 3.174, which shows a trend of decrease.
Fischer's test shows that there is a significant difference in pixel scores among the groups (P < 0.001) [Table 2].
On applying Tukey's test, the P value for normal control in comparison to well-differentiated OSCC is 0.001 and in poorly differentiated squamous cell carcinoma is 0.000 [Table 3].
> DiscussionOral cancer is the 10th most common cancer globally. OSCC is particularly common in the developing world, mostly in older males. In the last 30 years, the 5-year survival rate of patients with OSCC has not improved despite advances in diagnostic techniques and improvements in treatment modalities. There is concern about an ongoing increase in younger patients and in women as well as in the oropharynx.[22],[23]
Many studies in this field have focused on the development of specific markers that will bring out the biological properties of these tumors that can be used in early detection, disease monitoring, and determining the prognosis of oral cancer patients.
Cell adhesion molecules (CAM) form structural linkages between the cell cytoskeleton and the extracellular matrix or between cells and function as signaling receptors, transducing signals initiated by cellular interactions that regulate many diverse processes, including cell division, migration, and differentiation.[24] The development of malignant epithelial neoplasms is associated with aberrant expression of CAM leading to disruption of cell-to-cell and cell-to-matrix adhesion. Altered expression of these molecules has been frequently found in OSCC, where loss of CAM expression is often seen in poorly differentiated lesions. The major groups of CAMs are syndecans, cadherins, catenins, laminins, selectins, integrins, and molecules such as CD44.
Syndecans are a family of four cell surface heparan sulphate proteoglycans.[25] Syndecan-1 is the main syndecan expressed by epithelial cells, syndecan-2 is present mainly on cells of mesenchymal origin, syndecan-3 is primarily expressed by neuronal tissue and cartilage, whereas syndecan-4 is expressed by most tissues. Syndecan-1 binds cells via its heparan sulphate chains to a variety of components of the interstitial matrix, including types I, III, and V collagen, fibrillar collagen fibronectin, and tenascin. In keratinocytes, syndecan-1 is localized at the cell membrane, especially in the suprabasal cell layers. Syndecan-1 is also present on immature B lymphocytes, plasma cells, myeloma cells, and Leydig cells.
Syndecan-1 core protein consists of three major domains: a long extracellular domain that bears the HS and chondroitin sulfate chains at distinct sites that serves attachment extracellularly to adjacent cell or matrix, a short transmembrane domain, and a cytoplasmic domain present intracellularly that is highly conserved among different syndecans.
It is shown to mediate cell adhesion to several extracellular matrix molecules and to act as a co receptor for fibroblast growth factor and potent angiogenic growth factors, which is involved in differentiation. There is a marked decrease in syndecan-1 expression in transformed epithelium in comparison to its expression in normal oral squamous cell epithelium. A decreased expression of syndecan-1 is associated with low histological grade of differentiation. Also the expression of syndecan-1 is known to suppress the level of matrix metalloproteinase-9. Loss of cell membrane syndecan-1 diminishes adhesion of the epithelial cells and finally loosens the attachment to extracellular matrix causing migration and invasion of tumor cells.[26]
Many studies have shown that syndecan-1 plays an important role in cancer progression, and also can be used as cancer biomarker.[27] Other than expression of syndecan-1 has been linked with low differentiation in squamous cell carcinoma of the head and neck, it shows expression in the bone marrow of the patients suffering from the multiple myeloma and endometrial cancers.
In the present study, immunohistochemical staining was carried out on 52 paraffin-embedded tissue comprising histological sections of normal oral mucosa along with histologically diagnosed well-differentiated squamous cell carcinoma, moderately differentiated squamous cell carcinoma, and poorly differentiated squamous cell carcinoma. A quantitative analysis was done using “TissueQuant” that works on the principle of scoring pixels of an image based on a reference color representing a specific biological component. The positively stained area and the average intensity of the positively stained specimen can be obtained using this software. The software provides facility to adjust color settings to choose various color shades of a particular color to be considered for analysis as positively stained area in the image. In this study, the reference color was selected to quantify brown staining indicating positivity for syndecan-1.
The results obtained from our study showed statistically significant difference between the intensity of expression of syndecan-1 in different grades of OSCC (P < 0.001). The staining was seen to be more intense in normal and well-differentiated squamous cell carcinoma in comparison to moderately and poorly differentiated squamous cell carcinoma.
> ConclusionSyndecan-1, a member of the family of cell surface proteoglycans, is known to bind cell-to-cell and cells-to-interstitial matrix. Interaction between cells and their microenvironment is mediated by adhesion molecules that participate in fundamental biological process, including embryonic development and cell migration. The down regulation of syndecan-1 may offer the cell a possibility to detach and to invade. As the differentiation of cancer decreases, the expression of syndecan also decreases; hence, its expression may be high in well-differentiated tumor, but is suppressed in poorly differentiated squamous cell carcinoma.
Thus, from the present study, we can conclude that there is a gradual decrease in staining intensity of syndecan-1 from well-differentiated to poorly differentiated squamous cell carcinoma. Therefore, the expression of syndecan-1 in OSCC may be a useful parameter to evaluate the extent of tumor cell invasion, tumor aggressiveness, and probably metastasis. Further molecular and biochemical studies are needed to determine the detailed mechanism of the syndecan-1 and their related molecules in OSCC, which may lead to the development of new diagnostic and therapeutic applications.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
> References
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