Biomarker Potential of Vimentin in Oral Cancers

Oral carcinogenesis is a multistep process. As much as 5% to 85% of oral tumors can develop from potentially malignant disorders (PMD). Although the oral cavity is accessible for visual examination, the ability of current clinical or histological methods to predict the lesions that can progress to malignancy is limited. Thus, developing biological markers that will serve as an adjunct to histodiagnosis has become essential. Our previous studies comprehensively demonstrated that aberrant vimentin expression in oral premalignant lesions correlates to the degree of malignancy. Likewise, overwhelming research from various groups show a substantial contribution of vimentin in oral cancer progression. In this review, we have described studies on vimentin in oral cancers, to make a compelling case for vimentin as a prognostic biomarker

Prognostic role of Oct4, CD44 and c-Myc in radio-chemo-resistant oral cancer patients and their tumourigenic potential in immunodeficient mice

In the present study, we have investigated the prognostic value of known stem cell-associated molecules such as Oct4, CD44 and c-Myc in patients with oral SCC who had received post-surgery radio- and/or chemotherapy. Immunohistochemistry was performed to analyse the expression of Oct4, CD44 and c-Myc in 87 tumour tissues, and the expression profile obtained was correlated with clinicopathological parameters of the patients with oral cancer. Tumourigenic potential of these molecules was also evaluated by in vivo studies. Our results showed significant correlation of Oct4 (OS, p = 0.003; DFS, p = 0.001) and c-Myc (OS, p = 0.01; DFS, p = 0.03) with overall survival and disease-free survival independently. Furthermore, all the three markers in combinations of two markers each, i.e. Oct4 + CD44 (OS, p = 0.003; DFS, p = 0.001), Oct4 + c-Myc (OS, p = 0.0001; DFS, p = 0.0001), CD44 + c-Myc (OS, p = 0.008; DFS, p = 0.02) and in combinations of three markers each, i.e. Oct4 + CD44 + c-Myc (OS, p = 0.0001; DFS, p = 0.0001) also significantly correlated with overall survival and disease-free survival. Univariate and multivariate analyses further established the independent prognostic value of Oct4. Oct4-, CD44- and c-Myc-enriched populations independently induced sarcomatoid carcinomas whereas primary keratinocytes developed poorly differentiated carcinomas in immunodeficient mice. Oct4 and c-Myc independently as well as in combination with CD44 might be useful for the prediction of local recurrence and poor survival of patients with oral cancer which is the novel finding of this study. Oct4, c-Myc and CD44 can be used to predict local recurrence and the outcome of treatment in oral cancer patients. In addition, these molecules may find use as molecular targets for effective therapy

Establishment of 3D Co-Culture Models from Different Stages of Human Tongue Tumorigenesis: Utility in Understanding Neoplastic Progression

<div><p>To study multistep tumorigenesis process, there is a need of <i>in-vitro</i> 3D model simulating <i>in-vivo</i> tissue. Present study aimed to reconstitute <i>in-vitro</i> tissue models comprising various stages of neoplastic progression of tongue tumorigenesis and to evaluate the utility of these models to investigate the role of stromal fibroblasts in maintenance of desmosomal anchoring junctions using transmission electron microscopy. We reconstituted <i>in-vitro</i> models representing normal, dysplastic, and malignant tissues by seeding primary keratinocytes on either fibroblast embedded in collagen matrix or plain collagen matrix in growth factor-free medium. The findings of histomorphometry, immunohistochemistry, and electron microscopy analyses of the three types of 3D cultures showed that the stratified growth, cell proliferation, and differentiation were comparable between co-cultures and their respective native tissues; however, they largely differed in cultures grown without fibroblasts. The immunostaining intensity of proteins, viz., desmoplakin, desmoglein, and plakoglobin, was reduced as the disease stage increased in all co-cultures as observed in respective native tissues. Desmosome-like structures were identified using immunogold labeling in these cultures. Moreover, electron microscopic observations revealed that the desmosome number and their length were significantly reduced and intercellular spaces were increased in cultures grown without fibroblasts when compared with their co-culture counterparts. Our results showed that the major steps of tongue tumorigenesis can be reproduced <i>in-vitro</i>. Stromal fibroblasts play a role in regulation of epithelial thickness, cell proliferation, differentiation, and maintenance of desmosomalanchoring junctions in <i>in-vitro</i> grown tissues. The reconstituted co-culture models could help to answer various biological questions especially related to tongue tumorigenesis.</p></div

Validation of endogenous lineage phenotype.

<p>Immunohistochemistry of normal OC showing keratin (b) and vimentin (f) staining in the epithelium and stromal fibroblasts respectively and staining of integrin β4 (d) in the basement membrane as seen in native tissues (a, c, e). The experiments were performed in triplicates.</p

Ultrastructural analysis of altered desmosomal assembly.

<p>Electron micrographs depicting desmosomes (D) and intercellular spaces (ICS) in native tissues (a-c) along with Fib+ OCs (d-f) and Fib- OCs(g-i). Native tissue and Fib+ OCs show similar ultrastructure having widened intercellular spaces (ICS) and reduced number of desmosomes as disease stage progressed, whereas Fib- OCs showed complete collapse of ultrastructure with more widened ICS and reduced desmosomes even at normal stage. Normal desmosome structure was seen in normal-native tissues as well as Fib+ OCs (a’, d’) but in normal-Fib- OCs desmosomes were found to be in aggregated forms (g’). Desmosome structures were confirmed by immunogold labeling in normal native tissue (a”) normal Fib+ OC (d”) and normal Fib- OC (g”). Bars 1 μm.</p

Quantitative analysis of alterations in desmosomal assembly using iTEM software.

<p>Graphical representation showing variation in desmosome number (A), their length (B) and intercellular spaces (C) in all three OC models along with their respective native tissues. Statistical anaylsis was drawn in between Fib+ and Fib- OCs and represented as p values. The experiments were performed at least three times and data represented here as mean ± standard errors.</p

Cell proliferation and differentiation staining pattern in <i>in-vitro</i> grown tissues.

<p>Immunohistochemical staining for cell proliferation and differentiation specific proteins in Fib+ and Fib- OCs reconstructed from normal, dysplastic and malignant tongue tissues along with their respective native tissues: PCNA nuclear staining was seen in the basal proliferating cells of normal- native tissue, Fib+ and Fib- OCs (a, b, c) while cytoplasmic involucrin staining was seen in the upper differentiated cells of normal- native tissue, Fib+ and Fib- OCs (d, e, f). PCNA nuclear staining was seen in the basal as well as supra basal cells of dysplastic-native tissue, Fib+ and Fib- OCs (g, h, i) while cytoplasmic involucrin staining was seen in the native dysplastic tissue but not in Fib+ and Fib- OCs (j, k, l). PCNA staining was observed throughout the epithelium of malignant-native tissue, Fib+ and Fib- OCs (m, n, o), while involucrin staining was seen in the differentiated cells of malignant-native tissue but not in the Fib+ and Fib- OCs (p, q, r). The experiments were performed at least three times.</p

List of antibodies used in immunohistochemistry /immunofluorescence/immunogold staining protocols.

<p>List of antibodies used in immunohistochemistry /immunofluorescence/immunogold staining protocols.</p

Analysis of immunohistochemistry staining intensity using ImageJ.

<p>Graphical representation of percentage of positive staining intensity of desmoplakin (A), desmoglein (B) and plakoglobin (C). IHC staining intensity score was calculated using IHC profiler plugin in ImageJ software. Statistical analysis was done between cultures grown with and without fibroblasts using two-tailed unpaired <i>t-test</i> and represented as p values. The experiments were performed in triplicates and data are presented here as mean ± standard errors.</p

Isolation and characterization of primary keratinocytes and fibroblasts from tongue tissue.

<p>Representative phase contrast microscopic images showing the growth of primary keratinocytes/fibroblasts from human normal tongue tissues at different time points–day 2 (a, f), day 6 (b, g) and day 9 (c, h). Immunofluorescence stained images showed the positive expression (green) for cytokeratin (d) and negative expression for vimentin (e) in normal keratinocytes while, fibroblasts showed negative expression for keratin (j) and positive expression (red) for vimentin (i). Nuclei are counterstained with DAPI (blue). Bars-50 μm.</p