| Abstract|| |
Background: The extracellular matrix (ECM) is a dynamic tissue that provides nutrition and support to overlying epithelium. During tumorigenesis, the tumor microenvironment (TME) dysregulates the ECM. This is reflected by morphological changes seen in collagen and elastic fibers and is thought to facilitate metastasis. Aim: To study the degradation of elastic fibers in different grades of oral squamous cell carcinoma (OSCC) and in oral epithelial dysplasia (OED) using histochemistry and to correlate it to the TNM stage of OSCC. Methods: Tumor cores from 38 cases of OSCC (well-differentiated , moderately differentiated , and poorly differentiated ) and 15 incisional biopsies of OED were analyzed. Hematoxylin-eosin and Verhoeff's–Van Gieson (VVG) stains were used. The stained sections were assessed for morphological changes in elastic fibers. Statistical Analysis: Data were analyzed using Statistical Package for Social Sciences (SPSS) version 22 software. Fisher's exact, Kruskal–Wallis, one-way ANOVA, and Turkey post hoc tests were used to establish significance (P ≤ 0.05). Spearman's correlation test was used to correlate elastin fiber degradation with TNM stage of Results: All grades of OSCC showed absence of elastic fibers around the tumor islands. Elastic fiber degradation (fragmented and clumped type fibers) increased proportionately with the grade and TNM stage of OSCC. In OED, A significant reduction in the amount of elastic fibers with increasing grade was noted. Conclusion: A positive correlation was noted between elastin degradation and grade and stage of OSCC. Therefore, it may be implicated in tumor progression of OSCC.
Keywords: Elastic fibers, oral epithelial dysplasia, oral squamous cell carcinoma, tumor microenvironment
|How to cite this URL:|
Prabhudesai SA, Carvalho K, Dhupar A, Spadigam A. Elastin remodeling: Does it play a role in priming the malignant phenotype of oral mucosa?. Indian J Pathol Microbiol [Epub ahead of print] [cited 2022 Jul 2]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=345876
| Introduction|| |
The extracellular matrix (ECM) is a specialized connective tissue component, which is mesenchymal in origin and derived from the neural crest cells. It is a matrix composed of various glycosaminoglycans and fibrillar proteins, namely, collagen, elastic, and reticulin fibers. The elastic fibers of the ECM comprise elastin, a core protein surrounded by fibrillin-rich microfibrils that confer elasticity to tissues. Elastogenesis post birth is minimal with an estimated turnover rate of about 1% per decade. With a half-life of about 70 years, elastin remains almost constant throughout life. However, under special circumstances such as those seen in malignancies like oral cancer, specific proteases and matrix metalloproteinases (MMPs), capable of degrading the highly insoluble elastin protein are produced, subsequently yielding bioactive products called the “elastin-derived peptides” (EDPs) or “elastokines.”,
The malignant oral mucosal cells orchestrate and induce catabolic changes within the underlying ECM i.e., the emerging tumor microenvironment (TME). This, in turn, allows for disruption of the basement membrane and encourages tumor progression. Apart from the tumor cells, it is composed of altered stromal fibers, blood vessels, and host inflammatory cells. Changes within the TME can possibly reflect tumor growth potential. Studies quoted in literature assessing the role of elastic fibers in determining malignant transformation of oral mucosa and progression of OSCC are limited. The current study aims to highlight the potential role of elastic fiber degradation in driving tumorigenesis in oral mucosa and its impact on the grade and stage of oral cancer.
| Materials and Methods|| |
A retrospective analytical study was undertaken using the archival tissue blocks from the Department of Oral and Maxillofacial Pathology. Thirty-eight previously diagnosed cases of oral squamous cell carcinoma (OSCC) [well-differentiated (15), moderately differentiated (14), and poorly differentiated (9)-graded using Anneroth's Grading system] and 15 previously diagnosed cases of oral epithelial dysplasia (OED) [5 of each grade] were included in the study. Seven cases of normal oral mucosa were taken as control. Cases of OSCC with a history of receiving chemotherapy and/or radiotherapy were excluded.
Formalin-fixed and paraffin-embedded tissue blocks were sectioned at a 4 μm thickness. Sections were stained with freshly prepared Verhoeff's–Van Geison (VVG) stain and changes in elastic fiber morphology and arrangement were analyzed by two independent and blinded oral pathologists to avoid bias.
Changes in the distribution pattern of elastic fibers were observed within the TME, peripheral ECM, and around the muscle bundles. For the purpose of measurement, the connective tissue stroma measured from the advancing tumor front, up to 200 μm toward the epithelium was regarded as the TME (i.e., ECM in immediate vicinity of the tumor islands), whereas the rest of the stroma was regarded as the peripheral ECM. Distribution of elastic fibers was assessed both semiquantitatively and quantitatively.
1] Semiquantitative assessment: The distribution of elastic fibers was classified as: absent, sparse, moderate, and abundant within the TME, peripheral ECM, and around the muscle bundles in OSCC and within the connective tissue stroma of OED, respectively. Elastic fibers were further categorized as long, short/fragmented, clumped, or a combination of these based on morphological appearance.
2] Quantitative assessment: Each VVG stained section was scanned under 100× view. Three representative fields were selected and photomicrographs of standard size were captured at 400× view using ProgRes Microscope Camera and ProgRes Capture Pro Software (2L02722). These were analyzed using the ImageJ Fiji (Version 2.1.0) Image Analysis Software and the area fraction occupied by the degraded elastic fibers (fragmented and/or clumped) in a field of known dimensions was calculated., A correlation between elastin remodeling and the clinical TNM stage [as per the American Joint Committee on Cancer (AJCC)- Eight Edition] was established in different grades of OSCC.
Statistical analysis: Data were analyzed using Statistical Package for Social Sciences (SPSS) version 22 software. Application of the Shapiro–Wilk test revealed that the data was not normally distributed. Descriptive statistics were used to obtain results in the form of frequencies and percentages. The variables between groups were compared using Fisher's Exact Test at a 95% confidence interval. Kruskal–Wallis test was used for intergroup comparison between the mean areas showing elastin degradation in OSCC, whereas one-way ANOVA test followed by Tukey's post hoc test was used in case of OED as the data was normally distributed. Spearman correlation test was used to correlate elastin fiber degradation with the TNM stage of OSCC. Statistical significance was accepted at a value of P ≤ 0.05.
| Results|| |
The elastic fibers were stained black, collagen fibers pink, the nuclei blue-black, and the other components such as the epithelium, tumor islands, and muscle yellow-green. An absence of elastic fibers was noted within the TME, juxtaposition to tumor islands in all grades of OSCC (100%) [Figure 1]. Similarly, an absence of elastic fibers was noted within the dense lymphocytic infiltrate in 89.4% of OSCC cases. The amount of elastic fiber degradation within the peripheral ECM and around the muscle bundles increased with the grade of OSCC (P < 0.05) [Table 1] and [Table 2]. The mean area fractions occupied by degrading elastic fibers in well-, moderately, and poorly differentiated OSCC were found to be 12.14%, 18.28%, and 31.62%, respectively.
|Figure 1: Photomicrographs [100×, 400× -vvg stain] showing loss of elastic fibers within tme and corresponding elastic fiber degradation (black) with respect to grade and stage of oscc|
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|Table 1: Comparing elastic fiber degradation and arrangement within the peripheral ECM and muscle bundle|
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|Table 2: Kruskal-Wallis test for intergroup comparison of mean area in micrometers|
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With respect to the arrangement of elastic fibers, long and parallel fibers were more common in well-differentiated; short, fragmented in moderately differentiated, whereas haphazardly arranged, clumped fibers were predominant in poorly differentiated OSCCs (P < 0.05) [Table 1]; [Figure 1]. Moreover, a significant increase in the amount of elastic fiber degradation was noted within the peripheral ECM of OSCCs with increasing clinical TNM stage. A strongly positive correlation between the elastic fiber degradation and clinical TNM stage of OSCC was obtained using the Spearman correlation test (P value = 0.74).
In dysplasias, no predominant pattern in the arrangement was noted between grades (P > 0.05). However, a decrease in elastic fibers was noted with increasing grades of dysplasia (P < 0.05) [Table 3]. A significant difference in mean area (in μm) occupied by elastic fibers in the different grades of OED was observed using one way ANOVA test, followed by Tukey's post hoc test (P = 0.009) [Table 4] and [Table 5].
|Table 3: Arrangement and distribution of elastic fibers within the stroma of different grades of OED|
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|Table 4: One-way ANOVA test for intergroup comparison of mean area (in μm) in a field of known dimensions|
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|Table 5: Tukey's post hoc test for pairwise comparison of mean area following one-way ANOVA|
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| Discussion|| |
Elastic fibers are primarily of three types, oxytalan (lack protein core), elastin, and elaunin fibers (possess protein cores). Elastin is formed from its precursor, tropoelastin (a soluble monomomeric protein) during fetal period and synthesized by various mesenchymal cells including fibroblasts and endothelial cells. The synthesis of tropoelastin is controlled and regulated by the tropoelastin gene ELN, which is expressed prenatally and for a few years postnatally.
In oral carcinogenesis, the TME paves the way for tumor growth and invasion in deeper stromal tissues via epithelial-mesenchymal transition (EMT). EMT is a reciprocal inductive effect induced by epithelium on the underlying ECM and vice versa, which results in a loss of function in one or both tissues. Elastin remodeling and degradation is an event mediated by EMT.
In line with the findings noted by Patankar et al., the current study demonstrated uniform loss of elastic fibers around the tumor islands in all grades of OSCC. In another study, Ma et al. noted a decrease in the amount of dermal elastic fibers in epidermal cancer. Dineshshankar et al. noted a decrease in elastic fibers in well-differentiated and poorly differentiated OSCCs and a corresponding decrease with increasing grade of OED. Zhang et al. reported a considerable decrease in elastic fibers in early invasive carcinomas and SCC of the tongue as compared with OEDs.
Zainab et al. showed increased desmoplasia in well-differentiated and moderately differentiated OSCC followed by a complete loss of stromal fibers in poorly differentiated OSCC. This could be explained by the fact that during initial tumorigenesis, the stroma undergoes desmoplastic changes with an increase in collagen and decrease in elastic fibers, attaining a more fibrotic phenotype, whereas, during late tumorigenesis, loss of stromal fibers allows for tumor invasion. Tumor cells together with tumor-associated fibroblasts (TAFs) modulate ECM composition from a fairly elastic to a more fibrous phenotype. TGF-β and fibroblast growth factor (FGF) promote differentiation of TAFs to myofibroblasts that increasingly synthesize collagen fibers causing desmoplasia. This is accompanied by corresponding degradation of elastic fibers. Elastin is a protease-resistant, highly insoluble protein, however, cancer cells of epithelial origin release specific elastases, which are capable of degrading elastin. These include serine proteinases [serine neutrophil elastase (Ela-2), cathepsin G, and proteinase-3], cysteine proteinases [cysteine cathepsin family-L, S, K, and V], and metalloproteinases [MMP-2, 7, 9, and 12]. There is also sufficient evidence supporting the role of matrix stiffness and rigidity in driving tumor formation, invasion, and distant metastasis. Rigidity of the TME is mediated by mechanotransduction pathways, which get activated in response to tensile, compressive, and/or shear stresses. This allows redistribution of forces within the stroma. Increased stromal stiffness promotes the stabilization of disseminated tumor cells.
Li et al., in their study done in human colorectal cancer cases found that tumor cells could potentially lose their epithelial markers and express mesenchymal markers after incubation with elastin recombinant protein. The effect of EMT by malignant oral epithelium is mediated by TGF-β at the initiation of tumorigenesis. This results in a loss of E-cadherin in oral epithelial cells and a break in the basement membrane caused by lytic action of matrix metalloproteinases (MMPs), primarily MMP 2 and 9 (i.e. elastin degrading MMPs). Subsequently, TGF-β promotes the synthesis of Twist-related protein 1 (Twist1), a protein that is translocated to the cell nucleus of OSCC tumor cells. MicroRNA 200 and 205 downregulate the expression of transcriptional repressors of E-cadherin, thus allowing reexpression of E-cadherin in disseminated tumor cells., The cells also begin to express integrins and other cell adhesion molecules (CAMs) including claudins, desmogleins, and occludins, thus restabilizing cell junctions and forming large aggregates at sites of invasion, forming clinically palpable “tumor masses”., This facilitates individual or collective tumor cell migration (small clusters, nests, cords, or islands) within the TME.
In concurrence with the findings of Agrawal et al., the study also demonstrated minimal and sometimes absent elastic fibers within areas of dense lymphocytic infiltrate. This is explained by the fact that under hypoxic conditions prevailing within the tumor milieu (intratumoral hypoxia), nuclear factor-kappa B (NF-kB) is activated within the tumor infiltrated neutrophils (TILs), lymphocytes, and other inflammatory cells, subsequently releasing a cascade of proinflammatory cytokines, chiefly the interleukins (IL-1,6) and tumor necrosis factor α (TNFα), which, in turn, release various MMPs and proteases causing degradation of stromal fibers.,
In contrast to the findings of Kardam et al., a shift in the pattern of elastic fiber arrangement was noted with increasing grade of OSCC, ranging from more long and parallel fibers to more short/fragmented, clumped, and haphazardly arranged fibers. Similar findings were reported by John et al. in their study. The degree of clumping was highest in the case of poorly differentiated OSCCs in the current study, thus indicating increased activity of elastases and elastin degradation. A corresponding increase in degraded elastic fibers was noted within the peripheral ECM and around the muscle bundles with increasing OSCC grade and TNM stage.
A school of thought proposed to explain the fragmented, clumped haphazardly arranged elastic fibers is the production of elastin-derived peptides (EDPs) due to elastic fiber degradation. These soluble elastin fragments act as chemotactic agents to facilitate tumor cell aggregation.,,
In this study, the degree of fragmentation and clumping in elastic fibers increased with the cancer stage. This is indicative of increased elastin degradation, which can be justified by similar findings in other malignancies such as melanomas, colorectal cancers, and breast cancers.,, Elastin degradation products promote carcinogenesis, which is supported by various studies. Scandolera et al. emphasized the role of EDPs/elastokines (including kappa elastin) in driving tumorigenesis. EDPs are generated by the catabolic action of MMPs released by inflammatory and malignant tumor cells. These EDPs binds to specific receptors expressed on tumor cell surface, which include the elastin receptor complex (ERC), Galectin-3, and integrins αvβ3 and αvβ5.
The ERC is a heterotrimer, which is primarily composed of three domains, namely, a peripheral domain called the elastin binding protein (EBP) to which EDPs bind, the second called the protective protein/cathepsin A (PPCA) domain to which EBP binds and the third called as the neuraminidase 1 (Neu-1) domain, a membrane-bound protein that mediates signal transduction. This ligand-receptor type of binding between EDPs and ERC is thought to promote chemotactism, migration, angiogenesis, cell survival, and tumor cell proliferation within the tumor milieu. Various signaling pathways including the phosphatidylinositol 3 kinase (PI3K)/Protein kinase B (Akt)/Nitric oxide (NO) synthase and NO/cyclic guanosine monophosphate (cGMP)/extracellular signal-regulated kinase 1/2 (Erk1/2) pathways regulate the EDP-EBP binding. Elastokines promote activation of both PI3-kinase p110 subunit, Akt and Erk1/2, thus promoting the upregulation of MMPs, mainly membrane type 1 MMP (MT1 MMP) and downregulation of tissue inhibitor of metalloproteinase 2 (TIMP 2) facilitating peri tumoral proteolysis.
Pocza et al. found that EDPs containing the hexapeptide sequence VGVAPG and VAPG served as a potent chemotactic target for melanoma cells and concluded that EDPs bind to Galectin-3 receptors and encourage invasion and metastasis. In line with the results of these studies, Blood et al., also stated that EDPs could drive tumor cell migration in response to its chemotactic action in lung cancer.
Fortuna Costa et al., showed that the Galectin-3 receptor interacts with EDPs and other ECM proteins resulting in Galectin-3-dependent integrin activation. This results in enhanced tumor cell migratory capacity via a synergistic effect of Galectin-3 via Src kinase-dependent Caveolin 1 (Cav 1) and RhoA/Rho-associated protein kinase (ROCK) pathways. Integrins αvβ3 and αvβ5 have a high affinity for tropoelastin resulting in a number of tumor-promoting synergistic effects along with Galectin-3 receptor. An enhanced expression of MMP 2 and 3 as well as vascular endothelial growth factor C (VEGF C), a lymphangiogenic peptide has been proved to increase the invasive potential of tumor cells and support lymphatic metastasis.
Hence, the current study proposes a flow chart elucidating the role of EDPs in driving the tumorigenesis in oral mucosa [Figure 2].
|Figure 2: Proposed flow chart summarizing the molecular events and pathways promoted by elastin remodeling in the three stages of tumorigenesis, namely, initiation, promotion and progression|
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To our knowledge, this is the first study to quantify and correlate elastic fiber degradation to the clinical TNM stage of oral cancer.
The demonstration of elastic fibers in OSCC using special stains could serve as an adjuvant to histopathological grading and aid in prognosticating OSCC, however, it lacks validation due to paucity of research in this field. The increased elastic fiber degradation with increasing TNM stage of OSCC could indicate a pivotal role played by elastic fibers in the progression of oral cancer. The evaluation of negative resected tumor margins (i.e. possible controls) against positive resected tumor margins could add to the existing body of knowledge on elastin remodeling in OSCC. Further research in elastin remodeling within the TME of oral cancer could establish new avenues in oral cancer therapeutics.
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Conflicts of interest
There are no conflicts of interest.
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Sonal A Prabhudesai,
Goa Dental College and Hospital, Bambolim, Goa
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]