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ORIGINAL ARTICLE Table of Contents  
Ahead of print publication
Tumor microenvironment in oral squamous cell carcinoma: Special stains and scanning electron microscopic study


1 Department of Oral and Maxillofacial Pathology and Microbiology, D Y Patil University, School of Dentistry, Navi Mumbai, Maharashtra, India
2 Department of Oral and Maxillofacial Pathology and Microbiology, D.Y Patil Deemed to be University School of Dentistry, Navi Mumbai, Maharashtra, India

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Date of Submission09-Mar-2022
Date of Decision16-Apr-2022
Date of Acceptance15-Jun-2022
Date of Web Publication14-Nov-2022
 

  Abstract 


Background: Effect of the oral cancerous epithelial cells on the extracellular matrix (ECM) and vice versa is also responsible for the growth and progression of cancer. The effect of inflammation on the stroma and its association with progression has also been stated in literature. Materials and Methods: The control group consisted of 10 patients with apparently normal mucosa with no history of habits, the study group consisted of 30 patients with oral squamous cell carcinoma (OSCC) confirmed by examination and incisional biopsy. H and E staining, Masson's Trichrome Staining, and Verhoeff's Van Gieson staining were used to study the inflammation, collagen fibers, and elastic fiber, respectively. A few selected specimens were studied under the scanning electron microscope. The grade of inflammation was statistically correlated with collagen and elastic fibers. Observation: Grade of inflammation was closely associated with lymphadenopathy and the density of collagenous and elastic components of the ECM. The scanning electron microscopic evaluation of elastic fibers revealed irregular fragmented elastic fibers. Conclusion: The stroma is the key to complete removal and treatment of OSCC; however, more studies are needed to define the role of each component, therefore improving the prognosis of a patient.

Keywords: Collagen fibers, elastic fibers, inflammation, scanning electron microscope, squamous cell carcinoma, Verhoeff's van Gieson


How to cite this URL:
Chandni S, Tamgadge S, Tamgadge A, Pereira T, Mahajan M, Kumar S, Jadhav A. Tumor microenvironment in oral squamous cell carcinoma: Special stains and scanning electron microscopic study. J Microsc Ultrastruct [Epub ahead of print] [cited 2022 Dec 1]. Available from: https://www.jmau.org/preprintarticle.asp?id=361119





  Introduction Top


Oral cancer is a serious and global problem in many parts of the world. When grouped together with pharyngeal cancers, it is the sixth-most common cancer globally[1] In India, it is one of the most common cancers and an important public health problem.[2]

The understanding of cancer and its growth and development is closely associated with the molecular mechanism involved in the initiation and progression of cancer. Hanahan D, Weinberg RA proposed six hallmarks of cancer, which were distinctive and complementary capabilities that enable tumor growth and metastatic dissemination.[3] However, in the year 2011, these authors also concluded that, tumors are more than insular masses of proliferating cancer cells, instead they are complex tissues composed multiple distinct cell types participating in heterotrophic interactions with one another.[4]

This has led to the conclusion that the biology of tumors can no longer be understood by simply enumerating the traits of cancer but instead must encompass the contributions of “tumor microenvironment” to tumorigenensis.

Recent research has since identified multiple simultaneous mechanisms that cause the evolution of localized cancer to diffuse dissemination of cancerous cells which is ultimately fatal; authors have now begun to realize that the extracellular matrix (ECM) is not a passive entity in the disease process and interacts dynamically with the invading front of the tumor. Abnormal ECM promotes cellular transformation and metastasis, but more importantly facilitates tumor-associated angiogenesis and inflammation, leading to the generation of tumorigenic microenvironment. Factors in the microenvironment such as ECM proteins, growth factors, and host immune response play a role in the extension, invasion, and metastasis of the tumor. Alterations in the matrix may play a role in the invasion of tumor cells.[5]

Changes in ECM and ECM matrix proteins have been studied in the metastatic progression of colon, lung, prostate, and cervical cancer. It has also been reported that the ECM produced by transformed cells differs from normal cells. Characterization of these changes has mostly been based on immunohistochemical and genetic studies.[6] However, scanning electron microscope (SEM) studies on oral squamous cell carcinoma (OSCC) have been rarely reported except for a few normal tissues.[7],[8],[9]

In this study, the fibrous component of ECM has been studied for changes associated with squamous cell carcinoma of the oral cavity and correlations have been made between the grade of inflammation and the clinical parameters of disease progression using special stains and SEM.


  Materials and Methods Top


The study was carried out at DYPATIL University School of Dentistry Navi Mumbai in 2011. Ethical approval was considered from the institutional ethical board.

All the study participants were given clear explanations about the objective of the study and a written informed consent was taken after a standard questionnaire interview.

Patients with a biopsy-proven malignant oral ulcer/mass were constituted the study group (n = 30).

Specimens were received in 10% neutral-buffered formalin for light microscopy.

They were fixed with 10% neutral-buffered formalin, dehydrated through an increasing ethanol series and xylene, and embedded in paraffin. A representative series of sections, 3-μm thick, were cut from each specimen and stained with hematoxylin and eosin (H and E). Histopathological diagnosis of OSCC was confirmed, graded by Broder's grading system under Leica research microscope: (Model No. DM1000 LED).

Grading of inflammation was done via grid analysis. The grid analysis was performed via clicking the pictures of all parts of the tissue under ×40 and superimposing a 6 × 6 grid. In each square was designated a value I-for inflammatory, F-for fibrous, and X-for cannot be assessed. Percentage for inflammatory and fibrous components was calculated for each case[10] [Figure 1].
Figure 1: H and E staining with a grid. The grids are analyzed and marked F for fibrous, I-for inflammatory and X-not assessed. (a)This grid is mostly composed of fibrous tissue. (b) This grid is mostly composed of inflammatory cells

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Each slide at a high power field was assessed with this grid and the total number was counted for both the inflammatory and the fibrous tissue. The total was calculated and the result was displayed in percentage.



After calculating the percentage of these components grading was done and Scored as 1= 0-50 and 2= 51-100

Sections were studied with the Verhoeff's-Van Gieson method for demonstration of elastic fibers, and Masson's trichrome stain to identify collagen. The density and structure of fibers were recorded for each slide [Figure 2] and [Figure 3].
Figure 2: (a) Masson's Trichrome staining for control slides at ×10. The collagen fibers are stained blue within the connective tissue. INSET (×40): The higher magnification. (b) Verhoeff's Van Geison staining for control slides at ×10. The elastic fibers are stained black within the connective tissue. INSET (×40): the higher magnification

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Figure 3: (a) H and E staining for well-differentiated carcinoma at ×10. Connective tissue shows areas with fibrous connective tissue which appear eosinophilic. INSET (×40): The higher magnification. (b) Masson's Trichrome staining for well-differentiated carcinoma at ×10. The collagen fibers are stained blue within the connective tissue. INSET (×40): Fibers appear fragmented and sparse. (c) Verhoeff's Van Gesion staining for well-differentiated carcinoma at ×10. The elastic fibers are stained black within the connective tissue. INSET (×40):-fibers appear continuous and dense

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The presence of ECM changes was compared with the grade of tumor and lymph node metastasis and the grade of inflammation.

The areas of the slides containing elastic fibers were noted and recorded under Verhoeff's Van Gieson stain. For ultrastructural analysis, these slides were demounted and cleared in xylene. The section was then brought to water and preserved and processed it. The electron microscopic examination was done under the Quanta 200 ESEM (3 nm resolution, ×300,000, Secondary, and Backscattered Imaging, Elemental Analysis (Microanalysis) Boron-Uranium) after drying the slides, and the same areas were focused under the electron microscope.


  Results and Observations Top


Clinical parameters

Lymphadenopathy was seen in 18 (60%) and lymph node enlargement was absent in 12 (40%) of the participants.

H and E parameters

On comparison of Broders grade of disease with grade of inflammation, (P = 0.007) Broders grade of disease was not associated with inflammation. Broders grade of disease and lymphadenopathy were not correlated.

On the evaluation of the grids – In the control group, we saw that 2 (20%) of the controls had the high grade of inflammation, while 8 (80%) had low grade of inflammation. While in the study group 20 (67%) participants showed a high grade of inflammation and 10 (33%) participants showed a low grade of inflammation [Table 1] and [Graph 1].
Table 1: The results of the grid analysis done for the hematoxylin- and eosin-stained slides

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The study group was also studied for an association between lymphadenopathy and grade of inflammation, we found that out of 18 cases with lymphadenopathy 16 (52%) had high grade of inflammation and 2 (6%) cases had low grade of inflammation, on the other hand out of 12 cases with no lymphadenopathy 4 (13%) had high grade of inflammation and 8 (29%) cases had low grade of inflammation. There was a significant association between lymphadenopathy and grade of inflammation (P = 0.003).

Masson's trichrome staining

On comparison of the density of fibers and grade of inflammation, a significant correlation was found and it was seen that higher the grade of inflammation lower is the density of fibers (P = 0.001) [Table 2] and [Graph 2].
Table 2: Comparison of density of collagen fibers and grade of inflammation

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However, no significant correlation was found between the density of collagen fibers and the Broder's classification or lymphadenopathy.

Verhoeff's van Gieson Staining

Ten out of 30 cases showed the presence of elastic fibers.

The density of elastic fibers correlated with the grade of inflammation (P = 0.002). It was seen that the higher the grade of inflammation lower is the density of fibers [Table 3] and [Graph 3].
Table 3: Comparison of grade of inflammation and presence of elastic fibers

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However, no significant correlation was found between the density of elastic fibers and the Broder's classification or lymphadenopathy.

Scanning electron microscopic analysis

  1. Control slides showed regular fibers with the absence of remodeling [Figure 4]
  2. Nine out of the 10 slides studied showed disintegrated irregular fibers. Radiolucent edges were also seen these are indicative of remodeling of the fibers
  3. Five sections also showed a ballooning of the fibers which is also a feature of remodeling [Figure 5], [Figure 6], [Figure 7], [Figure 8].
Figure 4: Scanning electron microscopic image of elastic fibers in a control section (×2000). The fibers are continuous and do not show ballooning or irregular edges. There is no evidence of radiolucent edges that are indicative of remodeling. INSET (×3000): The elastic fiber bundle in higher magnification

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Figure 5: Scanning electron microscopic image of elastic fibers in a well-differentiated squamous cell carcinoma (×2000). The fibers are fragmented and show ballooning of the fibers (arrow). There is evidence of radiolucent edges that are indicative of remodeling. INSET (×3000): The elastic fiber bundle in higher magnification

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Figure 6: Scanning electron microscopic image of elastic fibers in a well-differentiated squamous cell carcinoma (×2000). The fibers are fragmented and multiple fibers show ballooning (arrow). There are several fibers with radiolucent edges that are evident and these are indicative of remodeling. INSET (×3000): The elastic fiber bundle in higher magnification

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Figure 7: Scanning electron microscopic image of elastic fibers in a moderately differentiated squamous cell carcinoma (×2000). The fibers continuous and there is no evidence of ballooning (arrow). Several fibers with radiolucent edges are evident and these are indicative of remodeling. INSET (×3000): The elastic fiber bundle with irregular and radiolucent edges in higher magnification

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Figure 8: Scanning electron microscopic image of elastic fibers in a moderately differentiated squamous cell carcinoma (×2000). The fibers sparse and fragmented, also there is no evidence of ballooning (arrow). INSET (×3000): The elastic fiber bundle with irregular and radiolucent edges in higher magnification

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  Discussion Top


Lymphadenopathy

Lymphadenopathy was seen in association with 58% of the study group, while the other 42% of the study group has no lymphadenopathy in association with the disease process. This particular finding is significant because it has been seen in previous reports by Patel and Shah and Bhargava et al. that the involvement of lymph nodes is always associated with poor prognosis, approximately 50% poorer than for patients with equivalent tumors and no nodal metastasis.[11],[12]

H and E Findings

On the evaluation of the H and E slides, it was found that the connective tissue of 64.5% of the patients showed a higher grade of inflammation and only 34.5% of the cases showed lower grades of inflammation and 5 (15%) participants showed a high grade of fibrosis and 25 (85%) participants showed a low grade of fibrosis.

This indicates that in OSCC there is the abnormal and excessive response of inflammatory cells and it is associated with chronic inflammation. Increased inflammation is also been previously observed in association with cancer.[13],[14]

The grade of fibrosis is much lower than the control group.[14],[15] have concluded in their reviews that this loss of the fibers may be due to the disintegration of the structure under constant attack from the cancer cells and their signaling to the inflammatory cells.[13],[15]

The histopathological evaluation revealed that 22 (73%) participants had well-differentiated carcinoma, while 8 (27%) had moderately differentiated carcinoma according to the Broder's classification.

On comparison of the grade of disease with the grade of inflammation, we found that most cases showed well-differentiated carcinoma and high grade of inflammation about 45%, but the association between these parameters is not significant. This is consistent with the findings of Anneroth et al.[16] and Bryne et al.[12],[17],[18],[19],[20] who have given in their classification that in more differentiated carcinomas the grade of inflammation is higher.[17] However, it has recently been postulated by Zamarron and Chen, that inflammation though present is unable to deter the progression of cancer and at times, chronic inflammation may actually perpetuate molecules that cause degradation of matrix and spread of cancer.[21]

When the grade of disease was assessed against lymphadenopathy, it was found that there was no association between these parameters, and this was in agreement with the findings of various authors such as Jakobsson et al., Annernoth, Bryne who have concluded that Broders's grading of disease is not a sufficiently predictive marker for the prognosis of the disease and does not associate well with clinical parameters of the ailment.[16],[17],[19],[22],[23]

On comparison of grade of inflammation and lymphadenopathy, it was found that most of the cases showing lymphadenopathy (16 out of 18) showed higher grade of inflammation and most of the cases without lymphadenopathy (9 out of 13) showed lower grade of inflammation. This is in agreement with several authors Hanahan and Weinberg[3] who in their review of hallmarks of cancer have given inflammation importance and stated that the presence of inflammatory cells is significantly associated with cancer. Schottenfeld and Beebe-Dimmer stated in their review that chronic inflammation caused the disintegration of ECM that in turn led to the invasion and progression of cancer[21],[24] who in his review has stated that the presence of inflammatory cells and their mediators such as cytokines and interleukins cause disintegration of the matrix.[14]

Masson's trichrome stain

The slides studied by Masson's trichrome revealed the presence of collagen fibers in all 30 slides. On comparison of the grade of inflammation with the density of collagen fibers, it was found that most of the cases showing loosely arranged fibers also showed a higher grade of inflammation. According to Lu et al.,[25] this may be attributed to the presence of chronic inflammation which in turn, elaborates factors that drive tumor growth, survival, and angiogenesis. George et al.[26] studied the stroma of OSCC and reported that the collagen fibers appear to undergo degeneration within the stroma and if present may help to the wall of the tumor invasion, Lu et al.[25] described the tumor microenvironment as a dynamic niche, in which the cancer cells cause dysregulation of the matrix causing tumor-associated angiogenesis and inflammation.[5]

In the study it was seen, well-differentiated carcinoma 18 (61%) cases had loosely arranged fibers, while 4 (13%) cases had densely arranged fibers while we found that out of 8 cases showing moderately differentiated carcinoma 4 (13%) had loosely arranged fibers, while 4 (13%) cases had densely arranged fibers.

There was no association between these parameters which is also reported by Anneroth et al. and Bryne.[16],[20]

In the study, it was seen, 18 cases that showed lymphadenopathy 14 (47%) cases had loosely arranged fibers, while 4 (13%) cases had densely arranged fibers, while we found that out of 12 cases showing no lymphadenopathy 8 (27%) had loosely arranged fibers while 4 (13%) cases had densely arranged fibers.

There is no association between the density of fibers and lymphadenopathy.

Verhoeff's van Gieson stain

On the evaluation of the slides stained with Verhoeff's Van Gieson stain, it was observed that only 10 out of the total 31 cases showed the presence of elastic fibers.

On comparison between grade of inflammation and presence of elastic fibers, it was observed that most of the slides which were positive for elastic fibers (9 out of 10) were showing low grades of inflammation and those without elastic fibers showed higher grades of inflammation. This was in accordance with authors who suggested that the presence of chronic inflammation will lead to degradation of the ECM components. Hou et al. studied tongue carcinoma in hamsters and concluded that there was the disintegration of elastic fibers and the associated increase in collagen deposition.[27]

Similarly, on comparison of the presence of elastic fibers with Broder's Grade of Disease and lymphadenopathy, there was no association in these parameters. Although several authors have stated that the presence of elastic fibers or elastosis of the connective tissue is associated with the lower spread of disease and clinically absence of lymphadenopathy our finding did not agree with studies conducted by Agrawal et al. and therefore showed no association between the presence of elastic fiber component and lymphadenopathy.[6] We conclude that this finding might be due to the absence of elastic fibers at the invading front of the tumors and their density observed might not be sufficient to limit the advancing edges of the tumor.

Scanning electron microscope analysis

Very few studies have been done on SEM studies on elastic fibers in OSCC.[7],[28] The scanning electron microscopic analysis showed the presence of elastic fibers that seen to be densely packed small length fibers on close examination and seemed to have jagged outlines as well as they appeared electron-dense on examination.[6]

The fibers also appeared irregular and shortened. Moreover, the presence of irregular radiolucent edges was also observed.[8],[9],[29],[30]


  Conclusion Top


Tumor microenvironment is a complex system composed of a largely altered ECM with different cell types that determine tumor progression. The highly dynamic nature of the ECM plays a crucial role in disease progression. The ECM is essential for normal wound healing processes, but excessive deposition – as is observed in the case of fibrotic and degenerative diseases – can lead to organ dysfunction. Similarly, the ECM plays a key role in the development of cancer, modulating processes such as tumor cell invasion and metastasis, among others.

In this context, while treating any case of oral cancer it becomes important to study the tumor matrix and identify the presence of certain molecules or cells that may cause rapid progression of the disease.

As immunity is so adversely affected and also adversely affects carcinoma a clinician might want to resort to immunoediting as a means of limiting the degeneration caused by the inflammatory cells in the cancer stroma.

The stroma is, therefore, the key to complete removal and treatment of OSCC; however, more studies are needed to define the role of each component, therefore improving the prognosis of a patient.

Further research is warranted to determine the mechanism of destruction and the molecules most affected so that therapies that target these specific molecules and prevent their dissociation can be considered to increase the quality of life of patients and reduce mortality associated with this disease.

Acknowledgement

Authors would like to acknowledge Mr. Kiran Bhotkar, Assistant Manager, for helping with SEM analysis of histological sections, (Address: Icon House,1st Floor, Plot No 52, Sector 6, Service Industrial Area, Sanpada, Navi Mumbai – 400705. Tel: +91 22 6965. [email protected]).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Sandhya Tamgadge,
Department of Oral and Maxillofacial Pathology and Microbiology, D Y Patil University School of Dentistry, Nerul Navi Mumbai Sector 7, 400 706, Maharashtra
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmau.jmau_19_22



    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

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    -  Chandni S
    -  Tamgadge S
    -  Tamgadge A
    -  Pereira T
    -  Mahajan M
    -  Kumar S
    -  Jadhav A


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