Year : 2010 | Volume
: 21 | Issue : 2 | Page : 260--265
Immunohistochemical evaluation of mast cells and angiogenesis in oral squamous cell carcinoma
Bhushan Sharma, G Sriram, TR Saraswathi, B Sivapathasundharam
Department of Oral and Maxillofacial Pathology, Meenakshi Ammal Dental College, Chennai, India
Department of Oral and Maxillofacial Pathology, Meenakshi Ammal Dental College, Chennai
Objectives : Angiogenesis is a complex event mediated by angiogenic factors released from cancer cells and immune cells. It has been reported to be associated with progression, aggressiveness and metastases of various malignant tumors including oral squamous cell carcinoma (OSCC). Similarly, mast cells have also been reported to play a role in tumor progression and metastases by promoting angiogenesis. The present study aims at comparison of microvascular density (MVD) and mast cell density (MCD) in normal oral mucosa (NM) and among various grades of OSCC.
Materials and Methods : MVD was assessed immunohistochemically using anti-Factor VIII related von Willebrand factor, and MCD using anti-mast cell tryptase in a study sample of 30 cases of OSCC and 10 cases of clinically normal oral mucosa.
Results : The mast cells in normal oral mucosa and oral squamous cell carcinoma strongly expressed mast cell tryptase. The density of mast cells and micro vessels were significantly higher in OSCC compared to normal oral mucosa. The MCD and MVD were higher in moderately differentiated OSCC than in well differentiated OSCC ( P > 0.05) and normal oral mucosa ( P < 0.05). Pearson«SQ»s correlation revealed a positive correlation between MCD and MVD ( r=0.33; P=0.077).
Conclusion : These findings indicate that mast cells may play a role in up regulation of tumor angiogenesis in OSCC probably through mast cell tryptase.
|How to cite this article:|
Sharma B, Sriram G, Saraswathi T R, Sivapathasundharam B. Immunohistochemical evaluation of mast cells and angiogenesis in oral squamous cell carcinoma.Indian J Dent Res 2010;21:260-265
|How to cite this URL:|
Sharma B, Sriram G, Saraswathi T R, Sivapathasundharam B. Immunohistochemical evaluation of mast cells and angiogenesis in oral squamous cell carcinoma. Indian J Dent Res [serial online] 2010 [cited 2021 Feb 24 ];21:260-265
Available from: https://www.ijdr.in/text.asp?2010/21/2/260/66655
Oral squamous cell carcinoma (OSCC) is a malignant neoplasm arising from the mucosal epithelium of the oral cavity. OSCC constitutes approximately 90 % of all oral malignancies and remains serious problem of oral health worldwide.  In spite of improvement in diagnostic methods and meticulous execution of radical surgery with or without adjuvant chemo radiotherapy, the overall five-year survival rate of patients with OSCC has improved only slightly.  Molecular biological markers of OSCC are extensively studied to aid in the prevention and prognosis of OSCC. However, no marker is universally accepted so far.
Angiogenesis or neovascularization, the formation of new microvasculature, is an important component in many biological processes, both in physiological conditions, such as proliferating endometrium and embryogenesis, and in pathological conditions, such as rheumatoid arthritis and neoplastic disease.  Angiogenesis has been known to aid progression and metastasis of many malignant tumors including tumors of the lung,  breast,  esophagus[5,6] and oral cavity.,,,,,,
The process of angiogenesis involves degradation of the basement membrane of the parent vessel and extracellular matrix, locomotion of endothelial cells toward a tumor implant, mitosis, lumen formation, development of sprout loops and of a new basement membrane, and finally, recruitment of pericytes.  The induction of angiogenesis is mediated by several stimulatory and inhibitory molecules released by both tumor and host cells and depends on a net balance between the stimulatory angiogenic and inhibitory anti-angiogenic factors. 
Among the various host immune cells, mast cells have been implicated in tumor progression by promoting angiogenesis. , Of the several angiogenic factors found in mast cells, mast cell tryptase has been reported to be a potent angiogenic factor.  To examine the relationship between angiogenesis, mast cells and the histological grade of OSCC, we immunohistochemically analyzed the microvascular density (MVD) and mast cell density (MCD) in OSCC using anti-factor VIII related von Willebrand factor and anti-mast cell tryptase respectively.
Materials and Methods
Formalin-fixed, paraffin-embedded tissue specimens of 30 cases of OSCC (17 well differentiated OSCC, 12 moderately differentiated OSCC, and one poorly differentiated OSCC) were obtained from the Department of Oral Pathology at Meenakshi Ammal Dental College, Chennai. Of the 30 cases, 20 were from males and the remaining from females. As controls, 10 clinically normal oral mucosa tissue specimens were obtained from people who had surgical removal of impacted tooth; these individuals neither smoked nor drank alcohol. All the tissue samples were collected after obtaining an informed consent.
MVD and MCD were assessed using mouse monoclonal anti-factor VIII related von Willebrand factor antibody (BioGenex, San Ramon, CA) and mouse monoclonal anti-mast cell tryptase antibody (BioGenex, San Ramon, CA) respectively. In brief, 4 ΅m sections of formalin-fixed, paraffin blocks were placed on silane coated slides and deparaffinized with xylene and rehydrated with graded alcohols. To retrieve the antigens, the slides were heated in a microwave oven in 10 mM of sodium citrate buffer at pH 6.2 and boiled thrice for five minutes and were allowed to remain at room temperature for 30 min and were washed in Tris buffer solution at pH 7.6. Endogenous peroxidase activity was blocked by incubating the slides in 3% hydrogen peroxide with methanol for 10 min at room temperature. To decrease the background staining, the slides were incubated with universal blocking solution (BioGenex, San Ramon, CA) rinsed in PBS, and incubated with mouse monoclonal anti-factor VIII related von Willibrand factor antibody (BioGenex, San Ramon, CA) for one hour or mouse monoclonal anti-mast cell tryptase antibody (BioGenex, San Ramon, CA) for two hours. Then the slides were incubated with secondary antibody - super enhancer (BioGenex, San Ramon, CA) and poly-horse raddish peroxidise (BioGenex, San Ramon, CA) for 30 minutes at room temperature. After being washed in two changes of phosphate buffered saline the slides were incubated with 0.1% 3, 3'-diaminobenzidine (BioGenex, San Ramon, CA) for 20 min. Finally, the sections were counterstained with Harris hematoxylin for five minutes.
Sections of neurofibroma and pyogenic granuloma were used as positive control for mast cells and endothelial cells respectively. Negative controls consisted of sections of normal mucosa and OSCC that were treated in the same manner as the study groups expect that the primary antibody was omitted.
Quantification of microvascular and mast cell densities
The number of micro vessels and mast cells in normal mucosa and OSCC in 4-5 fields at a magnification of x400 under an ocular grid (0.0625 mm 2 ) at the 'hot spots' were counted under light microscope, and the average per mm 2 was determined using the formula given below.[INLINE:1]
Any endothelial lined vessel lumen or endothelial cell cluster appearing reddish brown and clearly separate from an adjacent cluster was considered to be a single countable micro vessel. Any cluster of mast cell granules appearing reddish brown and clearly separate from an adjacent cluster was considered to be a single mast cell. All counts were performed by a single investigator with the knowledge of clinical or histopathological variables, to eliminate interobserver variation.
The MVD and MCD between OSCC and normal oral mucosa was compared using independent student 't' test. The MVD and MCD among well differentiated OSCC (WDOSCC), moderately differentiated OSCC (MDOSCC) and normal oral mucosa was analyzed using Post-ANOVA Tukey HSD test. The statistical correlation between MVD and MCD in OSCC was analyzed using Pearson's correlation coefficient.
Mast cell density
Tryptase-positive mast cells were characterized by the presence of numerous reddish-brown granules in the cytoplasm. The mast cells were round, oval, spindled or stellate shaped. Analysis of consecutive sections showed that the mast cells were distributed around micro vessels and appeared to accumulate in areas of increased vascularity (hot spots).
We used the independent 't'-test and found the MCD significantly higher in OSCC (241.87 ± 75.6) than in normal oral mucosa (115.2±24.5; P=0.000); using Tukey HSD test, MCD was found to be higher in MDOSCC (260.67±60.32) compared to WDOSCC (234.12±83.56; P=0.541) and normal oral mucosa (115.2±24.5; P=0.000). Similarly, the MCD was significantly higher in WDOSCC (234.12±83.56) compared to normal oral mucosa (115.2±24.5; P=0.000) [Table 1].
Micro vessel density
Von-Willebrand factor-positive endothelial cells were stained reddish brown and they were clearly demarcated from the connective tissue background and the negative controls showed no staining. The independent 't'-test showed the MVD to be significantly higher in OSCC (240.53±92.3) compared to normal oral mucosa (64.4±13.53; P=0.000). Tukey HSD analysis showed the MVD to be higher in MDOSCC (277.33±70.25) compared to WDOSCC (210.12±98.5; P=0.0625) and normal oral mucosa (64.4±13.53; P=0.000). Similarly, the MCD was significantly higher in WDOSCC (210.12±98.5) compared to normal oral mucosa (64.4±13.53; P=0.000) [Table 2].
Correlation between mast cell density and microvascular density
As shown in [Figure 1] and [Figure 2] the Pearson's correlation showed a positive correlation between MVD and MCD (r=0.33; P=0.077) [Table 3].
Sustained tumor growth requires a positive balance between tumor cell proliferation and cell death or apoptosis. Using an experimental animal model, it has been shown that the initiation of angiogenesis appears concomitantly with a decrease in tumor cell apoptosis, while the levels of tumor cell proliferation remain constant, thus leading to net tumor growth.  Preinvasive malignant cells are known to remain dormant until they become angiogenic, and this is followed by a phase of rapid tumor growth. ,
Angiogenesis is the outcome of an imbalance between positive and negative angiogenic factors produced by both tumor and host cells.  Among the host cells, which produce and release pro-angiogenic and angiogenic factors are mast cells.  Mast cells are an important source of several proangiogenic and angiogenic factors, such as histamine, heparin, chymase, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-b) and others. 
Tumor angiogenesis and tumor growth have been reported to be less in mast cell deficient mice compared with mice with normal mast cell numbers.  Moreover, mast cells were shown to induce neovascularization through the carcinogenesis of squamous cells.  Angiogenic factors including VEGF, bFGF, and platelet-derived growth factors have been reported to stimulate mast cell migration.  Hypoxia might induce tumor cells to release angiogenic factors which in turn could chemo attract the mast cells to migrate into the hypoxic areas of the tumor. After migration into the hypoxic areas, mast cells might produce angiogenic products that stimulate the infiltration of even more mast cells. 
Various angiogenic factors secreted by mast cells either directly promote angiogenesis by stimulating the migration and/or proliferation of mast cells or indirectly through degradation of extracellular matrix.  Tryptase is a serine endopeptidase that is released in abundant quantities from mast cells in a bound form with heparin.  Both heparin and tryptase are potent angiogenic factors, but the angiogenic potential of heparin per se is low compared to tryptase.  Secondly, the angiogenic effect of heparin requires tryptase to be catalytically active, as synthetic tryptase inhibitors abolish the angiogenic effect of heparin.  Tryptase acts as a mitogen for dermal endothelial cells,  fibroblasts,  smooth muscle cells  and epithelial cells.  Tryptase also activates latent metalloproteinases and plasminogen activator, which degrade the extracellular matrix, , which is important for the initial stages of angiogenesis. 
Density of mast cells in a tissue has been studied using histochemical stains like toluidine blue,  alcian blue  and immunohistochemically  using mast cell tryptase, heparin, chymase, and carboxypeptidase A. In the present study, mouse monoclonal anti-mast cell tryptase antibody was used to quantitate the presence of mast cells. In this study, MCD was found to be significantly higher in MDOSCC compared to WDOSCC and in WDOSCC compared to normal buccal mucosa. These findings were similar to those reported by previous studies on various tumors. ,,,
However, Oleiveira-Neto HH et al.  found MCD to be lower in OSCC and premalignant lesions compared normal controls. They attributed it to migration failure of mast cells, which possibly reflect a modification in the microenvironment during tumor initiation and progression. Certain researchers have shown antitumor functions of mast cells, including natural cytotoxicity and the release of antitumor compounds. 
Tomita M et al.  have put forth two reasons for such conflicting reports on the role of mast cells. The cytotoxic functions of mast cells that suppress tumor activities might be present initially when the mast cells infiltrate the tumor tissue. However, after infiltration, the tumor cells might promote the angiogenic properties of mast cells while suppressing their cytotoxic functions, thereby leading to tumor angiogenesis.  Secondly, cell-mediated cytotoxic effects of mast cells have been reported, with mast cell: tumor ratios greater than 20:1.  Conversely, cytotoxic effects of mast cells were nullified and tumor progression was found to be enhanced when the mast cell-tumor ratios were increased to 10:1 to 1:100.  Hence, the effect of mast cells against cancer cells might depend on the concentration of mast cell products in the microenvironment. Based on these findings, Tomita M et al.  hypothesized that reversing this process, i.e., enhancing the cytotoxic functions of mast cells and suppressing their angiogenic functions, could lead to a new anti-cancer treatment strategy. Furthermore, the mast cell heparin inhibitors, protamine and platelet factor 4, have been reported to inhibit angiogenesis. 
Density of micro vessels in the connective tissue has been studied by various staining and quantification methods. Micro vessels can be studied using various immunohistochemical stains like antibodies against VEGF,  factor VIII related antigen (FVIII Ag),  CD 31,  CD 34  and vimentin.  Vascularity is commonly quantified by assessing the density of micro vessels as the number of blood vessels per unit area. However, using vascular density as a parameter and above mentioned immunohistochemical targets, it is not possible to distinguish between resting and angiogenic vessels; avb3 has been reported as specific marker of angiogenic vessels. But Pazouki et al.  have reported the expression avb3 in the vasculature of oral tissues not necessarily specific for angiogenic vessels. In the present study, we have used monoclonal antibody against FVIIIAg to quantify the density of micro vessels in the tissue. Gleich LL et al.  compared the staining ability of FVIIIAg and CD 31 and reported FVIIIAg to be uniform and easy to interpret.
In the present study, MVD was found to be significantly higher in MDOSCC compared to WDOSCC and in WDOSCC compared to normal buccal mucosa. Similar results were reported by earlier workers on OSCC. ,,,,, Pazouki et al.,  and Iamaroon et al.  demonstrate a significant increase in vascularity during transition from normal tissue through different degrees of dysplasia to early and late carcinoma. However, Tae et al.,  found significantly higher MVD in normal mucosa compared to SCC, and they attributed it to the increase in vascularity in SCC being parallel to the increase in tumor volume. Similarly, Jin et al.,  found higher MVD in WDOSCC compared to MDOSCC and PDOSCC. Conflicting results may be due to subjective variation in the classification of OSCC and in the use of different pan-endothelial markers that cannot distinguish between resting and angiogenic vessels.
In the present study, correlation between MVD and MCD revealed a linear increase in MVD as the MCD increased, suggesting a positive correlation. However, if presence of mast cells was the key factor in the angiogenesis, there would have been an exponential increase rather than a linear one, indirectly suggesting the role of other factors that modulate the angiogenesis. Higher density of dermal mast cells have been reported as a predisposing factor for the development of basal cell carcinoma and melanoma of the skin.  However, a similar correlation has not been reported for patients with squamous cell carcinoma. Immune response to foreign bodies, chemicals and tumor cells are mediated through various epithelial and dermal cells types.
The Langerhans cells and keratinocytes are largely responsible for initiating immune activation, whereas the dermal fibroblasts, dendritic cells, mast cells and endothelial cells maintain and mediate immune responses.  The number of CD1a positive oral mucosal Langerhans cells (CD1a+LC) have been reported to be increased in lip and lateral border of the tongue of smokers with and without OSCC.  Whereas in smokeless tobacco users the number of CD1a+LCs were reported to be lower in the same sites.  Hence exposure to tobacco products could directly alter the function of Langerhans cells that could alter the function of mast cells or recruit more number of mast cells that could contribute to the increased number of mast cells found in OSCC. The increase in mast cells in OSCC could be due to chemo attractants produced by tumor cells or normal connective tissue cells in response to the tumor, or as an allergic reaction to various tobacco products diffusing into the connective tissue, or as a response to cellular injury brought about by the tobacco products.
The role of mast cells in angiogenesis in OSCC and the role of angiogenesis on tumor progression need to be further validated using larger samples that include recurrent cases and follow up studies. The various factors (tobacco-related and tumor-related) that lead to an increase in mast cells in OSCC needs to be validated. Large scale multi-institutional studies would provide us with a baseline data of the MCD and MVD in different grades of OSCC and may aid us in classifying the individuals with OSCC as high-risk or low- risk individuals, and also in planning and/or development of various adjuvant therapeutic strategies like anti-angiogenesis therapy and vascular targeting of anticancer gene therapy.
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