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Ann Thorac Surg 2000;69:1686-1690
© 2000 The Society of Thoracic Surgeons

Original articles: General thoracic

Effect of mast cells on tumor angiogenesis in lung cancer

Masaki Tomita, MDa, Yasunori Matsuzaki, MDa, Toshio Onitsuka, MD, PhDa

a Department of Surgery II, Miyazaki Medical College, Miyazaki, Japan

Address reprint requests to Dr Tomita, Department of Surgery II, Miyazaki Medical College, Kihara 5200, Kiyotake, Miyazaki, 889-1692 Japan
e-mail: mtomita{at}post.miyazaki-med.ac.jp


   Abstract
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 Abstract
Introduction
 Material and methods
 Results
 Comments
 References
 
Background. We conducted a retrospective study to clarify the effect of mast cells on tumor angiogenesis in lung cancer patients.

Methods. Formalin-fixed and paraffin-embedded tumor sections were used in this study. Parenchymal mast cells were stained with Alcian blue and safranin O. The number of mast cells per ten fields at a magnification of 200x was counted under light microscopy, and the average count was determined. To highlight the microvessels, endothelial cells were stained with anti-human factor VIII antibody. After the microvessel count was determined, the microvessels were further stained with Alcian blue and safranin O to show areas of mast cell infiltration. Expression of vascular endothelial growth factor was assessed using a polyclonal antibody.

Results. We found a significant correlation between mast cell count and microvessel density. This correlation was also observed in patients with adenocarcinoma (p < 0.001) as well as in patients with squamous cell carcinoma (p < 0.01). Double staining of the microvessels showed highly angiogenic areas densely populated with mast cells. Although we detected a slight trend toward a correlation between vascular endothelial growth factor expression and microvessel density, it was not statistically significant. We found no association between vascular endothelial growth factor expression and mast cell count.

Conclusions. There appears to be a direct correlation between the number of mast cells and tumor angiogenesis in patients with lung cancer, and this relationship appears to be independent of vascular endothelial growth factor expression.


   Introduction
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 Abstract
 Introduction
Material and methods
 Results
 Comments
 References
 
Although a direct association between mast cells and cancer tumors is generally accepted, the exact nature of this relationship appears somewhat contradictory. Galli [1] has reported the general functions of mast cells in malignant diseases [1], but other researchers have shown antitumor functions of mast cells, including natural cytotoxicity [2, 3] and the release of antitumor compounds [4, 5]. These antitumor effects have been reported in studies on breast cancer [6] as well as colorectal neoplasias [7, 8], suggesting that mast cells have an effect on tumor cell cytotoxicity.

Other studies, however, have shown that mast cells enhance tumor proliferation in vitro [9–13]. Mast cell products such as histamine [14, 15], basic fibroblast growth factor [16], and heparin [17] have been shown to promote tumor angiogenesis. Recently, mast cell tryptase was found to be a potent angiogenic factor [18]. These studies suggest that mast cells accelerate tumor growth.

Because, to date, researchers have not documented the exact relationship between mast cells and tumor angiogenesis in lung cancer, we conducted this retrospective study to clarify the effect of mast cells on tumor angiogenesis in lung cancer patients. To accomplish this goal, we counted mast cells and microvessels in tumor sections, determined the area of mast cell infiltration of microvessels, and assessed vascular endothelial growth factor (VEGF) expression, one of the pivotal mediators of tumor angiogenesis.


   Material and methods
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 Abstract
 Introduction
 Material and methods
Results
 Comments
 References
 
Patient profiles and surgical specimens
Fifty-five patients (36 men, 19 women) with primary lung cancer who were treated surgically at Miyazaki Medical College Hospital between 1994 and 1995 were included in this study. Patient ages ranged from 39 to 82 years (mean, 63.4 years). The follow-up period ranged from 39 to 55 months. Table 1 summarizes the clinical characteristics of these patients.


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  		Table 1. Clinical Characteristics

 
Surgical specimens previously fixed in formalin and embedded in paraffin were used in this study. Using one paraffin block per tumor, three sections were cut and subsequently analyzed to determine mast cell counts, microvessel counts, and VEGF expression. Microvessels were double stained to show areas of mast cell infiltration.

Mast cell counts
Sections (4 µm thick) were deparaffinized, rehydrated, and stained with Alcian blue (pH 0.3) and safranin O (pH 0.1). The number of mast cells in the lung parenchyma per ten fields at a magnification of 200x was counted under light microscopy, and the average was determined.

Immunohistologic studies
To determine microvessel density and VEGF expression, we used a specific monoclonal antibody against factor VIII (Dako Diagnostika, Hamburg, Germany) and a polyclonal antibody against VEGF (Santa Cruz Biotechnology, Santa Cruz, CA). Before staining, serial 4-µm-thick sections were deparaffinized in three changes of lemosol and rehydrated through a descending series of ethanol. These sections were immersed in 0.6% H2O2 in methanol for 20 minutes at room temperature to block endogenous peroxidase activity. After blocking nonspecific protein bindings by an overnight incubation with Block Ace (Dainippon, Inc, Osaka, Japan), the sections were incubated with primary antibodies against human factor VIII (1:50) and VEGF (1:50) at 4°C overnight. Subsequently, sections were incubated with the secondary antiserum (1:500) for 1 hour, followed by an incubation with peroxidase antiperoxidase (PAP) complex for 30 minutes at room temperature. The sections were visualized using a diaminobenzidine/Metal Concentration (10x) and Stable Peroxide Substrate Buffer (1x) system (Pierce, Rockford, IL). The VEGF sections were then washed with water and counterstained with hematoxylin. Factor VIII sections were also washed with water but were not counterstained. After the microvessel count was recorded, the microvessels were stained with Alcian blue and safranin O in order to observe areas of mast cell infiltration.

Determination of microvessel density and VEGF expression
Immunohistologic results were assessed semiquantitatively by two authors. Microvessel density was determined as described by Weidner and colleagues [19] in the area of the most intense vascularization (hot spot) of each tumor, and the average was determined. Expression of VEGF was evaluated as described by Inoue and associates [20]. Tumors in which more than 30% of the carcinoma cells stained more intensely than normal smooth muscle cells were judged to be VEGF-positive.

Statistical analysis
Using linear regression analysis, we assessed the statistical correlation between mast cell counts and microvessel counts. The relationships between VEGF expression and microvessel density and between VEGF expression and mast cell count were analyzed using Student t test. A p value less than 0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Material and methods
 Results
Comments
 References
 
Mast cell and microvessel counts
Mast cells often were observed in small groups around cancer cells and in the cancer parenchyma. At a magnification of 200x, the number (mean ± standard deviation) of mast cells was 25.85 ± 18.93, which was significantly higher than in the normal sections studied (4.3 ± 2.81, p < 0.01).

Intratumoral microvessels in endothelial cells were found in small groups at the margin as well as in the cancer parenchyma. At a magnification of 200x, the number of microvessels was 22.33 ± 11.90. They appeared as brown linear fragments, occasionally with a nucleus. Tiny lumens within these microvessels often resembled small circular or fusiform structures.

As shown in Figure 1, linear regression analysis found a significant relationship between mast cell counts and microvessel counts (r = 0.73, p < 0.001), indicating a high vascular grade with increased numbers of mast cells. This correlation was observed in patients with adenocarcinoma (r = 0.78, p < 0.001) as well as in patients with squamous cell carcinoma (r = 0.63, p < 0.01). This correlation was also observed in patients with stage I or II disease (r = 0.68, p < 0.001) as well as in patients with stage IIIA or IIIB disease (r = 0.71, p < 0.001).



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  		Fig 1. Linear regression showing a significant relationship between mast cell counts and microvessel counts (r = 0.73, p < 0.001).

 
VEGF expression
Tumors positive for VEGF were found in 36 of 55 patients (65.45%), with VEGF expression predominantly observed in the cytoplasm of the cancer cells. The mean microvessel counts for VEGF-negative and VEGF-positive tumors were 20.47 ± 14.17 and 23.31 ± 10.59, respectively. Figure 2A shows a definite trend toward an association between VEGF expression and microvessel count; however, it did not reach statistical significance (p = 0.09). The mean mast cell counts for VEGF-negative and VEGF-positive tumors were 22.07 ± 13.86 and 27.84 ± 21.03, respectively. As shown in Figure 2B, there was no significant association between VEGF expression and mast cell count (p = 0.228).



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  		Fig 2. Student t test showing (A) trend toward a correlation between vascular endothelial growth factor (VEGF) expression and microvessel density that did not reach statistical significance and (B) no correlation between vascular endothelial growth factor expression and mast cell count.

 
Figure 3 shows areas of mast cell infiltration consistent with those of intense vascularization (hot spots); that is, mast cells tended to populate highly angiogenic areas in the microvessels.



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  		Fig 3. Double staining of mast cells and factor VIII shows mast cell infiltration of microvessels. The areas of mast cell infiltration are consistent with those of intense vascularization. (Original magnification x200.)

 

   Comments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comments
References
 
We found a significant correlation between the number of mast cells and the number of microvessels in lung cancer tumor sections. These results could be from the presence of more vessels, allowing the infiltration of more mast cells, or the proangiogenic activity of mast cells, including proliferation, migration, and differentiation of endothelial cells. Based on these observations, mast cells could have an important effect in promoting angiogenesis by secreting angiogenic factors or enzymes that degrade extracellular matrix. Although mast cell products that promote angiogenesis in lung cancer have not been reported, it has been demonstrated that mast cell products, including histamine [14, 15], basic fibroblast growth factor [16], and heparin [17], might directly affect endothelial cells by stimulating their migration or proliferation or indirectly affect them by degrading the connective tissue matrix thereby providing space for neovascular sprouts to form [21]. We found a significant relationship between the presence of mast cells and tumor angiogenesis in lung cancer.

Although factors that induce mast cell infiltration of microvessels are not clearly defined, the condition of hypoxia could provide some clarification. Gruber and associates [21] reported that angiogenic factors including VEGF, basic fibroblast growth factor, and platelet-derived growth factor-AB stimulate mast cell migration. Mast cells might migrate into hypoxic areas chemotactically as a result of angiogenic factors released from cancer cells. Once in these hypoxic areas, mast cells might produce angiogenic products that stimulate the infiltration of even more mast cells. Since VEGF expression, one of the pivotal mediators of tumor angiogenesis, has been reported to be related closely to microvessel density [22], it seemed likely that in our study we would find a strong correlation between VEGF expression and microvessel density. However, only 36 of 55 patients (65.45%) were VEGF positive. Although those data indicate a definite trend toward an association between VEGF expression and microvessel density, it did not reach statistical significance, possibly because of the small size of our sample. More importantly, however, our results suggest that the angiogenic functions of mast cells may be independent of VEGF expression.

Because it is generally accepted that tumor angiogenesis is beneficial for tumor growth, mast cells, in the present study, seem to have an effect on accelerating tumor growth. However, in an earlier study, we found a direct correlation between mast cell count and patient prognosis in patients with pulmonary adenocarcinoma, indicating that mast cells had a cytotoxic rather than an angiogenic effect in tumors [23]. Although the reasons for our conflicting results remain unclear, a couple explanations must be considered. Initially, mast cells might infiltrate cancerous tissue to suppress tumor activities. After infiltration, however, cancer cells might promote the angiogenic properties of mast cells while suppressing their cytotoxic functions, thereby leading to tumor angiogenesis. Second, mast cell–mediated cytotoxicity was reported, with mast cell:tumor ratios greater than 20:1 [3, 9]. Conversely, when the mast cell-tumor ratios were chosen as 10:1 to 1:100, tumor proliferation was enhanced. Therefore, the effect of mast cells against cancer cells might depend on the concentration of mast cell products in the microenvironment. These findings lead to the hypothesis that reversing this process, ie, enhancing the cytotoxic functions of mast cells and suppressing their angiogenic functions, could lead to a new treatment for patients with lung cancer. In fact, the mast cell heparin inhibitors, protamine and platelet factor 4, have been reported to inhibit angiogenesis [24].

Further studies are required to determine whether mast cells enhance host immunity against cancer cells or promote tumor growth. These studies should take into account that several researchers have reported that the number of mast cells stained in formalin-fixed specimens is consistently less than in specimens fixed in Carnoy solution [25, 26]. In the present study, we used formalin-fixed specimens because we could not obtain specimens fixed in other solutions. Our results might have been different had we used specimens preserved in Carnoy solution.

In conclusion, we found a direct relationship between mast cell count and microvessel count in lung cancer. Further, mast cell count was not associated with VEGF expression. From these results, we surmise that mast cells might help enhance tumor angiogenesis in lung cancer. In addition, this activity may be independent of VEGF.


   Acknowledgments
 
We thank Mrs Yasuko Tobayashi for skillful technical assistance.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comments
 References
 

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Accepted for publication November 10, 1999.


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