The Role of Mast Cell Tryptase in Neoangiogenesis of Premalignant and Malignant Lesions of the Uterine Cervix

It has been well established that tumors require a blood supply for their expansive growth. Newly formed blood vessels comprise a complex network that allows adequate oxygen and metabolic distribution to neoplastic cells. Tumor angiogenesis is driven by positive and negative regulators of endothelial growth and is a potential target for therapy.

Among the many proangiogenic factors identified to date, the fibroblast growth factor (FGF) family (Czubayko et al. 1997) and the vascular endothelial growth factor (VEGF) (Nicosia 1998; Kraft et al. 1999) are the most widely studied in malignant growth. Recently, Kankkunen et al. (1997) found that tryptase, a serine proteinase contained in mast cells, is also a potent proangiogenic factor in malignancies. Although the role of mast cells in the development and progression of cancer has not been elucidated, there is evidence that the number of mast cells in various solid cancers increases with tumor progresssion (Yomita et al. 1999). It has been shown in experimental models that mast cells and their products are capable of inducing tumor angiogenesis (Kessler et al. 1976). The presence of mast cells in cancer of the uterine cervix has been investigated by various authors. Graham and Graham (1966) reported that the number of mast cells in cervical cancer diminishes with cancer progression. Jain et al. (1977) observed an increased number of mast cells only in areas of inflammation around the malignant cells, but a higher number in carcinoma in situ than in invasive cancer. In contrast, Pasternak and Jans (1986) observed an increment of mast cells with cancer progression. The aim of this study was to investigate the expression of FGF and tryptase in mast cells during development of neoangiogenesis in premalignant and malignant lesions of the cervix.

Paraffin-embedded cervical biopsies from 21 patients without cancer, 19 with cervical dysplasia, eight with carcinoma in situ, and 36 with squamous cell carcinoma in different clinical stages were studied. Sections 5 μm thick were mounted on poly-l-lysinecoated slides. Some were stained with routine H and E and Alcian blue-safranin O (ABSO) for heparin-containing mast cell granules. Other sections were processed for immunolocalization of factor VIII and CD105 as endothelial markers of neovascularization, and bFGF and tryptase for mast cell growth factors, using the following antibodies: mouse monoclonal anti-human factor VIII-related Ag; mouse monoclonal anti-CD105, and mouse antihuman monoclonal mast cell tryptase, all from DAKO (Glostrup, Denmark) and rabbit polyclonal FGF-2 from Santa Cruz Bio-Technology (Santa Cruz, CA). For staining, the DAKO ENVISION System was used. Blood vessels, number of mast cells, and expression of bFGF and tryptase were counted in five high-power fields (h.p.f.) (×400). We found that mast cells differ in their expression of ABSO granules, bFGF, and tryptase. In normal cervical samples, the number of ABSO-stained MCs was 12 ± 3, tryptase-positive MCs 17 ± 5, and bFGF MCs 11 ± 3 per h.p.f. In dysplasias and carcinoma in situ, tryptase-positive mast cells increased to 23 ± 8 and 26 ± 7 per h.p.f., respectively, and in invasive carcinoma they reached 31 ± 9 per h.p.f. In contrast, the numbers of MCs stained with ABSO and bFGF diminished from normal cervix to carcinoma in situ with a slight increase only in invasive cancer. Immunostaining for bFGF was positive in mast cell cytoplasm but was also found occasionally in the malignant cells. The histological location of MCs was in the underlying connective tissue and predominantly around small blood vessels growing into the epithelial lesion (Figure 1). Degranulation was common, and free ABSO and tryptase-positive granules were also seen. Blood vessels were counted only with the factor VIII immunoreaction because CD105 immunostaining showed weak and inconsistent results. The numbers of blood vessels in normal epithelium and in dysplasias were similar (average 9 ± 3 per h.p.f.). In carcinoma in situ and invasive carcinoma, a significant increase in the number of blood vessels was observed (13 ± 4 and 23 ± 6, respectively). Because tryptase-positive mast cells and vascularization showed a parallel increase with progression of the cervical lesions, it appears appropriate to suggest that the mast cell tryptase acts as a neoangiogenic factor in malignant lesions of the cervix rather than heparin, as suggested previously (Roche 1985). These findings confirm previous reports on the progressive increase of angiogenesis in cervical neoplasia (Dellas et al. 1997) and point towards the upregulation of angiogenesis by mast cells during carcinogenesis.

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Figure 1

Invasive carcinoma of the cervix with abundant capillaries in the stroma and tryptase-positive mast cells around blood vessels. Double immunocytochemical staining for Factor VIII and tryptase. Original magnification ×400.