Use of a Novel Technique for Visualizing the Microvascular Bed in Esophageal and Gastric Tumors.
Abstracts
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BACKGROUND: Tumor angiogenesis is the subject of intense study, but it remains unclear which, if any, of the standard methods for visualizing and quantifying the microvessels is optimal. The three dimensional structure of the tumor microvascular bed (MVB), in particular, remains incompletely investigated. Our previous studies showed that MVB of different experimental tumors (sarcoma 45, Pliss lymphosarcoma, Morris hepatoma, et al.) can be revealed three dimensionally with special Calcium-Adenosinetriphosphate (ATP) methods in which selective dyeing of MVB is achieved by precipitation of calcium with the phosphorus released from ATP on the vessel walls when incubated in solutions with pH 10.5-11.5. The aim of this study was to use these methods for the study of MVB in human esophageal and gastric tumors. METHODS: Ten gastric and esophageal adenocarcinomas and matching normal tissues removed during surgery were studied. After fixation in 4% neutral formaldehyde at 4 degrees C for 24-48 hours, frozen sections with thickness of 60, 100 and 150 microns were prepared. Visualization of the microvascular bed was achieved by exposing the section to different solutions in order to convert the non-visible calcium phosphate into lead phosphate and subsequently black lead sulfide. RESULTS: The MVB was optimally stained, with clear visualization of all vessels, in normal gastric mucosa at a tissue thickness of 30-60 microns. Vessels were also well visualized in some areas of all tumors, although staining of surrounding connective tissue structures was more prominent in the tumor sections. The Calcium-ATP method provided microvessel density counts at least double those obtained with immunohistochemical staining of the microvasculature using CD31 monoclonal antibody. CONCLUSION: Clear detection of the MVB of all gastric and some esophageal mucosae can be achieved using Calcium-ATP methods. Approximately half of the tumor microvasculature vessels are not visualized and thus uncounted after staining with conventional immunohistochemical methods. The three dimensional Calcium-ATP method developed offers a potentially superior technique for quantifying tumor angiogenesis. |
