[CANCER RESEARCH 53.4161-4163. September 15, 1993] Advances in Brief Relationship of Endothelial Cell Proliferation to Tlimor Vascularity in Human Breast Cancer Stephen B. Fox, Kevin C. Gatter, Roy Bicknell, James J. Going, Peter Stanton, Tim G. Cooke, and Adrian L. Harris NuffiM Department of Pathology /S. B. E, K. C. G.¡and Imperial Cancer Research Fund. Institute vf Molecular Medicine ¡R.B.. A. L. //./, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, ami Department of Surgery, University of Glasgow. Royal Infirmary. Queen Elizabeth Building. Glasgow G31 1ER ¡P.S., T. G. C.¡,and Department of Pathology, University of Glasgow, Royal Infirmary, Castle Street, Glasgow G4 OSF /J. J. G./, Uniled Kingdom Abstract Current studies of tumor angiogenesis rely on the concept that endo- thelium proliferates .'(I—10 times faster in tumors than in normal tissues. This evidence is based on histológica! autoradiographic data largely from animal studies. To assess endothelial cell proliferation in human cancer we used the more sensitive and specific technique of immunohistochemistry. We measured the frequency and distribution of endothelial cell prolifera tion and examined their relationship to tumor cell proliferation. For the first time, we also correlated endothelial and tumor cell proliferation with tumor vascularity. Twenty breast carcinomas from patients exposed to bromodeoxyuridine 3-8 h prior to surgery were double immunostained using antibodies to CD31 (as a marker of endothelium) and bromodeoxy uridine (as a marker of proliferation). The labeling index (LI) for both tumor and endothelial cells was determined and tumor vascularity was assessed by counting the number of CD31 positive vessels. Endothelial cell proliferation was predominantly at the tumor periphery while tumor cell proliferation occurred throughout the lesion. The mean Lis for endothe lium and tumor were 2.2% (range, 0.8-5.3) and 7.3% (range, 1.3-17.1), respectively. There was no correlation between tumor and endothelial cell LI (/* = 0.414) or between the tumor LI or endothelial cell LI and tumor vascularity (P = 0.08 and P = 0.39, respectively). These findings suggest that previous studies in animal tumors have significantly overestimated endothelial cell proliferation and that its importance in tumor angiogen esis may be related more to continual remodeling and migration of vessels than to proliferation alone. Introduction Angiogenesis is essential for tumor growth and metastasis (1). Endothelial cell matrix remodeling, migration, and proliferation are central to the angiogenic process (2). In marked contrast to the nu merous tumor cell kinetic studies there have been few examining endothelial cell proliferations during tumor angiogenesis (3-7). How ever, based on this small number of reports it has become generally accepted that endothelial cells proliferate 30-40-fold faster in tumor blood vessels than in the vasculature of normal tissue, irrespective of tumor type, growth rate, or size. However, this oft quoted figure was obtained from endothelial labeling indices derived from histological autoradiographs of animal tissues exposured to tritiated thymidine. To the best of our knowledge in humans only gliomas have been examined in detail (8). The iden tification of proliferating endothelium by tinctorial stains used in all of these studies permitted recognition only of larger caliber capillaries (a small proportion of the tumor vessels). This difficulty is likely to make the reported endothelial labeling indices inaccurate and also obscures patterns of endothelial cell proliferation and its relationship with the tumor vascularity. The weakness of previous studies and the paucity of human data warrant réévaluation of this question with more precise techniques. Using immunohistochemistry and antibodies to endothelium and Received 5/21/43; accepted 8/2/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. BrdUrd' we analyzed endothelial cell proliferation in a series of human breast adenocarcinomas. We report on the frequency and pat tern of endothelial cell proliferation and their relationship with tumor cell proliferation and, for the first time, tumor vascularity. Materials and Methods Tissue Specimens. Twenty invasive breast carcinomas resected between 1989 and 1991 were taken from the archival files of the Glasgow Royal Infirmary, Scotland. The tumors were derived from patients who had been given i.v. injections of 200 mg bromodeoxyuridine 3-8 h prior to mastectomy. Patients' ages ranges from 40 to 77 years (mean, 61.2 years); 17 were ductal carcinomas of no special type, 1 was a lobular carcinoma, 1 was a medullary carcinoma and 1 was an atypical medullary carcinoma. Of the ductal carcino mas 2 were grade I, 7 were grade II. and 8 were grade III (Nottingham modification of system of Bloom and Richardson) (9). Tumors ranged in diameter from 12 to 120 mm. Nine had histologically confirmed lymph node involvement by tumor. Immunohistochemistry. Four-fim sections were cut onto silane coated slides and dewaxed. Double immunostaining was performed on sections using streptavidin-biotin-peroxidase and alkaline phosphatase anti-alkaline phospha- tase techniques and the antibodies JC70 (Dako) (10) and Bu20a (Dako) (11), respectively (12). For optimal double staining it was necessary to pretreat sections with 12.5 mg protease type XXIV (Sigma)/10() ml phosphate buffered saline for 20 min at 37°Cfor JC70 and 2 NHCI for 5 min at 6()°Cfor Bu20a. After immunostaining a light hematoxylin counterstain was applied before mounting in aqueous medium. In 10 cases multiple tissue sections were stained. Single BrdUrd immunohistochemistry was performed in parallel sec tions for all cases in another laboratory. Morphometry. Labeling indices for both endothelial and tumor cells were determined by scanning the entire tumor section at X400. In 14 of 20 cases the entire cross-section of the tumor was examined and in 5 of 20, due to their size, at least one-half of the tumor diameter was studied. In one tumor, due to its size of 120 mm, a representative section had to be selected which included the invading tumor margin. A positive endothelial cell was identified on the basis of JC70 positive cytoplasm and/or cell membrane and Bu2()a positive nucleus. An endothelial cell was considered negative when an ¡mmunonegative nucleus was surrounded by JC70 positive cytoplasm and/or cell membrane. Occasional JC70 immunopositive macrophages and plasma cells were excluded on mor phological grounds. The LI of tumor cells was scored by selecting the maxi mally immunostained area. An average of 1860 endothelial cells and 1398 tumor cells (ranges, 689-2728 and 543-2742, respectively) were counted for each tumor. The vascularity of the tumors was assessed by averaging the number of JC70 positive vessels per mm- in the three most vascular areas (13). Statistical Analysis. Since the data skew and hence cannot be considered normal, a nonparametric Kendall's rank correlation was performed which makes no such assumptions, to assess the relationship between the variables. Results Endothelial cell BrdUrd labeling was almost restricted to the infil trating margin of the tumor with only occasional endothelial cell double immunoreactivity of microvessels within the tumor body. Mi- crovessels were often seen in the body of the tumor without an accompanying endothelial cell nucleus. Proliferating endothelium was 1The abbreviations used are: BrdUrd. bromodeoxyuridine; LI. labeling index. 4161 on March 18, 2016. © 1993 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from