Featured Article Angiogenic Profile of Breast Carcinoma Determines Leukocyte Infiltration Jessica C. A. Bouma-ter Steege, 1 Coen I. M. Baeten, 1 Victor L. J. L. Thijssen, 1 Sietske A. Satijn, 1 Inge C. L. Verhoeven, 1 Harry F. P. Hillen, 1 John Wagstaff, 1 and Arjan W. Griffioen 1,2 1 Angiogenesis Laboratory, Research Institute for Growth and Development, Department of Internal Medicine, and 2 Department of Pathology, University Hospital Maastricht, Maastricht, the Netherlands ABSTRACT To study the relationship between the angiogenic pro- file and leukocyte infiltration of tumors, single cell suspen- sions of archival frozen medullary and ductal breast cancer tissues were analyzed by flow cytometry. The amount of leukocytes and endothelial cells was measured, as well as the expression of intercellular adhesion molecule-1 (ICAM-1) on the endothelial cell fraction. A significantly higher number (3.2-fold) of infiltrating leukocytes was observed in medul- lary carcinoma. The composition of this infiltrate was sim- ilar to that seen in ductal carcinomas. The more intense infiltrate was explained by the 3-fold enhanced endothelial ICAM-1 expression in medullary carcinoma. The angiogenic profile of all tumors was assessed by quantitative real-time reverse transcription-PCR analysis. Vascular endothelial growth factor (VEGF)-C and VEGF-D, but not VEGF-A, basic fibroblast growth factor, placental growth factor, and angiopoietins 1, 2, and 3 showed a relatively higher level of expression in ductal carcinoma than in medullary carci- noma. In vitro, both VEGF-C and VEGF-D were found to decrease endothelial ICAM-1 expression in the presence of basic fibroblast growth factor. These data suggest that in vivo angiogenic stimuli prevent the formation of an effective leukocyte infiltrate in tumors by suppressing endothelial ICAM-1 expression. INTRODUCTION An intense leukocyte infiltrate in tumors is thought to be a sign of an effective antitumor immune response and is conse- quently often related to an improved clinical outcome (1–3). We and others have previously demonstrated, both in vitro and in vivo, that leukocyte vessel wall interactions are regulated by tumor derived angiogenic factors (4 – 8). These factors down- regulate the expression of endothelial adhesion molecules in- volved in leukocyte interactions, of which, intercellular adhe- sion molecule-1 (ICAM-1) is the most important one (9), ultimately leading to a suppressed infiltration and escape from immune surveillance. To study the relationship between angio- genesis, endothelial cell phenotype and leukocyte infiltration, we adapted the flow cytometric system which recently was described as a reliable and objective alternative for the assess- ment of microvessel density (10). This method was used to simultaneously determine the level and composition of leuko- cyte infiltrate in frozen breast cancer tissue, which allows ret- rospective analysis. To investigate the leukocyte infiltrate in tumor tissue, we selected breast carcinoma. Among these tumors, a distinct type of disease has been identified, namely medullary breast carci- noma, which is defined, among a number of other characteris- tics, by an intense infiltration of leukocytes (11, 12). Interest- ingly, medullary breast carcinoma has a better clinical prognosis, which is thought to be related to this inflammatory infiltrate We compared these rather rare tumors (5 to 8% of breast cancer) with the most common histologic diagnosis of breast cancer, which is not otherwise specified ductal carcinoma (60 to 70% of breast cancer; ref. 13). We demonstrate here, with flow cytometry, that a relatively lower level of leukocyte infiltration in ductal type of breast carcinoma than in the medullary type can be explained by the relatively lower level of ICAM-1 expression on tumor vessels in the former case. This suppressed ICAM-1 expression can be explained by a higher expression of angiogenic factors by ductal breast carcinoma tumor cells. This report describes the correla- tion between angiogenic potential endothelial adhesion mole- cules and leukocyte infiltrate in human breast cancer. MATERIALS AND METHODS Patient Characteristics. Frozen tumor tissues of ductal (not otherwise specified, n  14) and medullary breast tumors (n  9) were obtained from the archive of the Department of Pathology (University Hospital Maastricht). Mean age, tumor size, and percentage of patients with positive lymph nodes were comparable in both groups (60  14.55 versus 51  7.97 years, 21.8  5.31 versus 24.2  13.62 mm in diameter, and 43 versus 31% for medullary versus ductal breast carcinoma, respec- tively). Received 4/16/04; revised 7/23/04; accepted 8/3/04. Grant support: The Dutch Cancer Society Grant UM 2001-2529 (A. Griffioen), the Dutch Science Foundation (NWO)-Stichting Tech- nische Wetenschappen Grant MPG-5456, and by a grant from Reell Precision Manufacturing BV, Elsloo, the Netherlands. 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. Requests for reprints: Arjan W. Griffioen, Angiogenesis Laboratory, Department of Pathology, University Hospital Maastricht, P. O. Box 5800, 6202 AZ Maastricht, the Netherlands. Phone: 31-433874630; Fax: 31-433876613; E-mail: AW.Griffioen@path.unimaas.nl. ©2004 American Association for Cancer Research. 7171 Vol. 10, 7171–7178, November 1, 2004 Clinical Cancer Research