[CANCER RESEARCH 60, 1388 –1393, March 1, 2000] Heterogeneity of Angiogenesis and Blood Vessel Maturation in Human Tumors: Implications for Antiangiogenic Tumor Therapies 1 Anne Eberhard, 2 Sebastian Kahlert, 2 Valentin Goede, Bernhard Hemmerlein, Karl H. Plate, and Hellmut G. Augustin 3 Cell Biology Laboratory, Department of Gynecology and Obstetrics [A. E., S. K., V. G., H. G. A.] and Department of Pathology [B. H.], University of Go ¨ttingen Medical School, 37075 Go ¨ttingen, Germany, and Department of Neuropathology, Erlangen-Nu ¨rnberg University Medical School, 91054 Erlangen, Germany [K. H. P.] ABSTRACT Microvessel density (MVD) counting techniques have been widely used to assess the vasculature in tumors. MVD counts assess the presence of blood vessels but do not give an indication of the degree of angiogenesis and the functional status of the tumor neovasculature. To analyze angiogenesis and the functional status of the tumor vascular bed, we have quantitated endo- thelial cell proliferation and the recruitment of pericytes in human tumors [glioblastomas (n  30), renal cell carcinomas (n  22), colon carcinomas (n  18), mammary carcinomas (n  24), lung carcinomas (n  15), and prostate carcinomas (n  19)]. These findings were compared to the physi- ological angiogenesis in the cyclic bovine ovarian corpus luteum. Tissue sections were examined applying double-labeling immunohistochemical tech- niques to detect proliferating endothelial cells and to colocalize endothelial cells and pericytes. The following parameters were quantitated: (a) MVD count; (b) proliferating capillary index (PCI); (c) proliferating tumor versus endothelial cell index; and (d) microvessel pericyte coverage index (MPI). Based on endothelial cell proliferation, angiogenesis was found to be present in all tumors with characteristic and significant differences between the tumor types (glioblastomas, PCI  9.6  6.1%; renal cell carcinomas, PCI  9.4  5.2%; colon carcinomas, PCI  7.8  5.2%; mammary carcinomas, PCI  5.0  4.8%; lung carcinomas, PCI  2.6  2.5%; prostate carcinomas, PCI  2.0  1.4%). There was a considerable degree of heterogeneity in the intensity of angiogenesis within each tumor group, as indicated by large standard deviations. Even in the most angiogenic tumors, angiogenesis was found to be 4 to 20 times less intense as compared with the physiological angiogenesis in the growing ovarian corpus rubrum (PCI  40.6  6.2%). Varying degrees of pericyte recruitment to the tumor microvasculature were determined in the different tumor types (glio- blastomas, MPI  12.7  7.9%; renal cell carcinomas, MPI  17.9  7.8%; colon carcinomas, MPI  65.4  10.5%; mammary carcinomas, MPI  67.3  14.2%; lung carcinomas, MPI  40.8  14.5%; prostate carcinomas, MPI  29.6  9.5%). The data demonstrate distinct quantitative variations in the intensity of angiogenesis in malignant human tumors. Fur- thermore, the varying degrees of pericyte recruitment indicate differences in the functional status of the tumor vasculature in different tumors that may reflect varying degrees of maturation of the tumor vascular bed. INTRODUCTION Tumor growth and metastatic dissemination are critically depend- ent on the tumor’s supply of blood vessels (1–3). The angiogenesis dependency of tumor growth has led to the development of antian- giogenic therapies that are conceptually extremely appealing for a number of reasons (4 – 6): (a) as an oncofetal mechanism that is mostly down-regulated in the healthy adult, targeting of angiogenesis should lead to minimal side effects even after prolonged treatment; (b) tumor-associated angiogenesis is a physiological host mechanism; consequently, its pharmacological inhibition should not lead to the development of resistance (7); (c) each tumor capillary potentially supplies hundreds of tumor cells, and the targeting of the tumor vasculature should thus lead to a potentiation of the antitumorigenic effect; and (d) in contrast to the interstitial location of tumor cells, direct contact between the vasculature and the circulation allows efficient access to therapeutic agents. Despite the enormous efforts aimed at elucidating the molecular determinants of angiogenesis (8 –10) and the intense search for natural and synthetic angiogenesis inhibitors (4, 6), surprisingly little is known about the nature of the vascular bed in human tumors. Almost all of the studies that have assessed endothelial cell turnover in tumors were performed in experimental animal models with rapidly growing tumors whose growth kinetics are vastly different from the growth kinetics of human tumors (11, 12). In fact, the few endothelial cell turnover studies that have been performed in human tumors do sug- gest that endothelial cell proliferation in these tumors is detectable, albeit at a relatively low rate (13–16). Average tumor endothelial cell proliferation indices of 0.15% have been reported for prostatic carci- nomas (13). The endothelial cell labeling index in mammary carci- noma varies between 2.2% (14) and 2.7% (15), and a value as high as 9.9% has been reported for colorectal adenocarcinomas (16). As early as 1972, Brem et al. (17) proposed a microscopic angio- genesis grading system to assess the angiogenic status of the tumor vasculature. Based on the analysis of the vascular density, the number of endothelial cell nuclei, and the cytological properties of tumor- associated endothelial cells, an angiogenesis score was determined and used to establish an angiogenic rank order of different human brain tumors (17). In recent years, the vascular bed of human tumors has been characterized extensively by performing MVD 4 counting studies (18, 19). These studies have revealed that high MVD counts within vascular hot spots of tumors correspond with a poor prognosis for the patient. MVD studies using panendothelial cell markers reflect the vascular status of a tissue, i.e., the presence of blood vessels. However, they do not give an indication of the angiogenic status of a tissue vascular bed, i.e., the rate of ongoing angiogenesis and the functional status of tumor neovasculature. To more realistically assess the angiogenic status of the vasculature within human tumors, the present study was aimed at functionally analyzing the properties of the tumor vascular bed. Based on the analysis of tumor endothelial cell proliferation and pericyte recruitment, angiogenesis and the functional status of the tumor microvascular bed were quantitated in six different types of malignant human tumors. These findings were compared with the angiogenesis kinetics in the cyclic ovarian corpus luteum, one of the few organ sites in the adult with significant physiological angiogenesis. Received 3/22/99; accepted 1/4/00. 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. 1 Supported by Deutsche Krebshilfe/Mildred-Scheel-Stiftung Grant 10-0986-Au3 and Deutsche Forschungsgemeinschaft Grants SFB 500, C3 (to H. G. A.). 2 A. E. and S. K. contributed equally to this work. 3 To whom requests for reprints should be addressed, at Cell Biology Laboratory, Department of Gynecology and Obstetrics, University of Go ¨ttingen Medical School, Robert-Koch-Strasse 40, 37075 Go ¨ttingen, Germany. Phone: 49-551-396573; Fax: 49- 551-396711; E-mail: haugust@med.uni-goettingen.de. 4 The abbreviations used are: MVD, microvessel density; PCI, proliferating capillary index; PTE, proliferating tumor versus endothelial cell; MPI, microvessel pericyte cov- erage index; -SMA, -smooth muscle actin; vWF, von Willebrand factor; PCNA, proliferating cell nuclear antigen; BS-I, Bandeiraea simplicifolia I; AEC, amino ethyl carbazole; HPF, high power field. 1388 Research. on February 23, 2016. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from