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[CANCER RESEARCH 60, 1388–1393, March 1, 2000]

Heterogeneity of Angiogenesis and Blood Vessel Maturation in Human Tumors:Implications for Antiangiogenic Tumor Therapies1

Anne Eberhard,2 Sebastian Kahlert,2 Valentin Goede, Bernhard Hemmerlein, Karl H. Plate, andHellmut G. Augustin3

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 Gottingen Medical School,37075 Gottingen, Germany, and Department of Neuropathology, Erlangen-Nurnberg University Medical School, 91054 Erlangen, Germany [K. H. P.]

ABSTRACT

Microvessel density (MVD) counting techniques have been widely used toassess the vasculature in tumors. MVD counts assess the presence of bloodvessels but do not give an indication of the degree of angiogenesis and thefunctional status of the tumor neovasculature. To analyze angiogenesis andthe functional status of the tumor vascular bed, we have quantitated endo-thelial cell proliferation and the recruitment of pericytes in human tumors[glioblastomas (n 5 30), renal cell carcinomas (n 5 22), colon carcinomas(n 5 18), mammary carcinomas (n 5 24), lung carcinomas (n 5 15), andprostate carcinomas (n 5 19)]. These findings were compared to the physi-ological angiogenesis in the cyclic bovine ovarian corpus luteum. Tissuesections were examined applying double-labeling immunohistochemical tech-niques to detect proliferating endothelial cells and to colocalize endothelialcells and pericytes. The following parameters were quantitated: (a) MVDcount; (b) proliferating capillary index (PCI); ( c) proliferating tumor versusendothelial cell index; and (d) microvessel pericyte coverage index (MPI).Based on endothelial cell proliferation, angiogenesis was found to be presentin all tumors with characteristic and significant differences between thetumor types (glioblastomas, PCI 5 9.6 6 6.1%; renal cell carcinomas,PCI 5 9.4 6 5.2%; colon carcinomas, PCI 5 7.8 6 5.2%; mammarycarcinomas, PCI 5 5.0 6 4.8%; lung carcinomas, PCI 5 2.6 6 2.5%;prostate carcinomas, PCI5 2.06 1.4%). There was a considerable degree ofheterogeneity in the intensity of angiogenesis within each tumor group, asindicated by large standard deviations. Even in the most angiogenic tumors,angiogenesis was found to be 4 to 20 times less intense as compared withthe physiological angiogenesis in the growing ovarian corpus rubrum(PCI 5 40.66 6.2%). Varying degrees of pericyte recruitment to the tumormicrovasculature were determined in the different tumor types (glio-blastomas, MPI5 12.76 7.9%; renal cell carcinomas, MPI5 17.96 7.8%;colon carcinomas, MPI 5 65.4 6 10.5%; mammary carcinomas,MPI 5 67.3 6 14.2%; lung carcinomas, MPI 5 40.8 6 14.5%; prostatecarcinomas, MPI5 29.66 9.5%). The data demonstrate distinct quantitativevariations in the intensity of angiogenesis in malignant human tumors. Fur-thermore, the varying degrees of pericyte recruitment indicate differences inthe functional status of the tumor vasculature in different tumors that mayreflect 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 angiogenesisdependency of tumor growth has led to the development of antian-giogenic therapies that are conceptually extremely appealing for anumber of reasons (4–6): (a) as an oncofetal mechanism that ismostly down-regulated in the healthy adult, targeting of angiogenesisshould 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 thedevelopment of resistance (7); (c) each tumor capillary potentiallysupplies hundreds of tumor cells, and the targeting of the tumorvasculature should thus lead to a potentiation of the antitumorigeniceffect; and (d) in contrast to the interstitial location of tumor cells,direct contact between the vasculature and the circulation allowsefficient access to therapeutic agents.

Despite the enormous efforts aimed at elucidating the moleculardeterminants of angiogenesis (8–10) and the intense search for naturaland synthetic angiogenesis inhibitors (4, 6), surprisingly little isknown about the nature of the vascular bed in human tumors. Almostall of the studies that have assessed endothelial cell turnover in tumorswere performed in experimental animal models with rapidly growingtumors whose growth kinetics are vastly different from the growthkinetics of human tumors (11, 12). In fact, the few endothelial cellturnover 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 cellproliferation 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 as9.9% has been reported for colorectal adenocarcinomas (16).

As early as 1972, Bremet al. (17) proposed a microscopic angio-genesis grading system to assess the angiogenic status of the tumorvasculature. Based on the analysis of the vascular density, the numberof endothelial cell nuclei, and the cytological properties of tumor-associated endothelial cells, an angiogenesis score was determinedand used to establish an angiogenic rank order of different humanbrain tumors (17). In recent years, the vascular bed of human tumorshas been characterized extensively by performing MVD4 countingstudies (18, 19). These studies have revealed that high MVD countswithin vascular hot spots of tumors correspond with a poor prognosisfor the patient. MVD studies using panendothelial cell markers reflectthe vascular status of a tissue,i.e., the presence of blood vessels.However, they do not give an indication of the angiogenic status of atissue vascular bed,i.e., the rate of ongoing angiogenesis and thefunctional status of tumor neovasculature. To more realistically assessthe angiogenic status of the vasculature within human tumors, thepresent study was aimed at functionally analyzing the properties of thetumor vascular bed. Based on the analysis of tumor endothelial cellproliferation and pericyte recruitment, angiogenesis and the functionalstatus of the tumor microvascular bed were quantitated in six differenttypes of malignant human tumors. These findings were comparedwith the angiogenesis kinetics in the cyclic ovarian corpus luteum, oneof the few organ sites in the adult with significant physiologicalangiogenesis.

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 markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Deutsche Krebshilfe/Mildred-Scheel-Stiftung Grant 10-0986-Au3 andDeutsche 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 Gottingen Medical School,Robert-Koch-Strasse 40, 37075 Gottingen, Germany. Phone: 49-551-396573; Fax: 49-551-396711; E-mail: [email protected].

4 The abbreviations used are: MVD, microvessel density; PCI, proliferating capillaryindex; PTE, proliferating tumorversusendothelial cell; MPI, microvessel pericyte cov-erage index;a-SMA, a-smooth muscle actin; vWF, von Willebrand factor; PCNA,proliferating cell nuclear antigen; BS-I,Bandeiraea simplicifoliaI; AEC, amino ethylcarbazole; HPF, high power field.

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MATERIALS AND METHODS

Tissue Samples.Tissue specimens of formalin-fixed paraffin-embedded hu-man tumors were retrieved from the pathology archive. All tumors (1–4 blocks/tumor) were histopathologically evaluated by two investigators independently ofthe original pathology report. Tumor-free tissue adjacent to the tumor as well astumor-free specimen from necropsies served as normal control tissue. Specimensof bovine ovaries were retrieved from an established bank of tissues that has beenextensively analyzed for its angiogenesis status (20, 21).

Staining of Proliferating Endothelial Cells. A double-labeling immuno-histochemical technique was used to simultaneously stain nuclei of proliferatingcells and endothelial cells. Deparaffinized and rehydrated sections (4mm) weremicrowaved, endogenous peroxidase was blocked, and sections were incubatedwith an antibody to PCNA (clone PC10; 1:100 dilution; final concentration, 3.9mg/ml; Dako, Hamburg, Germany; 60 min, room temperature) or Ki67 (cloneMIB1; 1:10 dilution; final concentration, 20mg/ml; Dianova, Hamburg, Germany;60 min, room temperature). A biotinylated secondary antibody, streptavidin alka-line phosphatase complex, and nitroblue tetrazolium as a substrate (Zymed, SouthSan Francisco, CA) were used to visualize binding of the first antibody. Single-color-stained tissue sections were incubated with double-staining enhancer(Zymed) for 30 min, and then endothelial cells were stained for CD34 expression(human tissues; clone QBEnd/10; 1:25 dilution; Novocastra, Newcastle, UnitedKingdom; 2 h, room temperature; secondary antibody; Zymed) or binding of thelectin BS-I (bovine tissues; biotinylated BS-I; 10mg/ml; Sigma, Deisenhofen,Germany; 37°C, 2 h) using streptavidin-peroxidase as enzyme and AEC aschromogenic substrate (Zymed).

Staining of Mural Cells. To quantitatively assess the pericyte coverage ofmicrovessels, a double-labeling immunohistochemical technique was used tosimultaneously stain endothelial cells (CD34 or vWF) and mural cells (a-SMA).Of the analyzed tumors, 25% of archive-retrieved specimens were not suitable forthe CD34/a-SMA double-staining technique. Deparaffinized and rehydrated tissuesections were peroxidase-blocked, trypsinized, incubated with blocking serum, andthen double-stained fora-SMA expression to detect pericytes and smooth musclecells, followed by CD34 staining (human tumors) or vWF staining (bovineovaries) to label endothelial cells. Fora-SMA staining, sections were incubatedwith a monoclonal mouse antihumana-SMA antibody (clone 1A4; 1:400 dilution;final concentration, 20mg/ml; Sigma) for 2 h at room temperature. A biotinylatedsecondary antibody, streptavidin alkaline phosphatase complex, and nitrobluetetrazolium as substrate (Zymed) were used to visualize binding of thea-SMAantibody. Subsequent staining of endothelial cells was essentially performed asdescribed above using an antibody to CD34 to stain endothelial cells in humantumors and a polyclonal antiserum to vWF (polyclonal rabbit antihuman vWFantiserum; 1:200 dilution; final concentration, 28.5mg/ml; DAKO).

Quantitation of MVDs, PCI, and MPI. Sections were assessed for uni-formity of staining at low power (3100), and individual microvessel counts werethen performed in on a3400 field. To express MVD counts microscope-inde-pendent, counts were transformed and expressed as the number of microvessels/mm2 (1 HPF5 0.0681 mm2). Density counts of CD34-, BS-I-, or vWF-stainedmicrovessels were performed independently by three investigators, as describedpreviously (18, 20). At least five independent microscopic fields per tissue sectionwere analyzed by two independent investigators to count PCNA-positive tumorcells and endothelial cells. Tumor cell proliferation and endothelial cell prolifer-ation were quantitated in vascular hot spots that were identified by screening forthe areas with highest vessel density at low magnification. A PCI was determinedby calculating the ratio of the number of microvessels with proliferating endothe-lial cells:the total number of microvessels. A MPI was correspondingly establishedby quantitating the percentage of microvessels that colocalized endothelial cellstaining (CD34 or BS-I) and pericyte staining (a-SMA). For MPI quantitation, atleast five independent microscopic fields per section were independently analyzedby two investigators.

Statistical Analysis. Results were analyzed for statistical significance byan ANOVA and the Mann-WhitneyU test. Two-sided statistical calculationswere performed using the Statistica 5.1 program (StatSoft, Tulsa, OK) on anIBM-compatible personal computer.

RESULTS

Endothelial Cell Proliferation in Human Tumors. Angiogenesisand pericyte recruitment were assessed in six different types of human

tumors that were all histologically diagnosed as malignant tumors,namely, glioblastomas, renal cell carcinomas, colon carcinomas,mammary carcinomas, lung carcinomas, and prostate carcinomas (Ta-ble 1). To quantitate angiogenesis in these tumors, we used a double-labeling immunohistochemical technique, simultaneously staining en-dothelial cells for the expression of CD34 and detecting proliferatingcells by using the proliferation markers PCNA and Ki67. This ap-proach facilitated the detection of the relatively few proliferatingendothelial cells among the numerous tumor cells (Fig. 1). Countingthe CD34-positive microvessels in tumor vascular hot spots revealedrelatively uniformly high MVDs in the different tumor types, withglioblastomas and renal cell carcinomas having the highest averageMVDs (Fig. 2). Nevertheless, it is noteworthy that MVD counts in alltumors except for prostate carcinomas were lower than the MVDcounts for the corresponding normal tissues (brain tissueversusglio-blastomas, 128%; kidney tissueversusrenal cell carcinomas, 141%;colon tissueversuscolon carcinomas, 103%; mammary tissueversusmammary carcinomas, 135%; lung tissueversus lung carcinomas,348%; prostate tissueversusprostate carcinomas, 68%).

Both PCNA and Ki67 proved to be useful in assessing endothelialcell and tumor cell proliferation. On average, PCNA staining yielded1.31 6 0.23 (mean6 SD) times higher values than staining withKi67, confirming previously reported differences between PCNA andKi67 (22, 23). When quantitating a PCI reflecting the percentage ofcapillaries with PCNA-positive endothelial cell nuclei within vascularhot spots, significant differences were detected between the differenttumor types (Fig. 2B). Glioblastomas (mean6 SD, 9.6 6 6.1%;median, 8.6%) and renal cell carcinomas (mean6 SD, 9.46 5.2%;median, 8.3%) had significantly higher PCIs than mammary carcino-mas (mean6 SD, 5.0 6 4.8%; median, 3.4%), lung carcinomas(mean6 SD, 2.66 2.5%; median, 2.3%), and prostate carcinomas(mean6 SD, 2.06 1.4%; median, 1.9%;P , 0.005). Colon carci-nomas had intermediate PCI values of 7.86 5.2% (mean6 SD;median, 6.6%). In contrast, PCI values of all corresponding normaltissues were at the detection limit, with only an occasional PCNA-labeled endothelial cell being detectable (median of all tissues, 0%).The organs of the female reproductive system represent the only organsystem with significant physiological angiogenesis (20, 24). We con-sequently determined PCI values in the cyclic ovarian corpus luteum.The highest PCI values were determined in the growing corpusrubrum (mean6 SD, 40.66 11.2%; median, 37.8%; Fig. 2B). PCIvalues during corpus luteum angiogenesis were significantly higherthan the PCI values of all analyzed tumor groups (P , 0.005).

When comparing PCI values of individual tumors, a large degree ofvariation was seen (Fig. 3A). PCI values in glioblastomas, renal cellcarcinomas, and colon carcinomas varied over a wide range. Sometumors were found to have extremely high PCI values (.20%), whereasothers had PCI values that were not higher than those of the groups withlow PCI values (mammary, lung, and prostate carcinomas). With fewexceptions, PCI values in these tumors were consistently low.

To assess the relative angiogenesis-inducing capacity of differenttypes of tumor cells in relation to their own proliferative capacity, wedetermined an index of the overall ratio of proliferating tumor cells:proliferating endothelial cells (PTE index; Fig. 2C). Glioblastomas

Table 1 Summary of tumors

n Histology/grading

Glioblastomas 30 All grade IV astrocytomasRenal cell carcinomas 22 4 grade I and 18 grade II tumorsColon carcinomas 18 15 grade II and 3 grade III tumorsMammary carcinomas 24 19 grade II and 5 grade III tumorsLung carcinomas 15 11 grade II and 4 grade III tumorsProstate carcinomas 19 3 grade I, 15 grade II, and 1 grade III tumors

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and renal cell carcinomas had PTE indices around 10, colon andmammary carcinomas had PTE indices between 20 and 30, and lungand prostate carcinomas had PTE indices between 40 and 70.

Pericyte Coverage of the Neovasculature in Human Tumors.To quantitatively assess the functional status of the tumor neovasculature,we applied a double-labeling immunohistochemical technique to simul-taneously stain endothelial cells for CD34 expression and mural cells(pericytes/smooth muscle cells) with an antibody toa-SMA (21). Asso-ciation ofa-SMA with capillary endothelial CD34 expression was inter-preted to reflect pericyte staining, whereasa-SMA association withCD34 expression in arteries and veins was interpreted to reflect smoothmuscle cell staining (Fig. 4). A MPI was quantitated that reflects thepercentage of capillaries associated witha-SMA-positive pericytes. MPIswere determined for all tumor types. Glioblastomas and renal cell carci-nomas were identified as the tumor types with the lowest MPI values(glioblastomas, mean6 SD 5 12.76 7.9% and median5 9.7%; renalcell carcinomas, mean6 SD5 17.96 7.8% and median5 17.6%; Figs.2D and 3B). Mammary carcinomas had the highest MPI values(mean6 SD, 67.36 14.2%; median, 70.4%). Similarly, colon carcino-mas also had relatively high MPI values (mean6 SD, 65.46 10.5%;median, 67.7%). Lastly, lung and prostate carcinomas had intermediateMPI values of 40.86 14.5% (mean6 SD; median, 40.6%) and29.66 9.5% (mean6 SD; median, 29.3%), respectively.

The pericyte coverage data in the different types of malignanttumors were compared with the status of the neovasculature duringcyclic ovarian angiogenesis. Angiogenesis in the cyclic ovary is aphysiological event with a coordinated program of sprouting angio-genesis and mural cell recruitment. This is reflected by a MPI of60.2% even in the angiogenic corpus rubrum (Refs. 21; Figs. 2D and4G). The MPI of the mature midstage corpus luteum (62.4%) char-acterizes an overall immature neovasculature that rapidly undergoesregression at the onset of luteolysis.

DISCUSSION

Antiangiogenic targeting of the neovasculature within tumors isconsidered one of the most promising strategies in the search for novelantineoplastic therapies (5, 6). The concept that tumor vessels can beselectively targeted without affecting the quiescent organ vasculatureis based on the fact that the molecular phenotype of immature,angiogenic blood vessels is distinctly different from that of restingblood vessels. A number of molecular determinants of angiogenicendothelial cells have been identified in recent years (4) and are beingextensively explored for their suitability to target angiogenic bloodvessels. Correspondingly, numerous animal studies have shown that

Fig. 1. Histochemical detection of proliferating endothelial cells using a double-labeling technique to stain endothelial cells for CD34 expression (A–F) or lectin BS-I binding (G–I;red) and stain proliferating cells using PCNA as a proliferation marker (dark blue). The sections were not counterstained. Thus, all dark nuclei represent PCNA-positive tumor cellsand endothelial cells (arrows). The double-labeling technique was applied to different types of malignant human tumors. Representative pictures for each tumor type are shown.A,glioblastoma;B, renal cell carcinoma;C, colon carcinoma;D, mammary carcinoma;E, lung carcinoma;F, prostate carcinoma. Endothelial cell proliferation during tumor angiogenesiswas compared to the intensity of physiological angiogenesis in the cyclic ovary.G, early corpus rubrum, reflecting the most intense phase of ovarian angiogenesis in the first few daysafter ovulation (days 1–4 after ovulation);H, mature midstage corpus luteum with detectable turnover of endothelial cells (days 12–18 after ovulation);I, luteolysis with regressionof the corpus luteum and all of its vasculature (days 18–24 after ovulation).

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the activated, angiogenic neovasculature can be selectively targetedwithout affecting the normal organ vasculature (7, 25–27).

Essentially all of the antiangiogenic animal studies have used experi-mental models with a very high intensity of angiogenesis. These includerapidly growing tumor models such as the Lewis lung carcinoma (7, 25),the rabbit cornea assay with an implanted angiogenic factor (26), or the

naturally occurring angiogenic processes in the female reproductive sys-tem (27). Contrasting these experimental models with a high intensity ofangiogenesis, little is known about the degree of active angiogenesis andthe functional status of the vasculature within human tumors. Vesseldensity counting studies have used panendothelial cell markers thatfacilitate the quantitation of the number of blood vessels within tumors(17, 18). These studies have demonstrated that high vessel densities intumors correspond with poor prognosis. However, the use of panendo-thelial cell markers, such as CD31 or CD34, facilitates the assessment ofthe vascular status of a tumor but does not give an indication of thetumor’s angiogenic status. In fact, recent histomorphological studiesindicate that some tumors may be vascularized without significant an-giogenesis, probably by using the preexistent organ vasculature (28) oreven by forming vascular channels on their own through a nonendothelialcell process designated as vascular mimicry (29). Recent studies havetried to circumvent this problem by using marker molecules that areup-regulated during angiogenesis, such as CD105 (30) and the integrinavb3 (31).

In this study, we have quantitatively assessed the rate of angiogenesis(based on the proliferation of endothelial cells) as well as the functionalstatus of the neovasculature (based on the recruitment ofa-SMA-positivepericytes) in six different types of malignant human tumors. The resultsindicate that there is active angiogenesis in human tumors, albeit at amuch lower rate compared with the physiological cyclic angiogenicprocesses in the ovarian corpus luteum. Furthermore, the varying degreesof pericyte recruitment indicate differences in the functional status of thetumor vasculature in different tumors that may reflect varying degrees ofmaturation of the tumor vascular bed.

Studies performed as early as 1972 have attempted to establish aprocedure for the assessment of the angiogenesis status of tumors (17).To date, however, no standardized scheme is available to reliably assessthe angiogenesis status of a given tissue or a tumor. Despite the increas-ingly recognized distinct phenotypic properties of angiogenic endothelialcells (4, 32), proliferation of endothelial cells may still be considered asthe single most reliable parameter to quantitate angiogenesis. Few endo-

Fig. 3. Endothelial cell proliferation and pericyte recruitment in individual malignanthuman tumors.A, PCIs (Prol. Cap. Index) in glioblastomas, renal cell carcinomas, andcolon carcinomas vary over a wide range. In contrast, with few exceptions, mammarycarcinomas, lung carcinomas, and prostate carcinomas have uniformly low PCI values.B,MPIs (Peric. Cov. Index) in the different tumor types varied over a much smaller rangethan the PCIs.

Fig. 2. Quantitative analysis of endothelial cellproliferation and pericyte recruitment during patho-logical tumor angiogenesis and physiological ovarianangiogenesis. All data are expressed as mean6 SE.A, colon carcinomas, mammary carcinomas, lung car-cinomas, and prostate carcinomas have similarly highMVD counts. Glioblastomas and renal cell carcino-mas have higher average MVD counts (P , 0.005).B, a PCI (Prol. Cap. Index) was quantitated assessingthe percentage of microvessels with proliferating en-dothelial cells (see “Materials and Methods”). Aranked order of the intensity of angiogenesis in thedifferent types of tumors was determined as follows:glioblastomas. renal cell carcinomas. colon car-cinomas. mammary carcinomas. lung carcino-mas. prostate carcinomas. PCI values in malignanthuman tumors were compared with the intensity ofangiogenesis in the cyclic bovine corpus luteum [CR;early corpus rubrum (angiogenesis)], midstage corpusluteum (CL; maturation), and regressing corpus lu-teum (REG-CL; regression).C, a quantitative ratio ofthe total number of proliferating tumor cells in alltumors:the total number of endothelial cells (Prol.Tumor/EC Index) was determined for each type oftumor to establish a relative parameter that reflects theper tumor cell angiogenic capacity of the tumors.D, aMPI (Peric. Cov. Index) was quantitated assessing thepercentage of microvessels that are associated witha-SMA-positive pericytes (see “Materials and Meth-ods”). MPI indices of the different types of malignanthuman tumors were compared with the MPI valuesof the transient neovasculature during ovarian angio-genesis.

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thelial cell proliferation studies in human tumors have been reported(13–16). These studies describe tumor endothelial cell proliferation indi-ces between 0.15% (13) and 9.9% (16). Much of the variation in theliterature data may be attributed to differences in methodology. Thus, oneof the goals of the present study was to standardize one technique and tocomparatively apply this technique to different tumors. When assessingangiogenesis based on endothelial cell proliferation in different types ofhuman tumors, we identified significant differences in the degree ofactive angiogenesis between different types of tumors as well as withinone tumor type. Glioblastomas, renal cell carcinomas, and colon carci-nomas were identified as the most angiogenic types of tumors. There wasa high degree of variation among the individual tumors, indicative of alow rate of active angiogenesis in a subgroup of these tumors. In contrastto the intense angiogenesis in some types of tumors, lung carcinomas,prostate carcinomas, and most of the mammary carcinomas had relativelylow endothelial cell proliferation indices. Nevertheless, these low PCIvalues (around 2%) are still indicative of active angiogenesis in thesetumors. Endothelial cell turnover in the corresponding normal tissues wasbelow the detection limit, with only single PCNA- or Ki67-positiveendothelial cells being detectable. A very careful analysis of endothelialcell turnover in normal tissue has identified around 0.1% proliferatingendothelial cells in normal tissues (12), which is still lower than the PCIvalues for prostate and lung carcinomas by a factor of 20. When com-paring tumor PCI values to angiogenesis in the cyclic ovary, it becameapparent that angiogenesis in human tumors is operative, albeit at a much

lower rate than in the corpus luteum. Angiogenesis in the growing corpusluteum in the first few days after ovulation was found to be fourfold totwentyfold more intense than the angiogenesis in the different malignanthuman tumors (PCI. 40%), corresponding to previous reports on thehigh intensity of angiogenesis in the female reproductive system (33, 34).

In addition to assessing tumor endothelial cell proliferation, we quan-titated the recruitment of mural cells (pericytes, smooth muscle cells) tothe tumor neovasculature. The identification of the angiopoietins (35–37)and the phenotype of platelet-derived growth factor-b-deficient mice(inability to recruit pericytes) (38, 39) has focused attention on themolecular mechanisms of blood vessel maturation mediated by the re-cruitment of pericytes. The mature phenotype of the quiescent organvasculature in most organs is characterized by an extensive coverage withpericytes that appear to play a role in controlling the quiescent endothelialcell phenotype. Correspondingly, it has long been speculated that thetumor vasculature is characterized by a distinct maturation defect that isat least partially responsible for the irregular, tortuous, and leaky bloodvessels found within tumors (40, 41). It was recently shown that androgenablation therapy of prostate tumors leads to a down-regulation of vascularendothelial growth factor within the tumor, leading selectively to theregression of immature tumor microvessels that were not covered bypericytes (42). In line with these findings, we determined in the presentstudy that only one-third of the vasculature within prostate carcinomas iscovered by pericytes, despite the fact that prostate tumors were identified

Fig. 4. Histochemical analysis of pericyte recruitment using a double-labeling technique to stain endothelial cells for CD34 expression (A–F) or vWF expression (G–I;red) and stainmural cells (pericytes, smooth muscle cells) fora-SMA expression (dark blue).a-SMA staining of microvessels was considered to reflect pericyte staining, whereasa-SMA of largervessels indicated smooth muscle cell staining. Representative pictures for each tumor type are shown.A, glioblastoma;B, renal cell carcinoma;C, colon carcinoma;D, mammarycarcinoma;E, lung carcinoma;F, prostate carcinoma;G, early corpus rubrum;H, mature midstage corpus luteum;I, regressing corpus luteum.

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as not very angiogenic tumors based on the assessment of endothelial cellproliferation.

The degree of pericyte recruitment to the neovasculature in the differ-ent tumor types varied significantly. The neovasculature in mammary andcolon carcinomas had the highest rate of pericyte coverage, with as manyas 70% of all microvessels being in contact with mural cells. In contrast,glioblastomas and renal cell carcinomas had pericyte coverage indicesbetween 10% and 20%, indicating that most microvessels did not estab-lish mural cell contact. These quantitative differences in mural cellrecruitment could reflect varying degrees of vessel maturation of thetumor vascular bed. There is good evidence to suggest that pericytecoverage is a correct functional reflection of the degree of microvesselmaturation (38, 39, 42, 43). However, it should be noted that pericytecoverage is not the only mechanism of vessel maturation, as indicated bythe fact that the quiescent organ vasculature in some organs such as thelungs is not extensively covered by pericytes.

In summary, the present study has demonstrated that malignant humantumors are characterized by varying degrees of angiogenesis and pericyterecruitment. Furthermore, they indicate that the degree of angiogenesis inhuman tumors varies widely and may be very low in some types oftumors. Despite the fact that little is known about the mechanism ofaction of most angiogenesis inhibitors, the data suggest that the suitabilityof tumors for antiangiogenic therapies may differ between different tumortypes and even within one type of tumor. Tumors with a low intensity ofangiogenesis may not benefit much from antiangiogenic therapies thatdepend on the rate of endothelial cell proliferation. This stresses theimportance that techniques such as those described in this study need tobe implemented in clinical practice to assess the angiogenic status of apatient to identify those who will benefit most from antiangiogenictherapy. Furthermore, the data also indicate that the vasculature in mosttumors is not very extensively covered by pericytes, which may reflect afunctional immaturity of the tumor vascular bed. Not only may pericytecoverage of microvessels control vessel maturation, but it has also beenshown to define a plasticity window for blood vessel remodeling (43).Thus, our data provide support for the concept that in addition to anti-angiogenic therapies, angioregressive therapies could be developed thatare capable of selectively inducing the regression of the immature tumorvasculature with an open plasticity window (42).

ACKNOWLEDGMENTS

We thank Renate Dietrich for excellent technical assistance.

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2000;60:1388-1393. Cancer Res Anne Eberhard, Sebastian Kahlert, Valentin Goede, et al. TherapiesHuman Tumors: Implications for Antiangiogenic Tumor Heterogeneity of Angiogenesis and Blood Vessel Maturation in

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