[CANCER RESEARCH 62, 560–566, January 15, 2002]

Hemodynamics in Vasculogenic Mimicry and Angiogenesis of Inflammatory BreastCancer Xenograft1

Kazuo Shirakawa, Hisataka Kobayashi, Yuji Heike, Satomi Kawamoto, Martin W. Brechbiel, Fujio Kasumi,Toshihiko Iwanaga, Fumio Konishi, Masaaki Terada, and Hiro Wakasugi2

Pharmacology Division [K. S., Y. H., H. W.] and Genetics Division [M. T.], National Cancer Center Research Institute, Tokyo 104-0045, Japan; Hitachi Medical Co.-chairedDepartment of Diagnostic and Interventional Imagiology, Kyoto 606-8507, Japan [H. K., S. K.]; Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch,National Cancer Institute, NIH, Bethesda, Maryland 20892 [M. W. B.]; Department of Surgery, Japanese Foundation for Cancer Research, Tokyo 170-8455, Japan [F. Ka.];Department of Veterinary Anatomy, Hokkaido University, Sapporo 060-8638, Japan [T. I.]; and Department of Surgery, Omiya Medical Center, Jichi Medical School, Omiya 330-0834, Japan [K. S., F. Ko.]

ABSTRACT

In the present study, we examined hemodynamics in vasculogenicmimicry (VM) and angiogenesis of inflammatory breast cancer (IBC)xenografts (WIBC-9), having previously reported on the unique histolog-ical features and molecular basis of these processes (K. Shirakawa et al.,Cancer Res., 61: 445–451, 2001). Histologically, the WIBC-9 xenograftsexhibited invasive ductal carcinoma with a hypervascular structure (an-giogenesis) in the tumor margin and VM without endothelial cells, centralnecrosis, or fibrosis in the tumor center. Results of molecular analysisindicated that WIBC-9 had a vasculogenic phenotype, including expres-sion of Flt-1 and Tie-2. Comparison of WIBC-9 with an establishednon-IBC xenograft (MC-5), using time-coursed dynamic micromagneticresonance angiography analysis (with our newly developed intravascularmacromolecular magnetic resonance imaging contrast agent), electromi-croscopy, and immunohistochemistry, demonstrated blood flow and aVM-angiogenesis junction in the central area of the WIBC-9 tumor. It haspreviously been considered impossible to prove a connection between VMand angiogenesis using angiography, because there are no intravascularmacromolecular magnetic resonance imaging contrast agents that do notexhibit significant leakage through the vascular wall. In the present study,laser-captured microdissection was performed in regions of WIBC-9 tu-mors that exhibited VM without endothelial cells, central necrosis, orfibrosis, revealing expression of human-Flt-1 and human-Tie2 and theabsence of human-CD31, human-endothelin B receptor, and human-throm-bin receptor. These facts led us to hypothesize that the VM of WIBC-9involves hemodynamics that serve to feed WIBC-9 cells, and this in turnsuggests a connection between VM and angiogenesis.

INTRODUCTION

Tumors require a blood supply for growth and hematogenousmetastases. Most attention has been focused on the role of angiogen-esis, the recruitment of new vessels into a tumor from preexistingvessels. Previously, we and others reported the presence of VM3 [acondition in which tumors (IBC and melanoma) may have the poten-tial to feed themselves without angiogenesis] in the tumor-bearingstate (1–7). The interface between erythrocytes and tumor cells in VMhas been visualized by electromicroscopy and immunohistochemistry(1). Using semiquantitative RT-PCR with species-specific primers,

we have found a molecular basis for VM (1). The generation ofmicrovascular channels by genetically deregulated, aggressive tumorcells has been termed “vasculogenic mimicry” to emphasize their denovo generation, without participation of ECs and independent ofangiogenesis. This mechanism, by which an aggressive tumor gener-ates its own network of pseudo-vascular channels, may challenge theassumption that angiogenesis and related mechanisms are the onlymeans by which a tumor acquires a blood supply. However, thehemodynamics of VM have never been observed. In this study, weinvestigated the hemodynamics of VM and angiogenesis of IBC,using WIBC-9 xenografts and dynamic micro-MRA analysis with ournewly developed intravascular macromolecular MRI contrast agent.The results of LCM in the VM regions of WIBC-9 strongly suggestthe existence of hemodynamics that serve to feed WIBC-9 cells, andthis in turn suggests a connection between VM and angiogenesis.

MATERIALS AND METHODS

Establishment of WIBC-9, MC-5, MC-2, and MC-18. The animal pro-tocols for all experiments were approved by the Animal Use Committee of theNational Cancer Center. Tumor specimens from patients with IBC and non-IBC cancer were obtained immediately after surgery and processed as reportedpreviously (1). The tumor xenografts were subsequently serially transplantedover a period of more than 3 years, with up to 15 transplants, and stable seriallytransplantable xenografts (WIBC-9, MC-5, MC-2, and MC-18) were success-fully established in BALB/c nude mice (CLEA Japan, Tokyo, Japan; Refs. 1).When the tumors had reached 10 mm in diameter, they were examined bydynamic micro-MRA.

Dynamic Micro-MRA with an Intravascular Contrast Agent. We per-formed dynamic micro-MRA analysis, using our newly developed intravascu-lar macromolecular MRI contrast agent [G6D-(1B4M-Gd)256 (Mr 240,000),which consistently showed no significant leakage through the vascular wallafter remaining in circulation for more than 30 min] to evaluate the physio-logical properties of the vascular channels in the xenografted tumors. Themethods used for synthesis and preparation of the contrast agent and forobtaining dynamic MR images, along with a discussion of the agent’s prop-erties in relation to visualizing microvasculature, have previously been pub-lished (8). MR angiography of the mice was performed with injection of 0.066mmol Gd/kg of G6D-(1B4M-Gd)256, using a 1.5-tesla superconductive magnetunit (Signa; General Electric Medical System, Milwaukee, WI). We usedfemale 8-week-old BALB/c nude mice bearing either WIBC-9 or MC-5 tumorxenografts. All of the images were obtained with dual phased-array 3-inchround surface coils, fixed at 3-cm intervals by an in-house-constructed mouseand coil holder. The mice were anesthetized with 1.15 mg of sodium pento-barbital (Dinabot, Osaka, Japan), and placed at the center of the coils. Thethree-dimensional-fast spoiled gradient echo technique (efgre3d; TR/TE, 10.5/2.7; flip angle, 30°) with chemical fat-suppression was used for all mice. Theimages were acquired before injection of the contrast agents and at 0 (imme-diately postinjection), 1, 2, 3, 5, 8, 10, 15, and 30 min postinjection. Thecoronal images were reconstructed with 1.0-mm section thickness and 0.5-mmoverlap. The FOV was 8 � 4 cm, and the size of the matrix was 256 � 128.This procedure was performed with mice bearing WIBC-9 and MC-5 tumors(n � 3, for each).

Morphological Analysis and Immunohistochemistry. In addition to dy-namic micro-MRA, we performed the following analyses on the specimens,

Received 6/13/01; accepted 11/12/01.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 with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grants-in-Aid for the Second-Term Comprehensive 10-year Strategyfor Cancer Control from the Ministry of Health and Welfare of Japan, by Grants-in-Aidfor Cancer Research from the Ministry of Health and Welfare of Japan, and by Grants-in-Aid for Cancer Research from the Ministry of Education, Culture, Sports, Science andTechnology, Japan.

2 To whom requests for reprints should be addressed, at National Cancer CenterResearch Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan. Phone: 81-3-3542-2511; Fax: 81-3-3542-1886.

3 The abbreviations used are: VM, vasculogenic mimicry; RT-PCR, reverse transcrip-tion-PCR; MRA, magnetic resonance angiography; ROI, region(s) of interest; LCM,laser-captured microdissection; VEGF, vascular endothelial growth factor; Ang, angio-poietin; MRI, magnetic resonance imaging; EC, endothelial cell; IBC, inflammatorybreast cancer; HUVEC, human umbilical vein endothelial cell.

560

on June 1, 2018. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

using conventional methods: H&E staining, Giemsa staining and electronmicroscopy. Frozen and paraffin sections of the nonresected tumor samplesthat had been examined by dynamic micro-MRA were embedded in O.C.T.Compound (Mile’s Sankyo, Tokyo, Japan), and assayed using the immunoper-oxidase procedure. Antimurine CD31 (Pharmingen, San Diego, CA) was usedas the primary antibody. The reaction was visualized using streptavidin-biotin(Pharmingen) techniques.

LCM. Paraffin-embedded WIBC-9 and MC-5 xenograft specimens werecut into serial 5-�m sections and mounted on uncoated slides treated withdiethyl pyrocarbonate. The VM in WIBC-9 and MC-5 cancer cells wasexamined in coverslipped H&E-stained sections. One specimen was selectedfor collection of 2000 individual cells for mRNA isolation and nested PCRanalysis. Sections to be used for LCM were taken from 4°C storage andimmediately immersed in xylene (three times, 10 min each time) and ethanol(in 100% ethanol three times, 10 min each time; and in 75% ethanol for 10min). Slides were rinsed in H2O, stained with filtered Meyer’s hematoxylin for30 s, rinsed in H2O again, stained with bluing reagent for 30 s, washed in 70and 95% ethanol for 1 min each, stained with eosin Y for 30 s, and finallydehydrated in 95% ethanol (twice, for 1 min each time), 100% ethanol (overa molecular sieve three times, 1 min each time), and xylene (three times, 10min each time). Slides were air-dried under a laminar flow for 10 min, and thenimmediately processed for LCM. Diethyl pyrocarbonate-treated, autoclaved,distilled water was used to prepare all of the solutions. LCM was performedwith a PixCell II Microscope (Arcturus Engineering, Inc., Mountain View,CA) and a 7.5-�m laser beam at 50 mV. Target cells in the specimens wereeasily identified and captured; tumor cells immediately adjacent to necroticareas and blood cells were avoided.

RT-PCR Conditions and Reagents. Total RNA was isolated from LCM-collected cells and culture cell lines (SK-BR-3, MCF-7 and HUVEC; AmericanType Culture Collection, Manassas, VA, and Sankojunyaku Co., Tokyo, Japan)using the guanidinium thiocyanate-phenol-chloroform extraction method. PCRwas designed to amplify specific mRNAs, using published sequences. Primersequences for VEGF were as follows: sense, 5�-GGCCAGCACATAGGAGAG-3�; antisense, 5�-TGCAGGAACATTTACACG-3� (amplicon size, 157 bp). Prim-ers for Flt-1 were as follows: sense, 5�-TTTTACCGAATGCCACCTC-3�; anti-sense, 5�-GCGTGCTAGCTGGATGTCTT-3� (amplicon size, 159 bp). Nestedprimers for Flt-1 were as follows: sense, 5�-GGGCGACAGCAGCACTCT-3�;antisense, 5�-GCCCCGACTCCTTACTTTT-3� (amplicon size, 122 bp). Primersfor KDR were as follows: sense, 5�-GAACCAAATTATCTCCATCTT-3�; anti-sense, 5�-GCACTCCAATCTCTATCAGC-3� (amplicon size, 180 bp). Nestedprimers for KDR were as follows: sense, 5�-TGTGGCATCTGAAGGCTCAA-3�;antisense, 5�-GCACTCCAATCTCTATCAGC-3� (amplicon size, 125 bp). Prim-ers for Ang-1 were as follows: sense, 5�-ACCGAGCCTATTCACAGTAT-3�;antisense, 5�-CAAGCATCAAACCACCATCCT-3� (amplicon size, 180 bp).Primers for Ang-2 were as follows: sense, 5�-TTCTAAACATCCCAGTCCAC-3�;antisense, 5�-CCCGTCAGCACCGAGCACAC-3� (amplicon size, 132 bp).Nested primers for Ang-2 were as follows: sense, 5�-TCTAAACATCCCAGTC-CAC-3�; antisense, 5�-CCCGTCAGCACCGAGCACAC-3� (amplicon size, 131bp). Primers for Tie-2 were as follows: sense, 5�-GTCCCGAGGTCAAGAG-GTG-3�; antisense, 5�-CAAGTCATCCCGCAGTAGG-3� (amplicon size, 177bp). Nested primers for Tie-2 were as follows: sense, 5�-TGCGCTGGATGGC-CATCG-3�; antisense, 5�-CAAGTCATCCCGCAGTAGG-3� (amplicon size, 127bp). Primers for CD31 were as follows: sense, 5�-AGTGGTTATCATCG-GAGTG-3�; antisense, 5�-TCATTTATTGGTTTCATT-3� (amplicon size, 222bp). Nested primers for CD31 were as follows: sense, 5�-AGTGGTTATCATCG-GAGTG-3�; antisense, 5�-CTGCTGGCCTGGACATTTC-3� (amplicon size, 110bp). Primers for Thrombin receptor were as follows: sense, 5�-GCCAACCGCAG-CAAGAAGTC-3�; antisense, 5�-GGTGGAAGTGTGAGAAAGGA-3� (ampli-con size, 129 bp). Nested primers for Thrombin receptor were as follows: sense,5�-GCCAACCGCAGCAAGAAGTC-3�; antisense, 5�-TGGGTCCGAAGCAA-ATGATG-3� (amplicon size, 79 bp). Primers for Endothelin B receptor were asfollows: sense, 5�-TTGTCTTTGCCCTCTGCTG-3�; antisense, 5�-GCAGTTTT-TGAATCTTTTGCTC-3� (amplicon size, 200 bp). Nested primers for EndothelinB receptor were as follows: sense, 5�-GCTTCCCCTTCACCTCAG-3�; antisense,5�-GCAGTTTTTGAATCTTTTGCTC-3� (amplicon size, 181 bp). Primers forG3PDH were as follows: sense, 5�-AATCCCATCACCATCTTCCAG-3�; anti-sense, 5�-AGGGGCCATCCACAGTCTTCT-3� (amplicon size, 361 bp).

PCR was performed using the following gene-specific annealing tempera-tures: 50°C for VEGF, Ang-1, endothelin B receptor, CD31, and Ang-2; 54°C

for Flt-1 and Tie-2; 51°C for KDR; 52°C for thrombin receptor; and 57°C forG3PDH. PCR was performed with 40 cycles, as follows: 95°C for 30 s,gene-specific annealing temperature for 15 s, 72°C for 30 s. Additionally,agarose gel electrophoresis of the PCR products, followed by staining withethidium bromide, was performed to confirm the specificity of the amplifica-tion.

Comparative Analyses. The following analyses were performed onWIBC-9 MC-5, MC-2, MC-18, HUVEC, SK-BR3, and MCF-7, to determinepatterns of VM and angiogenesis: dynamic MRA, histopathology, and post-LCM RT-PCR.

Statistical Analysis. All data are expressed as the mean � SD. StatViewcomputer software (ATMS Co., Tokyo, Japan) was used for statistical analysisof results for WIBC-9 and MC-5. Two-sided Ps less than 0.05 were consideredto indicate statistical significance.

RESULTS

Horizontal Scanning of WIBC-9 and MC-5 by Dynamic Micro-MRA with an Intravascular Macromolecular MRI ContrastAgent. To visualize blood flow in VM and neovascular vessels (an-giogenesis) of the xenografted tumor, we used dynamic micro-MRAanalysis with our newly developed intravascular macromolecular MRIcontrast agent [G6D-(1B4M-Gd)256 (Mr 240,000)]. In Fig. 1, we show2-mm-interval horizontal scans of the ventral area of anesthetizedxenografted mice. To compare tumor signal intensities between mice,the amount of G6D-(1B4M-Gd)256 was normalized at 0.066 mmol Gdper kg of mouse body weight. Loupe images and high-power H&Estaining images were also used for comparison, along with a side-by-side comparison of the MRA data. The tumor marginal area ofWIBC-9 and MC-5 exhibited a high-intensity signal that completelysurrounded the xenografted tumor, a result consistent with angiogen-esis (compare Fig. 1, A–E, with Fig. 1, F–J). The high-power H&Estaining images clearly showed neovascular vessels (compare Fig. 1,D and E, with Fig. 1, I and J). In the tumor center, WIBC-9 exhibitedmultiple high-intensity spots (�), a finding consistent with patholog-ical VM (Figs. 1, A–C, and 4), whereas MC-5 exhibited a low-intensity signal or a lack of signal (�), a finding consistent withcentral necrosis and disappearance of nuclei (Figs. 1, F–H, and 4;compare circled areas of Fig. 1, B–E with those of Fig. 1, G–J).

Time-coursed MRA of WIBC-9 and MC-5. Time-coursed mi-cro-MRA was performed to analyze hemodynamics in VM andangiogenesis. The images were acquired before injection of thecontrast agents and 0 (immediately postinjection), 1, 2, 3, 5, 10, 15,and 30 min postinjection. The tumor marginal area of WIBC-9 andMC-5 exhibited a signal that gradually increased in intensity, aresult consistent with time lag relative to the intensity recorded forthe heart (which reflects properties of blood) and the liver (a highlyvascularized organ). In the tumor center, WIBC-9 tumors exhibitedspots in which the signal increased in intensity (which is consistentwith the intensity observed at the tumor margin), whereas MC-5tumors exhibited a low-intensity signal or a lack of signal (com-pare circled areas of Fig. 2, A–H with those of Fig. 2, I–P). All ofthe data were obtained directly from the MRA analyzer and areshown in the graphs in Fig. 3.

Hemodynamics of VM and Angiogenesis of IBC and Non-IBCXenografts. To analyze hemodynamics in VM and in angiogenesis,we gated on three ROI in the central area and the marginal area of thexenografted tumors and counted time-coursed pixel numbers permm2. Three experiments were performed on these three gated ROI.All of the data in Fig. 3 were obtained directly from the MRA analyzerand are expressed as the mean � SD. To compare tumor signalintensities between mice, the amount of contrast agent per mouse wasnormalized, resulting in normalization of heart signal intensities be-tween mice. The time-coursed intensity of the tumor center (which

561

HEMODYNAMICS IN VM

on June 1, 2018. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Fig. 1. Horizontal scanning of WIBC-9 and MC-5 by dynamic micro-MRA with an intravascular macromolecular MRI contrast agent. Shown are 2-mm-interval vertical sectionsof the xenografted mice with loupe images and high-power H&E staining images. The tumor marginal area of WIBC-9 and MC-5 exhibited a high-intensity signal that completelysurrounded the xenografted tumors, a result consistent with angiogenesis (compare A–E with F–J). In the tumor center, WIBC-9 exhibited multiple high-intensity spots (�) consistentwith histological VM, whereas MC-5 exhibited a low-intensity signal or a lack of signal (�) consistent with central necrosis (compare circled areas of B–E with those of G–J).

562

HEMODYNAMICS IN VM

on June 1, 2018. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

corresponded to the hemodynamics of VM, as shown in Figs. 1 and 4)was consistent with the time-coursed intensity of the tumor margin(which corresponded to the hemodynamics of angiogenesis, as shownin Fig. 5). Examination of the hemodynamics of VM revealed bloodflow with two peaks of intensity and a statistically significant time lagrelative to the hemodynamics of angiogenesis. The rate of tumormargin angiogenesis in WIBC-9 was four times as great (or more) asthat observed in MC-5, a finding consistent with the microvascularintensities observed in immunohistochemical analysis (Fig. 4) and ourpreviously reported data, including results of immunohistochemicalanalysis using murine flt-1, murine tie-2, murine integrin �v�3 andmurine CD31 (1).

Morphology in the Central and Marginal Area of WIBC-9 andMC-5. In the central area of the WIBC-9 tumor, analysis by H&E,Giemsa staining, electromicroscopy, and immunohistochemistry withmurine CD31 revealed positivity for murine CD31 and blood poolingwithout an EC lining (Fig. 4, A–D). These features indicated that VM,which is associated with the absence of ECs, central necrosis, and centralfibrosis of IBC, xenografted the tumors. Transmission electromicroscopyclearly visualized the tumor cell-erythrocyte interface, which did notinclude EC structures (Fig. 4C; T indicates tumor cells). In contrast, H&Eand Giemsa staining of MC-5 revealed a medullary growth pattern,central necrosis, rare neovasculars with ECs, and positivity for murineCD31 and infiltration by mononuclear cells (Fig. 4, H–K). These featuresare consistent with the data obtained by MRA (Figs. 1–3).

In the marginal area and the overlying skin (Fig. 4F) of WIBC-9,macroscopic examination (Fig. 4E) and immunohistochemistry withmurine CD31 (Fig. 4G) revealed hypervascularity with a lining of ECs(angiogenesis). However, the central area of WIBC-9 rarely exhibitedangiogenesis, a finding consistent with the lack of a CD31 PCR band(Fig. 5B) and post-LCM observation of VM (Fig. 5A). MC-5 exhibitedless angiogenesis in the marginal area of the tumor (Fig. 4N) and theoverlying skin (Fig. 4M) than did WIBC-9 and exhibited central tumornecrosis (Fig. 4L, �). This strongly suggests that VM of WIBC-9involves hemodynamics that serve to feed WIBC-9 cells, and this inturn suggests a connection between VM and angiogenesis.

RT-PCR after LCM. LCM was performed in the regions of VM(Fig. 5, A and D) in WIBC-9 tumors (Fig. 5A). The regions examined didnot contain stromal cells or lymphocytes (Fig. 5, B and E). The HUVECcell line was used as the positive control, and interspecies primers(human-mouse) of angiogenic factors were used. All of the primers usedwere for EC-associated genes. For comparison of post-LCM RT-PCR

Fig. 2. Time-coursed MRA of WIBC-9 and MC-5. The images were acquired before injection of the contrast agent and 1, 2, 3, 5, 10, 15, and 30 min postinjection. The tumor marginalarea of WIBC-9 and MC-5 exhibited a signal that gradually increased in intensity, a result consistent with time lag relative to the intensity recorded for the heart (which reflects propertiesof blood) and liver (a highly vascularized organ). In the tumor center, WIBC-9 tumors exhibited spots in which the signal gradually increased in intensity (consistent with the intensityobserved at the tumor margin), whereas MC-5 tumors exhibited a low-intensity signal or a lack of signal (compare circled areas of A–H with those of I–P).

Fig. 3. Hemodynamics of VM and angiogenesis of IBC and non-IBC xenografts. Toanalyze hemodynamics of VM and angiogenesis, in the three gated ROI in the central areaand the marginal area of the xenografted tumors, the number of time-coursed pixels permm2 was counted. The time-coursed intensity of the tumor center was consistent with thetime-coursed intensity of the tumor margin. Examination of the hemodynamics of VMrevealed blood flow with two peaks of intensity and a statistically significant time lagrelative to the hemodynamics of angiogenesis. The rate of tumor margin angiogenesis inWIBC-9 was four times as great (or more) as that observed in MC-5, a finding consistentwith results of immunohistochemical analysis using murine CD31.

563

HEMODYNAMICS IN VM

on June 1, 2018. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

results (Fig. 5B), we used post-LCM regions of WIBC-9 tumors (Fig. 5,C and F) and several other breast cancer cell lines, including SK-BR3 andMCF-7. Gene expression of VEGF, Flt-1, Ang-1, Ang-2, and Tie-2 wasobserved in samples exhibiting VM. This finding is consistent with thoseof our previous study, in which we used species-specific primers (1). Of

particular interest is the gene expression of Flt-1 and Tie-2 in the absenceof CD31, thrombin receptor, and endothelin B receptor. In addition,post-LCM regions of WIBC-9 tumors and whole tumors of WIBC-9,MC-5, MC-2, and MC-18, all of which included ECs, showed expressionof all of the genes assayed for. These facts suggest that VM of WIBC-9

Fig. 4. Morphology in the central and marginal area of WIBC-9 and MC-5. In the central area of the WIBC-9 tumor, analysis by H&E (A), Giemsa staining (B), electromicroscopy(C; T, tumor cells) and immunohistochemistry with murine CD31 (D) revealed multiple blood pooling without EC lining. In contrast, H&E (H) and Giemsa (I) staining of MC-5 revealeda medullary growth pattern, central necrosis (�), neovascular vessels with ECs (black arrow and black arrowhead), and infiltration by mononuclear cells (J, K). Scale bar: 1 cm (A,J); 50 �m (B–F, I, K, L, O); 10 �m (G, H, M, N). In the marginal area and overlying skin of WIBC-9 (F), macroscopic examination (E) and immunohistochemistry with murine CD31(G) revealed hypervascularity with a lining of ECs. MC-5 exhibited less angiogenesis in the marginal area of the tumor (L) and the overlying skin (M, N) than did WIBC-9, and exhibitedcentral tumor necrosis (L, �).

564

HEMODYNAMICS IN VM

on June 1, 2018. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

without ECs has a vasculogenic phenotype (Flt-1 and Tie-2), and this, inturn, suggests that VM occurs in the central area of WIBC-9 and feedsWIBC-9 cells.

DISCUSSION

The ubiquity of the association of angiogenesis with tumors indi-cates the extent to which tumor development and metastasis aredependent on neovascularization, and suggests that this relationshipmight involve angiogenic growth factors that are specific to neo-

plasms (9–22). We and others have proposed that, in certain circum-stances, tumors could feed themselves without neovasculars (e.g.,exploration of preexisting vessels or VM), suggesting the potential forresistance to antiangiogenic treatment (1–7, 23). Previously, it wasconsidered impossible to prove a connection between VM and angio-genesis using angiography and the hemodynamics of VM, becausethere were no intravascular macromolecular MRI contrast agents thatdidn’t exhibit significant leakage through vascular walls and becausethere were no transplantable animal models that exhibited VM. Thegreat advantage of the newly developed contrast agent used in the

Fig. 5. RT-PCR after LCM. In A, LCM was performed in the regions ofVM (a and d) in WIBC-9 tumors. The regions examined did not containstromal cells or lymphocytes (b and e). For comparison of results of post-LCM RT-PCR (B), we used post-LCM regions in WIBC-9 tumors (c and f)and post-LCM regions of MC-5 cancer cells and other breast cancer celllines, including SK-BR3 and MCF-7. Gene expression of VEGF, Flt-1,Ang-1, Ang-2, and Tie-2 was observed in samples exhibiting VM. Geneexpression of Flt-1 and Tie-2 in the absence of CD31, thrombin receptor, andendothelin B receptor was observed. Post-LCM regions of WIBC-9 tumorsand whole tumors of WIBC-9, MC-5, MC-2, and MC-18 exhibited expres-sion of these genes.

565

HEMODYNAMICS IN VM

on June 1, 2018. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

present study, compared with conventional agents, is that leakagethrough vascular walls is greatly reduced (8). To the best of ourknowledge, the present results, obtained from angiography and exam-ination of the hemodynamics of VM, represent the first confirmationof a connection between VM and angiogenesis. The established xe-nograft WIBC-9 preserves histological and biological characteristicsof human IBC. Erythema in the overlying skin, marked lymphaticpermeation, and a high rate of metastasis are commonly seen in bothWIBC-9 and human IBC (1, 24–26). WIBC-9 has two unique histo-logical features: blood pooling without a lining of ECs, and tube-likestructures and loops in the central tumor nests (1). Electron micros-copy revealed that the tumor cell-erythrocyte interface lacks necrosisand fibrosis. These tubules, produced by VM, are lined externally withtumor cells, and no ECs are found in them.

As shown in Fig. 3, examination of the hemodynamics of VMrevealed blood flow with two peaks of intensity and a statisticallysignificant time lag, relative to the hemodynamics of angiogenesis,after injection of the reagent into the tail vein of WIBC-9-xenograftedmice. In addition, in the marginal area and overlying skin of WIBC-9tumors, macroscopic examination and immunohistochemistry withmurine CD31 (reported previously: murine flt-1, murine tie-2, andmurine integrin �v�3) revealed hypervascularity with a lining of ECs(angiogenesis). In the central area of the tumor, WIBC-9 exhibitedVM in the absence of ECs, central necrosis, and fibrosis. MC-5exhibited less angiogenesis in the marginal area of the tumor and theoverlying skin than WIBC-9, and exhibited central tumor necrosis.This fact suggests that VM of WIBC-9 involves hemodynamics thatserve to feed WIBC-9 cells, and this in turn suggests a connectionbetween angiogenesis and VM.

The fact that the central area of WIBC-9 tumors rarely exhibitedangiogenesis is consistent with the lack of a CD31 PCR band (Fig. 5B)and the VM revealed by LCM (Fig. 5B). MC-5 exhibited less angio-genesis in the marginal area of the tumor and the overlying skin thandid WIBC-9, and exhibited central tumor necrosis. Results of post-LCM RT-PCR analysis indicated that WIBC-9 had a vasculogenicphenotype, including expression of Flt-1 and Tie-2. These resultssuggest de novo formation of vascular channels by tumor cells in thecenter of the tumor as a result of putative hypoxia and induction ofangiogenic factors. They also suggest that vessel regression has notbeen occurring in these tumors, and that the blood from rupturedvessels has not filled tumor-lined lakes or channels. WIBC-9 showedno signs of fibrosis, central necrosis, or endothelial lining, whereasnon-IBC xenografts such as MC-2, MC-5, and MC-18 commonlyexhibit fibrosis and central necrosis as the tumor grows (1). Webelieve these findings may be related to the expression of certainhuman genes in WIBC-9. As shown in Fig. 5, LCM revealed geneexpression of VEGF, Flt-1, Ang-1, Ang-2, and Tie-2 in samplesexhibiting VM. These findings were consistent with those of ourprevious study, in which we used species-specific primers (1). Ofparticular interest is the gene expression of Flt-1 and Tie-2 in theabsence of CD31, thrombin receptor and endothelin B receptor. Thesefacts also suggest that VM of WIBC-9 results in a vasculogenicphenotype in which WIBC-9 cells are fed without ECs.

In the present study, we investigated the hemodynamics of VM andangiogenesis of WIBC-9, using time-coursed dynamic micro-MRAanalysis with our newly developed intravascular macromolecular MRIcontrast agent, and demonstrated the existence of a connection be-tween VM and angiogenesis. The observed gene expression may bethe cause of the observed endothelial-vascular phenotype and theputative de novo formation of vascular channels by tumor cells. Weare presently conducting experiments designed to further clarify theissues addressed by this article.

ACKNOWLEDGMENTS

We are grateful to Professor Masabumi Shibuya and to Assistant ProfessorMasaaki Hamaguchi for their advice. We are also grateful to Minako Taka-hashi and Takayuki Morikawa for their technical assistance.

REFERENCES

1. Shirakawa, K., Tsuda, H., Heike, Y., Kato, K., Asada, R., Inomata, M., Sasaki, H.,Kasumi, H., Yoshimoto, M., Iwanaga, T., Konishi, F., Terada, M., and Wakasugi, H.Absence of endothelial cells, central necrosis, and fibrosis are associated withaggressive inflammatory breast cancer. Cancer Res., 61: 445–451, 2001.

2. Maniotis, A. J., Folberg, R., Hess, A., Seftor, E. A., Gardner, L. M., Pe’er, J., Trent,J. M., Meltzer, P. S., and Hendrix, M. J. Vascular channel formation by humanmelanoma cells in vivo and in vitro: vasculogenic mimicry. Am. J. Pathol., 155:739–752, 1999.

3. Barinaga, M. New type of blood vessel found in tumors. Science (Wash. DC), 285:1475, 1999.

4. Folberg, R., Hendrix, M. J., and Maniotis, A. J. Vasculogenic mimicry and tumorangiogenesis. Am. J. Pathol., 156: 361–381, 2000.

5. Hess, A. R., Seftor, E. A., Gardner, L. M., Carles-Kinch, K., Schneider, G. B., Seftor,R. E., Kinch, M. S., and Hendrix, M. J. Molecular regulation of tumor cell vasculo-genic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2). Cancer Res., 61: 3250–3255, 2001.

6. Pezzella, F., Pastorino, U., Tagliabue, E., Andreola, S., Sozzi, G., Gasparini, G.,Menard, S., Gatter, K. C., Harris, A. L., Fox, S., Buyse, M., Pilotti, S., Pierotti, M.,and Rilke, F. Non-small-cell lung carcinoma tumor growth without morphologicalevidence of neo-angiogenesis. Am. J. Pathol., 151: 1417–1423, 1997.

7. Seftor, R. E., Seftor, E. A., Koshikawa, N., Meltzer, P. S., Gardner, L. M., Bilban, M.,Stetler-Stevenson, W. G., Quaranta, V., and Hendrix, M. J. Cooperative interactionsof laminin 5 �2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggres-sive melanoma. Cancer Res., 61: 6322–6327, 2001.

8. Kobayashi, H., Sato, N., Kawamoto, S., Saga, T., Hiraga, A., Haque, T. L., Ishimori,T., Konishi, J., Togashi, K., and Brechbiel, M. W. Comparison of the macromolecularMR contrast agents with ethylenediamine-core versus ammonia-core generation-6polyamidoamine dendrimer. Bioconjug. Chem., 12: 100–107, 2001.

9. Chambler, A. F. W., Drew, P. J., Hill, A. D. K., Darzi, A., and Monson, J. R. T.Inflammatory breast carcinoma. Surg. Oncol., 4: 245–254, 1995.

10. Folkman, J., and Klagsbrun, M. Angiogenic factors. Science (Wash. DC), 235:442–447, 1987.

11. Holash, J., Maisonpierre, P. C., Compton, D., Boland, P., Alexander, C. R., Zagzag,D., Yancopoulos, G. D., and Wiegand, S. J. Vessel cooption, regression, and growthin tumors mediated by angiopoietins and VEGF. Science (Wash. DC), 284: 1994–1998, 1999.

12. Ferrara, N., and Alitalo, K. Clinical applications of angiogenic growth factors andtheir inhibitors. Nat. Med., 5: 1359–1364, 1999.

13. O’Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E.,Birkhead, J. R., Olsen, B. R., and Folkman, J. Endostatin: an endogenous inhibitor ofangiogenesis and tumor growth. Cell, 88: 277–285, 1997.

14. Kendall, R. L., and Thomas, K. A. Inhibition of vascular endothelial cell growthfactor activity by an endogenously encoded soluble receptor. Proc. Natl. Acad. Sci.USA, 90: 10705–10709, 1993.

15. Alpaugh, M. L., Tomlinson, J. S., Shao, Z. M., and Barsky, S. H. A novel humanxenograft model of inflammatory breast cancer. Cancer Res., 59: 5079–5084, 1999.

16. Dinarello, C. A. Interleukin-1 and interleukin-1 antagonism. Blood, 77: 1627–1652,1991.

17. Cozzolino, F., Torcia, M., Aldinucci, D., Ziche, M., Almerigogna, F., Bani, D., andStern, D. M. Interleukin 1 is an autocrine regulator of human endothelial cell growth.Proc. Natl. Acad. Sci. USA, 87: 6487–6491, 1990.

18. Singh, P. K., Gutman, M., Reich, R., and Bar-Eri, M. Ultraviolet B irradiationpromotes tumorigenic and metastatic properties in primary cutaneous melanoma viainduction of interleukin 8. Cancer Res., 55: 3669–3674, 1995.

19. Koch, A. E., Polverine, P. J., Kunkel, S. L., Harlow, L. A., DiPietro, L. A., Elner,V. M., Elner, S. G., and Strieter, R. M. Interleukin-8 as a macrophage-derivedmediator of angiogenesis. Science (Wash. DC), 258: 1798–1801, 1992.

20. Suri, C., Jones, P. F., Patan, S., Bartunkova, S., Maisonpierre, P. C., Davis, S., Sato,T. N., and Yancopoulos, G. D. Requisite role of angiopoietin-1, a ligand for the TIE2receptor, during embryonic angiogenesis. Cell, 87: 1171–1180, 1996.

21. Brooks, P. C., Montgomery, A. M., Rosenfeld, M., Reisfeld, R. A., Hu, T., Klier, G.,and Cheresh, D. A. Integrin �v�3 antagonists promote tumor regression by inducingapoptosis of angiogenic blood vessels. Cell, 79: 1157–1164, 1994.

22. Maru, Y., Yamaguchi, S., and Shibuya, M. Flt-1, a receptor for vascular endothelialgrowth factor, has transforming and morphogenic potentials. Oncogene, 20: 2585–2595, 1998.

23. Hashizume, H., Baluk, P., Morikawa, S., McLean, J. W., Thurston, G., Roberge, S.,Jain, R. K., and McDonald, D. M. Openings between defective endothelial cellsexplain tumor vessel leakiness. Am. J. Pathol., 156: 1363–1380, 2000.

24. Weidner, N., Semple, J. P., Welch, W. R., and Folkman, J. Tumor angiogenesis andmetastasis-correlation in invasive breast carcinoma. N. Engl. J. Med., 324: 1–8, 1991.

25. Gasparini, G., and Harris, A. L. Clinical importance of the determination of tumorangiogenesis in breast carcinoma: much more than a new prognostic tool. J. Clin.Oncol., 13: 765–782, 1995.

26. Brooks, H. L., Mandava, N., Pizzi, W. F., and Shah S. Inflammatory breast carci-noma: a community hospital experience. J. Am. Coll. Surg., 186: 622–629, 1998.

566

HEMODYNAMICS IN VM

on June 1, 2018. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

2002;62:560-566. Cancer Res   Kazuo Shirakawa, Hisataka Kobayashi, Yuji Heike, et al.   Inflammatory Breast Cancer XenograftHemodynamics in Vasculogenic Mimicry and Angiogenesis of

  Updated version

  http://cancerres.aacrjournals.org/content/62/2/560

Access the most recent version of this article at:

   

   

  Cited articles

  http://cancerres.aacrjournals.org/content/62/2/560.full#ref-list-1

This article cites 26 articles, 13 of which you can access for free at:

  Citing articles

  http://cancerres.aacrjournals.org/content/62/2/560.full#related-urls

This article has been cited by 14 HighWire-hosted articles. Access the articles at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/62/2/560To request permission to re-use all or part of this article, use this link

on June 1, 2018. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from