Hyperproduction of Hyaluronan in Neu-Induced Mammary Tumor Accelerates Angiogenesis through Stromal Cell Recruitment

The American Journal of Pathology, Vol. 170, No. 3, March 2007 Copyright © American Society for Investigative Pathology DOI: 10.2353/ajpath.2007.060793

Tumorigenesis and Neoplastic Progression

Hyperproduction of Hyaluronan in Neu-Induced Mammary Tumor Accelerates Angiogenesis through Stromal Cell Recruitment Possible Involvement of Versican/PG-M

Hiroshi Koyama,*† Terumasa Hibi,‡ Zenzo Isogai,§ Masahiko Yoneda,¶ Minoru Fujimori,† Jun Amano,† Masatomo Kawakubo,储 Reiji Kannagi,‡** Koji Kimata,†† Shun’ichiro Taniguchi,* and Naoki Itano*‡ From the Department of Molecular Oncology,* Division of Molecular and Cellular Biology, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Nagano; the Department of Surgery,† Shinshu University School of Medicine, Nagano; Biomedical Research Center,储 Department of Laboratory Medicine, Shinshu University Hospital, Nagano; Core Research for Evolutional Science and Technology,‡ Japan Science and Technology Corporation, Saitama; the Division of Pharmacotherapy,§ Department of Advanced Medicine, National Center for Geriatrics and Gerontology, Aichi; Biochemistry and Molecular Biology Laboratory,¶ Aichi Prefectural College of Nursing and Health, Aichi; Program of Molecular Pathology,** Aichi Cancer Center, Research Institute, Aichi; and Institute for Molecular Science of Medicine,†† Aichi Medical University, Aichi, Japan

Elevated concentrations of hyaluronan are often associated with human breast cancer malignancy. Here, we investigated the roles of hyaluronan in carcinogenesis and cancer progression using the mouse mammary tumor virus (MMTV)-Neu transgenic model of spontaneous breast cancer. Conditional transgenic mice that express murine hyaluronan synthase 2 (Has2) by Cre-mediated recombination were generated and crossed with the MMTV-Neu mice. In expressing Cre recombinase under the control of the MMTV promoter , the bigenic mice bearing Has2 and neu transgenes exhibited a deposition of hyaluronan matrix and aggressive growth of Neu-initiated mammary tumors. Notably , forced expression of Has2 impaired intercellular adhesion machinery and elicited cell survival signals in tumor cells. Concurrent with these alterations of tumor cells , intratumoral stroma and microvessels were markedly induced. To reveal

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the molecular basis of hyaluronan-mediated neovascularization, various hyaluronan samples were examined for their ability to potentiate in vivo angiogenesis. In Matrigel plug assays, basic fibroblast growth factor-induced neovascularization was elevated in the presence of either hyaluronan oligosaccharides or a hyaluronan aggregate containing versican. Administration of hyaluronan-versican aggregates, but not native hyaluronan alone, promoted stromal cell recruitment concurrently with the infiltration of endothelial cells. Taken together, these results suggest that hyaluronan overproduction accelerates tumor angiogenesis through stromal reaction, notably in the presence of versican. (Am J Pathol 2007, 170:1086 –1099; DOI: 10.2353/ajpath.2007.060793)

Carcinogenesis is a multistep process accompanied by genetic alterations of precancerous cells and by simultaneous construction of the tumor microenvironment.1 Clinical and experimental evidence have evaluated the importance of interactions between cancer cells and the surrounding stroma to facilitate tumor progression.2,3 One of the cellular components in the intratumoral stroma is a subpopulation of fibroblasts.4 These activated fibroblasts, sometimes termed tumor-associated fibroblasts, are capable of modulating the tumor microenvironment during tumor development and progression. A specific contribution of the tumor-associated fibroblasts is to supSupported by grants from the Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency, the Aichi Cancer Research Foundation, and the Shinshu Association for the Advancement of Medical Sciences. Accepted for publication December 9, 2006. Address reprint requests to Naoki Itano, Ph.D., Department of Molecular Oncology, Division of Molecular and Cellular Biology, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto, Nagano 390-8621, Japan. E-mail: [email protected].

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ply a variety of cytokines, growth factors, and tissueremodeling enzymes, all of which facilitate tumor cell invasion and angiogenesis.5,6 Moreover, they synthesize many of the constituents of the stromal extracellular matrix (ECM) and provide supporting frameworks amenable to easy penetration by endothelial cells, leading to neovascularization.7 Hyaluronan (HA) is a major constituent of ECM linking proteoglycans and other binding molecules into macromolecular aggregates.8 HA-rich ECM provides a favorable microenvironment for cell proliferation and migration by maintaining the turgidity and hydration of tissues and also by activating intracellular signals through interaction with cell surface receptors.8,9 Increased synthesis of HA is often associated with malignant progression in certain types of human tumors, including breast cancer, where the level of HA is considered to be a reliable prognostic indicator.10 Ectopic expression of HA synthases and perturbation of endogenous HA function in several cancer cell lines have suggested that accumulated HA stimulates growth, survival, invasion, and metastasis of cancer cells.11–16 To date, however, there have been several arguments against the tumor-promoting effect of HA,17 and the governing molecular mechanism remains elusive. One such argument is whether the HA produced by cancer cells acts in an autocrine or a paracrine fashion to stimulate tumor growth. Furthermore, it remains unclear whether HA overproduction is sufficient for oncogenic malignant transformation and tumor initiation. A spontaneous cancer model was used in this study because it was more likely to improve understanding of the pathogenesis of this disease process. Because xenograft tumor models generally undergo immediate tumor formation without initiation or promotion, and because their growth seems to be largely independent on the interaction with host cells, this relatively sudden and autonomous event may not be representative of slow-onset human cancers. To address the role of HA in the sequential steps involving host-tumor interactions, we produced conditional transgenic (cTg) mice carrying the murine Has2 gene, which allows hyperproduction of HA in the spontaneous mammary tumors. Our findings indicate a critical role of HA in the formation of intratumoral stroma and acceleration of tumor angiogenesis through stromal reaction. We further demonstrate the possible involvement of HA-versican aggregates in the promotion of stromal cell recruitment.

Materials and Methods Materials Rooster comb native HA and its oligosaccharides were kindly provided by the Seikagaku Corp. (Tokyo, Japan). Streptococcus HA and human umbilical cord HA samples were purchased from EMD Bioscience Inc. (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO), respectively. The HA oligosaccharide was a mixed fraction of average molecular weight 6.8 ⫻ 103 composed of 16 disaccharide units. Assays for other glycosaminogly-

cans, proteins, nucleic acids, and endotoxins were negative. Biotinylated hyaluronan-binding region of aggrecan (b-HABP) and anti-human versican antibody [mouse monoclonal antibody (mAb); clone 2B1] were purchased from Seikagaku Corp. Antibody against mouse CD31 (rat mAb; clone MEC13.3) was from BD Pharmingen (San Diego, CA). Polyclonal antibodies against mouse ␣-smooth muscle actin (␣-SMA) and vimentin were from Lab Vision (Fremont, CA). Antibodies against fibronectin and type I collagen were from Dako Japan Co. Ltd. (Kyoto, Japan) and LSL Co. Ltd. (Tokyo, Japan), respectively. Antibodies against E-cadherin, ␤-catenin, and proliferating cell nuclear antigen (PCNA) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Polyclonal antibodies against total ErbB2 and Y877 phosphorylated ErbB2, Akt, and phosphorylated Akt were from Cell Signaling Technology Inc. (Beverly, MA). The secondary antibodies used in this study were as follows: horseradish peroxidase-linked anti-rabbit IgG, anti-mouse IgG from goat and horseradish peroxidase-conjugated streptavidin were from Dako Japan Co. Ltd.; Alexa Fluor 594 chicken anti-rabbit IgG, Alexa Fluor 594 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rat IgG, Alexa Fluor 488 chicken anti-rabbit IgG, Alexa Fluor 488 rabbit anti-goat IgG, and Alexa Fluor 488 streptavidin conjugate were from Invitrogen (San Diego, CA). In situ apoptosis kit and reverse transcription reagent were from Takara Biochemicals (Shiga, Japan). Hemoglobin B-test Wako was from Wako Pure Chemical Industries (Osaka, Japan). RNeasy total RNA isolation kit was from QIAGEN (Valencia, CA). TaqMan rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) detection reagents were from Applied Biosystems (Foster City, CA). Enhanced chemiluminescence (ECL) Western blotting detection system was from GE Health Care Bio-Sciences Corp. (Piscataway, NJ). Phenol red-free Matrigel (9 to 10 mg/ml) was from BD Biosciences (San Jose, CA). Unless specified, all other reagents were of the highest grade from Sigma-Aldrich.

Generation of Has2 Transgenic Animal and Backcross with Mammary Tumor Model FLAG-tagged murine Has2 cDNA18 was subcloned into multicloning sites of pCALNL5 expression plasmid.19 This plasmid is composed of the Cre-mediated activation transgene unit that includes CAG (chicken ␤-actin) promoter,20 a loxP sequence, Neo (neomycinresistance gene), SV40 poly(A) signal, and a second loxP sequence (Figure 1A). The CALNL5-Has2 unit was excised from the vector by SalI and SfiI digestion and purified by agarose gel electrophoresis. The purified CALNL5-Has2 fragment was microinjected into fertilized BALB/cCrSlc mouse eggs (Japan SLC, Inc., Hamamatsu, Japan). Potential founders were analyzed for the presence of the transgene by polymerase chain reaction (PCR) of mouse genomic DNA isolated from tail specimens using murine Has2 specific primers as described below. Of the generated Has2 cTg mice, one line (HA-99) with higher expression was expanded

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Inc., Wilmington, MA). B6129-TgN (MMTV-cre)4Mam (MMTV-Cre) mice expressing Cre recombinase (Jackson Laboratories, Bar Harbor, ME) were backcrossed for six generations to the MMTV-Neu mice. Has2:Neu bigenic mice bearing both Has2 and neu transgenes were intercrossed to Cre:Neu bigenic mice bearing both Cre and neu transgenes. Founder lineages with a different combination of three transgenes, MMTV-Neu (Neu), MMTV-Cre/MMTV-Neu (Cre:Neu), CAG-NeoHas2/MMTV-Neu (Has2⫹Neo), and CAG-Has2/MMTVCre/MMTV-Neu (Has2⌬Neo), were generated and genotyped by PCR analysis of genomic DNA. Genomic DNA was extracted from mouse tail by using the DNeasy tissue kit. The primer sequences of the oligonucleotides used for PCR were as follows: Has2 forward: 5⬘-GACCTGGTGAGACAGAAGAGTCCC-3⬘; Has2 reverse, 5⬘-TATATTAAAAGCCATCCAGTATCTCACG-3⬘; Cre forward, 5⬘-GCGGTCTGGCAGTAAAAACTATC-3⬘; Cre reverse, 5⬘-GTGAAACAGCATTGCTGTCACTT-3⬘; Neu forward, 5⬘-GGAACCTTACTTCTGTGGTGTGAC-3⬘; and Neu reverse, 5⬘-TAGCAGACACTCTATGCCTGTGTG-3⬘. The PCR condition for Has2 and neu transgenes was as follows: one cycle at 94°C for 2 minutes; 35 cycles at 94°C for 45 seconds, 59°C for 1 minute, and 72°C for 1 minute; and one cycle at 72°C for 7 minutes. The PCR condition for Cre transgene was as follows: one cycle at 94°C for 3 minutes; 35 cycles at 94°C for 30 seconds, 51°C for 1 minute, and 72°C for 1 minute; and one cycle at 72°C for 2 minutes. All animal care and experimentation were performed according to the study guidelines established by the Shinshu University ethics guidelines for animal care, handling, and termination.

PCR Analysis of Cre-Mediated Genomic DNA Recombination Figure 1. Generation of Has2 conditional transgenic mice. A: Schematic of the transgenic construct. FLAG-tagged murine Has2 cDNA was positioned downstream of the transgene unit, including CAG promoter (CAG Pro), a loxP sequence, the Neo-resistance gene (Neo), the SV40 poly(A) signal (pA), and a second loxP sequence. On recognition of the loxP site, Cre recombinase deletes the Neo cassette along with one of the loxP sequences and then joins the CAG promoter and Has2 cDNA, leading to expression of Has2 mRNA. White, gray, and black triangles represent CAG promoter, PGKNeo, and Has2-R7 primers, respectively. B: PCR analysis of Cre-mediated genomic DNA recombination. Genomic DNA samples were isolated from Neu-initiated mammary tumors. PCR screening (#1) with the CAG promoter and PGK-Neo primers gave the anticipated 360-bp DNA product (arrow) for the Has2⫹Neo tumor, but not for the Has2⌬Neo tumor. PCR screening (#2) with the CAG promoter and Has2-R7 primers gave the predicted 1880-bp DNA product (white arrowhead) for the Has2⫹Neo tumor and 670-bp product (black arrowhead) for the Has2⌬Neo tumor. This change in size of the amplified product demonstrated that deletion of the Neo cassette was successfully achieved by Cre-mediated transgene recombination in mammary tumors of Has2⌬Neo mice. All PCR products were not amplified for the genomic DNA from Cre:Neu and Neu tumors. C: HA matrices of the tumor cells established from tumor tissues of Has2⌬Neo and Has2⫹Neo mice. Tumor cells were surgically isolated from Neu-initiated mammary tumors and cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS. HA matrices surrounding the tumor cells were visualized by the particle exclusion assay as described previously.12 The HA matrix occupies the clear area (arrowheads) between the fixed erythrocytes and the tumor cells.

and backcrossed for nine generations to the mouse mammary tumor virus-Neu (MMTV-Neu) mammary tumor model (Charles River Laboratories International,

Has2 cTg mice bearing mammary tumors were sacrificed, and genomic DNA samples were obtained from the surgically removed Neu-initiated mammary tumors of Has2⌬Neo, Has2⫹Neo, Cre:Neu, and Neu. The mammary tumors were incubated at 55°C overnight in 500 ␮l of lysis buffer [50 mmol/L Tris-HCl, pH 8.0, 100 mmol/L EDTA, 100 mmol/L NaCl, 1% sodium dodecyl sulfate (SDS), and 500 ␮g/ml proteinase K]. The homogenates were extracted with phenol/chloroform, precipitated with ethanol, and then dissolved in TE (10 mmol/L Tris-HCl, pH 8.0, and 1 mmol/L EDTA). PCR screening was performed using CAG promoter, PGK-Neo and Has2-R7 primers (Figure 1A). CAG promoter forward, 5⬘-CTGCCGCAGGGGGACGGCTGCCTTCG-3⬘; PGK-Neo reverse, 5⬘-TGCCCAGTCATAGCCGAATAGCCTCTC-3⬘; and Has2-R7 reverse, 5⬘-CTTGGTACGCAGCGATGCAGAGTGCTAC-3⬘. The PCR condition was as follows: one cycle at 94°C for 2 minutes; 35 cycles at 94°C for 45 seconds, 59°C for 1 minute, and 72°C for 1 minute; and one cycle at 72°C for 7 minutes.

Particle Exclusion Assays Fixed erythrocytes were reconstituted in phosphate-buffered saline (PBS) to a density of 5 ⫻ 108 cells/ml and

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used for the particle exclusion assay as described previously.12 HA matrices were visualized by adding 1 ⫻ 107 erythrocytes to the growth medium of the mammary tumor cells and then analyzed using a Zeiss Axiovert 200 inverted phase-contrast microscope (Carl Zeiss GmbH, Jena, Germany).

Matrigel Plug Assay Eight-week-old male C57BL/6 mice were anesthetized and given a subcutaneous injection of sterile phenol-red free Matrigel (500 ␮l/injection). Matrigel containing bFGF (200 ng/ml; Progen Biotechnik GmbH, Heidelberg, Germany) served as the negative control. Rooster comb native HA, its oligosaccharides (a mixed fraction of average molecular weight of 6.8 kd), Streptococcus HA, or human umbilical cord HA was mixed in the Matrigel before injection. In another experiment, Matrigel containing 200 ng/ml bFGF was mixed with 1 ␮g/ml versican in the presence or absence of 1 ␮g/ml rooster comb native HA. Animals were sacrificed 7 days after injection, and the Matrigel plugs were dissected out. Hemoglobin content in the Matrigel plugs was measured at 540 nm using hemoglobin B-test Wako (Wako Pure Chemical Industries, Osaka, Japan) following the manufacturer’s instructions.

Immunoprecipitation and Western Blot Analysis Harvested mammary tumors were homogenized in radioimmunoprecipitation assay (RIPA) buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% SDS, 1% NP40, 0.5% sodium deoxycholate, 2 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 250 ␮g/ml sodium orthovanadate, 25 mmol/L NaF, and protease inhibitors, pH 7.5). Four hundred micrograms of each tumor lysate were mixed with anti-Akt antibody for 1 hour at 4°C. Protein A-Sepharose Fast Flow (GE Health Care Bio-Sciences Corp.) was then mixed with the protein-antibody mixture for 1 hour at 4°C. The bound fraction was precipitated by centrifugation at 12,000 ⫻ g for 20 seconds and washed three times with RIPA buffer. The pellet was suspended in sample buffer and heated to 60°C for 30 minutes. After centrifugation, the supernatant was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride nylon membrane. The transferred membranes were probed with antibodies specific for Akt and phosphorylated Akt using a goat anti-rabbit IgG/horseradish peroxidase conjugate as the secondary antibody and then visualized with an ECL Western blotting detection system. Quantitative analysis was performed by densitometric imaging of the digitized image using NIH Image (version 1.60; Bethesda, MD) software.

Mammary Tumor Analysis Only age-matched virgin female mice were bred with male FVB/N mice and then analyzed for tumor development. Mice were monitored weekly by palpation to determine the presence of mammary tumors. The length and width of tumors were measured daily with calipers until the tumors reached approximately 1 cm in diameter. Tumor volume was calculated as (length ⫻ width2)/2. Tumor growth was measured for seven Has2⌬Neo mice and eight control (Has2⫹Neo, Cre:Neu, and Neu) mice. Average values represent between six and eight individual values for each time point.

Histological and Immunohistochemical Analyses Harvested mammary tumors and Matrigel implants were immediately fixed in 10% formalin or in Tris-buffered zinc fixative, dehydrated, and embedded in paraffin wax. Deparaffined sections (5 ␮m thick) were rehydrated and stained with Mayer’s hematoxylin and eosin B, b-HABP, or antibodies against CD31, ␣-SMA, vimentin, fibronectin, type I collagen, E-cadherin, and ␤-catenin. The number of tumor microvessels was calculated by counting after immunohistochemical staining with anti-CD31 antibody. Staining for PCNA was performed as described previously.21 Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) using the in situ apoptosis detection kit according to the manufacturer’s instructions. Immunolocalization of the antigens was visualized by using standard immunoperoxidase detection under the microscope or by using Alexa Fluor-conjugated second antibodies under a Zeiss LSM 510 Meta confocal microscope or Zeiss Axiovert 200 fluorescent microscope.

Total RNA Preparation and Real-Time PCR Analysis Total RNA was isolated from the mammary tumors or Matrigel implants using the RNeasy Total RNA Isolation kit. Reverse transcription was performed by random priming using reverse transcription reagent. Real-time quantitative PCR for murine Has2 gene was performed as described previously.22 The sequences of the oligonucleotides for murine Has2 were 5⬘-CCTCGGAATCACAGCTGCTTATA-3⬘ (forward primer), 5⬘-CTGCCAATAACTTCGCTGAATA-3⬘ (reverse primer) and 5⬘-TCGCATCTCATCATCCAAAGCCTCTTTG-3⬘ (TaqMan probe). The relative amounts of GAPDH mRNA were measured using TaqMan rodent GAPDH detection reagents. TaqMan gene expression assays were used for the gene expression analyses of CD31, nonmuscle myosin heavy chain-B (SMemb), vascular endothelial growth factor-A (VEGF-A), hypoxia-inducible factor-1␣ (HIF-1␣), basic fibroblast growth factor (bFGF), and stromal cell-derived factor 1␣ (SDF-1␣)/CXCL12. Relative gene expression was determined by normalizing the amount of each mRNA divided by that of the GAPDH mRNA.

Determination of HA Concentrations by Competitive ELISA-Like Assay Equal amounts of mammary tumor were dissected from five individual mice. The tumors were immediately homogenized in 200 ␮l of 50 mmol/L Tris-HCl, pH 8, 100 mmol/L EDTA, 100 mmol/L NaCl, and after the addition of

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proteinase K (500 ␮g/ml, PCR grade; Roche Diagnostics Corp., Mannheim, Germany), samples were incubated overnight at 50°C. The protease was then heat-inactivated. After centrifugation at 10,000 rpm for 10 minutes, the HA content in the supernatants was determined by an HA assay kit (IBA Method; Seikagaku Corp.) according to the manufacturer’s instructions.

Determination of HA Molecular Mass by HighPerformance Liquid Chromatography Tumor homogenates were pooled and a 500-␮l aliquot of the sample was subjected to gel filtration chromatography using a Superose 6 HR 10/30 column (GE Health Care Bio-Sciences Corp.). HA content was determined in each fraction from the column using the IBA method as described above. HA with average masses of 146, 9.6, 5.5, 3.8 ⫻ 103 d were used as standards.

Purification of Human Versican Versican with high molecular mass was partially purified from human umbilical cord and the culture medium of human dermal fibroblasts by ion exchange chromatography, CsCl density gradient centrifugation, and gel filtration chromatography as described previously.23

Dot Blot Detection of HA-Versican Aggregates in Tumor Tissues Equal amounts (60 mg) of tumor tissues were homogenized in 0.5 mol/L guanidine hydrochloride, 50 mmol/L Tris-HCl, pH 8.0, 10 mmol/L EDTA, 1 mmol/L phenylmethanesulfonyl fluoride, and protease inhibitor cocktail (Roche Diagnostics Corp.) and extracted overnight at 4°C. Solid CsCl was added to give an initial density of 1.42 g/ml and then centrifuged at 106,000 ⫻ g, 10°C, for 72 hours. The solution in the tube was fractionated into six fractions (A1–A6) from the bottom. To detect versican and HA in each fraction, the samples were immobilized on a nitrocellulose membrane using a Bio-Dot SF microfiltration apparatus (Bio-Rad Laboratories Inc., Hercules, CA). Blots were blocked with 5% nonfat milk in Tris-buffered saline (TBS), 0.1% Tween 20, washed, and incubated with primary versican antibody or b-HABP in 3% bovine serum albumin in TBS for 1 hour at room temperature. After washing, the blot was incubated with horseradish peroxidase-linked anti-rabbit IgG or streptavidin for 30 minutes at room temperature, washed, and reacted with enhanced chemiluminescence reagent according to the manufacturer’s instructions. Chemiluminescent signals were detected by exposure to Fuji RX X-ray film (Fuji Photo Film Ltd., Tokyo, Japan). For detection of versican, we used two different primary antibodies, 6084 polyclonal antibody that was generated by immunizing a recombinant N-terminal domain of human versican (rVN; K. Hasegawa, M. Yoneda, H. Kuwabara, O. Miyaishi, N. Itano, A. Ohno, M. Zako, Z. Isogai, manuscript submit-

ted) and 2B1 anti-versican monoclonal antibody, which recognizes C-terminal epitope of human versican.23

Statistical Analysis All results were expressed as the means ⫾ SE. Significance of differences was determined with Student’s t-test and ␹2 test.

Results Generation and Characterization of Has2 Transgenic Mice That Develop Mammary Tumors In an effort to simulate the hyperproduction of HA, as observed in human breast cancer, a transgenic mouse model that allows overexpression of Has2 in the mammary glands under the control of Cre recombinase was generated (Figure 1A). Of the 65 founders, 12 animals containing the transgene were identified by PCR screening. All mice developed normally and were capable of reproduction. Recombination and expression of the Has2 transgene were then assessed using skin dermal fibroblasts that were surgically isolated and cultured in vitro. Cre recombinase expression in fibroblasts derived from seven founder lineages produced FLAG-tagged Has2 protein, as determined by Western blot analysis (data not shown). One line (HA-99) with higher expression was expanded and backcrossed for nine generations to the MMTV-Neu mammary tumor model, expressing rat neu proto-oncogene in mammary luminal epithelia. The Has2:Neu bigenic mice carrying both Has2 and neu transgenes were subsequently crossed with Cre:Neu bigenic mice that expressed both Cre recombinase and Neu under the control of the MMTV promoter. Founder lineages with a different combination of three transgenes, MMTV-Neu (Neu), MMTV-Cre/MMTV-Neu (Cre:Neu), CAG-NeoHas2/MMTV-Neu (Has2⫹Neo), and CAG-Has2/MMTVCre/MMTV-Neu (Has2⌬Neo), were generated and verified by PCR genotyping. To accelerate tumor development, age-matched virgin female mice were bred with male FVB/N mice. As demonstrated by PCR analysis using genomic DNA isolated from mammary tumors, deletion of the Neo cassette was successfully achieved by Cre-mediated recombination in mammary tumors of Has2⌬Neo mice (Figure 1B), and sequence analysis of the generated PCR product confirmed recombination. The formation of HA pericellular matrix was then assessed using cultured tumor cells that were surgically isolated from Neu-initiated mammary tumors. Particle exclusion assay demonstrated visible HA pericellular matrix formation around the Has2⌬Neo tumor cells (Figure 1C). Elevated levels of Has2 expression were found in the mammary tumors of Has2⌬Neo mice, as determined by real-time quantitative PCR. The relative expression of Has2 transcripts was 16- to 21-fold higher in mammary

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Figure 2. Histopathological and immunohistochemical analyses of Neu-initiated mammary tumors. Tumor sections from the indicated genotypes were stained with hematoxylin and eosin. By detection using b-HABP, intense HA staining (red) was observed at the intercellular boundaries of tumor cells and particularly in the tumor stroma of Has2⌬Neo (arrows). HA-rich matrix was also prominent in the perivascular elastic structure of angiogenic microvessels (arrowheads). An analogous stromal staining was observed for versican (green) as a HA-bound matrix component (arrows). In contrast, few deposition of HA and versican was observed in Has2⫹Neo mice.

tumors of Has2⌬Neo mice than in Has2⫹Neo or Cre:Neu mice. The amount of HA in mammary tumors was consistently sixfold higher in Has2⌬Neo mice (354.7 ⫾ 88.7 ng/mg tumor) than in Has2⫹Neo animals (62.7 ⫾ 25.8) and eightfold higher than in Cre:Neu animals (40.8 ⫾ 24.7). All above examinations confirmed the successful generation of cTg mice, as characterized by hyperproduction of HA in the spontaneous mammary tumors.

Massive Stromal Reaction and Impaired Formation of Intercellular Junction in Has2Overexpressing Tumors Histologically, Has2⌬Neo tumors were poorly differentiated adenocarcinoma with numerous loosely cohesive tumor cells (Figure 2). Examination of the tumor cells at

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epithelial-mesenchymal transition (EMT),26 therefore supporting previous notions showing that HA overproduction induces EMT.27,28

Acceleration of Tumor Growth and Promotion of Cell Survival by Has2 Overexpression

Figure 3. Decreased organization of intercellular junctions in Has2-overexpressing mammary tumors. Tissue sections from Has2⫹Neo and Has2⌬Neo tumors were immunostained with antibodies against E-cadherin (green) and ␤-catenin (red), and the cell nucleus were stained with DAPI (blue). The immunolocalization was visualized by fluorescent and confocal microscopy. Staining of E-cadherin and ␤-catenin at intracellular boundaries was observed in Has2⫹Neo tumors (arrowheads).

higher magnification showed them to exhibit an abundant foamy to granular cytoplasm and nuclear pleomorphism. The most prominent histological feature of the Has2overexpressing tumors was increased formation of intratumoral stroma. In contrast, Has2⫹Neo tumors had the characteristics of ductal carcinoma with much less stroma. Distribution of HA was then examined by using biotinylated HABP (b-HABP), a specific probe for HA. HA staining was detected at the intercellular boundaries of tumor cells and particularly in the tumor stroma of Has2⌬Neo mice (Figure 2). Staining with b-HABP indicated that HA-rich matrix was also prominent in the perivascular elastic structure of angiogenic microvessels. Only a low level of HA deposition was detected in Has2⫹Neo mice. Consistent with a previous observation,24 an analogous stromal staining was observed for versican (also called PG-M25) as an HA-bound matrix component. Since histological observations of Has2⌬Neo mice revealed numerous loosely associated tumor cells, we investigated the influence of HA overproduction on cell-tocell junctions by immunohistochemical studies of E-cadherin and ␤-catenin. E-cadherin and ␤-catenin were localized to the perimembrane region, as illustrated by a control Has2⫹Neo tumor section (Figure 3). In contrast, staining of these molecules at intercellular boundaries was less intense in Has2⌬Neo mammary tumors (Figure 3), suggesting that hyperproduction of HA disrupts formation of adherence junctions. Interestingly, increased nuclear staining of ␤-catenin was also observed in the Has2⌬Neo mammary tumors. The loss of E-cadherin and nuclear translocation of ␤-catenin are hallmarks of

To investigate the differences in tumor appearance associated with Has2 expression, the early stages of tumor development were examined. Tumors were detected in Has2⌬Neo animals after an average of 14.4 ⫾ 0.5 days postcoitus compared with an average of 17.1 ⫾ 4.9 days for controls (Has2⫹Neo, Cre:Neu, and Neu). The incidence of mammary tumors was significantly greater in Has2⌬Neo mice than in control mice. The ratio of tumorbearing mice per total mice of each group was 77.8% (7/9 animals) in Has2⌬Neo, 13.3% (2/15 animals) in Has2⫹Neo, 40.0% (4/10 animals) in Cre:Neu, and 28.6% (2/7 animals) in Neu mice. This acceleration of carcinogenesis was associated with significantly faster growth of the tumors after appearance (Figure 4A). However, signs of any visible tumors were not detected in over 1 year in Has2:Cre mice lacking the neu proto-oncogene. Taken together, these data indicate that tumor growth is accelerated by increased HA production in tumor cells and strongly suggest that carcinogenesis is not simply affected by expression of Has2 and HA overproduction alone. Immunohistochemistry for PCNA, a well-characterized marker for cellular proliferation, revealed a slightly increased number of PCNA-positive cells in Has2⌬Neo tumors (Figure 4B). Because HA is known to influence phosphatidylinositol 3 (PI3)-kinase/Akt cell survival signals,16,29 we performed TUNEL staining to measure DNA strand breakage, a hallmark of apoptosis, in mammary tumors. TUNEL analysis demonstrated that Has2⌬Neo tumors underwent a fivefold lower rate of cell death compared with control tumors (Figure 4C). This lower level of TUNEL staining in the Has2⌬Neo tumors was inversely correlated with the increased tumor size of these animals. We next explored whether Has2 overexpression promotes the activation states of the PI3-kinase/Akt cell survival signals in the mammary tumors. Tissue sections from Has2⫹Neo and Has2⌬Neo tumors were immunostained with anti-phospho-Akt antibody, which recognizes Ser473-phosphorylated epitope of Akt. Serine phosphorylation of Akt was predominantly detected in the Has2⌬Neo tumor cells near the stroma-like structures surrounded by HA-rich matrix (Figure 5A). Similar phosphorylation patterns of ErbB2 were detected in the tumors (data not shown). When tissue lysates of mammary tumors were analyzed by Western blotting, significantly increased levels of phosphorylated Akt were detected in Has2⌬Neo tumors (Figure 5B). Based on our observations, the Akt signaling may partially account for the more rapid growth of Has2⌬Neo tumors and enhanced cell survival. Next, we examined in vitro cell growth to establish whether the difference in tumor growth could be attributed to tumor cell proliferation. However, the in vitro cell proliferation of Has2-expressing cells was not substan-

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Figure 4. Growth acceleration of Neu-initiated tumor by Has2 overexpression. A: Growth of mammary tumors in Has2 transgenic mice. After discovery of tumor, the length and width were measured daily with calipers until the tumors reached approximately 1 cm in diameter. The mean tumor volume (length ⫻ width2 )/2 is shown as a function of elapsed time after first detection of the tumors of seven Has2⌬Neo mice and eight controls (Has2⫹Neo, Cre:Neu, and Neu). Data are presented as mean ⫾ SE (*P ⬍ 0.05). B: The percentage of PCNA-positive nuclei was calculated with the formula [(number of PCNA⫹ nuclei)/(total nuclei) ⫻ 100]. n ⫽ 30 fields (five random fields of six tumors per genotype). The data point for each field is shown by an open circle. Closed circles and bars represent means ⫾ SE (**P ⬍ 0.01). C: The percentage of TUNEL-positive nuclei was calculated by using the formula [(number of TUNEL⫹ nuclei)/(total nuclei) ⫻ 100]. n ⫽ 15 fields (five random fields of three tumors per genotype). The data point for each field is shown by an open circle. Closed circles and bars represent means ⫾ SE (**P ⬍ 0.01).

tially different from those of control cells in both 10 and 2% fetal bovine serum (FBS) medium (data not shown). This result prompted us to investigate whether the observed tumor growth could be partly attributed to neovascularization, because rapid tumor growth is often associated with hypervascularization induced by several angiogenic factors secreted from tumor and stromal cells in the tumor milieu.

Has2 Expression Enhances Tumor Angiogenesis and Stromal Cell Recruitment To investigate whether the increased tumor development in Has2⌬Neo mice was due to accelerated neovascularization, microvessels in mammary tumors were counted after immunohistochemical staining with an antibody specific for CD31. Although neovascularization in the transgenic mice proceeded in an expected fashion with a ring of endothelial cells (Figure 6A), the size distribution of the microvessels was smaller than in control tumors. Further-

more, the number of tumor microvessels with a size of less than 1000 ␮m2 was twofold greater in Has2⌬Neo mice compared with tumors of control mice (Figure 6B). Realtime quantitative PCR analysis also showed that CD31 mRNA expression was increased in the mammary tumors of Has2⌬Neo mice (Figure 6C), indicating elevated neovascularization. VEGF-A is a well-characterized angiogenic factor, so the real-time quantitative PCR was then performed to determine the expression levels of VEGF-A and its transcriptional regulator, HIF-l␣. Unexpectedly, their mRNA levels were not significantly different among the transgenic mice. These data let us expect the involvement of another factor in HA-enhanced angiogenesis. Consistent with the histological observations, Has2⌬Neo tumors exhibited prominent stromal reactions as demonstrated by intense staining of type I collagen and fibronectin (Figure 6A). Transcriptional up-regulation of SMemb, which is a marker for immature mesenchymal cells, was also detected in the mammary tumors of the Has2⌬Neo mice (Figure 6C), suggesting the recruitment of many stromal cells in the tumors. Because stroma-derived growth factors and chemokines are known to promote an angiogenic response,6 we analyzed the transcriptional levels of bFGF and SDF-1␣/CXCL12. A near twofold increase in these mRNA levels was detected in the Has2 overexpressing tumors, which is consistent with hyperneovascularization. Together with the immunohistochemical observation of microvessel accumulation within or near the stromal structures of tumors, these results support the idea that tumor-derived HA matrix promotes tumor angiogenesis via induction of stroma-derived angiogenic factors.

HA Acceleration of bFGF-Induced Angiogenesis Genetic manipulation of Has2 expression in the mammary tumors strongly suggested that HA plays a crucial role in the recruitment of stromal cells and concomitant promotion of angiogenesis, which may be explained by the well-known fact that HA degradation products of a specific size induce an angiogenic response.30,31 Indeed, Has2⌬Neo tumors contained significant amounts of small HA species as assessed by gel filtration chromatography of tumor homogenates (Figure 7A). In contrast, HA derived from control Has2⫹Neo tumor was mainly eluted in the void volume equivalent to a molecular mass of ⬎150 kd. This supports the idea that HA oligosaccharides influence tumor-induced angiogenesis. To further strengthen this notion, we performed in vivo Matrigel plug angiogenesis assays where Matrigel was injected subcutaneously into the mice. In this assay, angiogenic bFGF strongly induced infiltration of endothelial cells and neovascularization in the Matrigel plugs at a concentration of 500 ng/ml. However, plugs with 200 ng/ml bFGF were visibly clearer, with fewer blood vessels migrating into the Matrigel (referred to as a less angiogenic condition). Under the less-angiogenic conditions, various HA species from different sources and with different molecular sizes were tested for their ability to promote bFGF-induced angiogenesis. A low concentration

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Figure 5. Activation of cell survival signals by HA overproduction. A: Phosphorylation of Akt in Has2-overexpressing tumor. Tissue sections from Has2⫹Neo and Has2⌬Neo tumors were immunostained with anti-phospho-Akt, which recognizes the Ser473-phosphorylated epitope of Akt. HA was counterstained with b-HABP (green). Phosphorylation (red) of Akt was predominantly detected in Has2⌬Neo tumor cells near the stroma-like structures (arrowhead) that are surrounded by HA-rich matrix. B: Tumor homogenates were analyzed by Western blotting using anti-Akt (non-phospho) and anti-phospho-Akt antibodies, and each band was quantified by densitometric imaging as described in Materials and Methods. Four independent tumors from each group were used for the comparison. Data represent the mean ⫾ SE (*P ⬍ 0.05 versus control).

of HA oligosaccharide (16 disaccharide units, average molecular mass of 6.8 kd) markedly promoted the formation of functional veins as judged by hemoglobin content within the excised Matrigel implants (Figure 7B). In contrast, high molecular mass HA (Mr 1000 kd from rooster combs or 757 kd from Streptococcus) did not promote angiogenesis. These data are consistent with the previous notion that only HA degradation products of a specific size induce angiogenic responses. Surprisingly, high molecular mass umbilical cord HA enhanced the angiogenic response in a dose-dependent manner (Figure 7B). The HA sample restored a similar angiogenic response even after elimination of contaminated HA oligosaccharides by gel filtration chromatography (data not shown), suggesting the involvement of high molecular mass components in the promotion of angiogenesis. Although the physiological significance of HA oligosaccharides in the promotion of angiogenesis is well established, the current findings give rise to the question as to whether HA-rich matrices have a positive role in angiogenesis as well.

Effect of HA-Versican Aggregate on Angiogenesis A previous study reported that umbilical cord arteries contain chondroitin sulfate (CS) proteoglycan species that are immunologically reactive to decorin and versican.32 Indeed, commercially available umbilical cord HA contains a small proportion of CS, as described in the manufacturer’s data sheet. To determine the proteoglycan composition of the umbilical cord HA, we performed dot blot analysis of samples using an antibody specific for human versican. Strong reactivity indicated that ver-

sican was enriched as a HA-binding proteoglycan during the preparation of umbilical cord HA (Figure 7B, inset). As demonstrated in this and other studies, increased immunostaining of versican is associated with vascular and perivascular HA matrices in tumors.33 Therefore, we expected versican to act as an angiogenic modulator via interaction with HA. Dot blot analysis of tumor extracts clearly demonstrated sixfold higher accumulation of versican in Has2⌬Neo tumors than in controls (Figure 7C). To confirm the presence of HA-versican aggregates, tumor extracts were fractionated by CsCl density gradient ultracentrifugation under associative conditions (0.4 mol/L guanidine hydrochloride) and the sedimentation patterns of versican and HA were examined. The significant proportion of versican was recovered in the A1 fraction together with HA. Because free HA is sedimented at a buoyant density of more than 1.55 g/ml (in fraction A6), the cosedimentation of versican and HA in the A1 fraction suggests that they form aggregates in Has2-overexpressing tumors. Our findings that versican accumulated into Has2overexpressing tumors next prompted us to investigate whether versican exerted cooperative effects with high molecular mass HA in terms of potentiation of bFGFinduced angiogenesis. Human versican was therefore partially purified from both umbilical cord HA and conditioned medium of cultured dermal fibroblasts and applied in Matrigel plug angiogenesis assays. When CD31 gene expression was used as an index of angiogenesis, both umbilical cord and dermal versicans were observed to promote the infiltration of endothelial cells (Figure 7D). A similar response was also induced by the administration of aggregates comprising high molecular mass HA (1000 kd) and versican. Because the Matrigel itself contains HA at a concentration of ⬃70 ng/ml, the enhancing effect of

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versican could have been exerted by the in situ formation of HA-versican aggregate within the implant. When the mixture of HA oligosaccharides and versican was tested for the angiogenic activity using Matrigel plug assays, a high dose of HA oligosaccharide resulted in the slight inhibition of this versican-enhanced angiogenesis (data not shown), suggesting that the oligosaccharide perturbed such versican accumulation.

HA-Versican Aggregate Promotes Stromal Cell Recruitment After Matrigel implantation, cells having the morphology of stromal fibroblasts infiltrated from the edge of the plug toward the center of the implant. Consistent with the increased infiltration of CD31-positive endothelial cells, they displayed a markedly enhanced infiltration within plugs containing HA-versican aggregates compared with the control plugs (Figure 8, A and B). The majority of the infiltrating cells had the characteristics of myofibroblasts because they were stained with both ␣-SMA and vimentin antibodies (Figure 8C), and ␣-SMA-positive cells were virtually always surrounded by a HA-rich matrix, strongly suggesting that they are responsible for the HA synthesis. Interestingly, as detected by 2B1 antibody specific for human versican, exogenous human versican was predominantly incorporated and accumulated into the HA matrix surrounding the stromal cells (Figure 8C), suggesting that versican exerts cooperative effects by aggregating with HA in terms of the stromal cell recruitment.

Discussion The presence of stromal compartment in the most invasive breast cancers is well documented by extensive clinical evidence.34,35 In this study, we show that the stroma within Neu-initiated breast cancers are strongly induced by the assembly of HA-rich ECM, which in turn accelerates tumor angiogenesis. To our knowledge, this is the first reported demonstration that the accumulation of an extracellular structural constituent can elicit intratumoral stroma. Several hypotheses have emerged regarding the origin of intratumoral fibroblasts. Whereas some reports demonstrate they originate from local recruitment of fibroblasts in the surrounding connective tissues, others argue that mesenchymal stem cells (MSCs) are proFigure 6. HA-induced recruitment of endothelial and stromal cells. A: CD31 immunostaining of tumor sections showing the vascular density in tumors. Tissue sections from Has2⫹Neo and Has2⌬Neo tumors were stained with a rat antibody against murine CD31. Tumor microvessels of smaller size were more numerous in the Has2⌬Neo mice compared with Has2⫹Neo tumors. Stromal reaction was demonstrated by immunostaining of type I collagen (Col I) and fibronectin (FN). B: Microvascular density in tumor sections from Has2⌬Neo and control mice. The graph represents average number and the size distribution of the microvessels in five random (⫻200) fields of three tumors per genotype, showing that tumors had a significant increase in the number of microvessel with less than 1000 ␮m2. Data represent the mean ⫾ SE (*P ⬍ 0.05 versus control). C: Relative mRNA expression of angiogenesisrelated genes in mammary tumor. Total RNA from Has2⌬Neo and control tumors was transcribed into cDNA, and the transcriptional levels were analyzed by real-time quantitative PCR. All values are normalized to GAPDH mRNA. Data represent the mean ⫾ SE from more than three different tumors of each group (*P ⬍ 0.05, **P ⬍ 0.01 versus control).

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Figure 7. Effects of various HA samples on vascularization of Matrigel implants. A: Gel filtration chromatography of HA samples isolated from tumor tissues of Has2⌬Neo and Has2⫹Neo mice. HA-containing tumor homogenates were prepared as described in Materials and Methods and then separated by gel filtration chromatography using a Superose 6 HR 10/30 column. HA content was determined in each fraction from the column using the IBA method. Representative elution pattern is shown. B: The degree of angiogenesis in Matrigel plugs containing various HA samples. The Matrigel plug model of in vivo angiogenesis was conducted in C57BL/6 mice with the indicated treatments. The indicated concentration of HA oligosaccharides (Mr 6.8 kd, white column), rooster comb native HA (gray column), Streptococcus HA (hatched column), or human umbilical cord HA (black column) was mixed with the Matrigel and subcutaneously injected into the abdomen of mice. Matrigel plugs were removed 7 days after implantation, and the hemoglobin (Hb) concentration was quantified as described in Materials and Methods. Data represent the mean of two independent experiments. Dot blot detection of human versican in the umbilical cord HA sample was performed using 2B1 antibody specific for human versican (inset). C: Dot blot detection of HA and versican in mammary tumor extract (T) or in fractions (A1–A6) of CsCl density gradient ultracentrifugation. Tumor extracts from three different genotypic mice were fractionated into six fractions by CsCl density gradient ultracentrifugation under associative condition (0.4 mol/L guanidine hydrochloride) and examined for the sedimentation patterns of HA and versican. Significant amounts of HA and versican were recovered in the A1 fraction with lowest buoyant density. D: The degree of angiogenesis in Matrigel plugs containing rooster comb native HA and/or versican. Total RNA samples were isolated from Matrigel plugs supplemented with or without umbilical cord versican (V) and dermal versican (dV). The expression levels of CD31 mRNA were assessed as an index of angiogenesis by real-time quantitative PCR. The mRNA levels were normalized to GAPDH mRNA. Data represent the average from more than five different implants. Data represent the mean ⫾ SE from more than five different implants (*P ⬍ 0.05 versus PBS control).

genitors for tumor-associated fibroblasts.7 In fact, HA has been suggested to mediate the recruitment of MSCs into the sites of wounds and fibrosis.36 A yet third hypothesis favors EMT in the local formation of intratumoral fibroblasts from mammary epithelium,7 and recent studies have revealed that HA plays a central role in the EMT,27,28 implying that HA-mediated stromal reactions therefore may progress as consequences of complex

biological responses that include EMT and intratumoral recruitment of pre-existent fibroblasts and MSCs. Two hallmarks of EMT are loss of E-cadherin expression and nuclear translocation of ␤-catenin.26 The fact that HA overproduction resulted in the suppression of E-cadherin expression and nuclear translocation of ␤-catenin provides significant evidence that endogenous HA promotes EMT. Receptor tyrosine kinases (RTKs),

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which induce the activation of downstream Ras/mitogenactivated protein kinase and PI3-kinase/Akt signaling pathways, govern EMT in cooperation with transforming growth factor ␤ (TGF␤) signaling.26 Here, we demonstrated that constitutive activation of Akt survival signaling was consistent with increased cell survival in HAoverproducing tumors. All these findings support previous reports showing that HA overproduction induces EMT. Because EMT in breast cancer can provide a nonmalignant stroma,37 carcinoma cells that have undergone EMT may partly participate in the formation of intratumoral stroma in HA-overproducing tumors. One of the main cellular components in intratumoral stroma is myofibroblasts.4 Sometimes termed activated fibroblasts, these cells are capable of modulating the tumor microenvironment during invasion and angiogenesis. A specific contribution of these cells to the stromal microenvironment is the supply of angiogenic growth factors.5,6 Here, we demonstrated that stroma-derived angiogenic factors such as bFGF and SDF-1␣/CXCL12 were transcriptionally up-regulated in Has2-overexpressing tumors. The accumulation of microvessels within or near the stromal structures of tumors supports the idea that tumor-derived HA matrix promotes tumor angiogenesis via induction of stroma-derived angiogenic factors. The present study also demonstrates that extracellular accumulation of HA in mammary carcinomas accelerates the intratumoral infiltration of such host cells. Because of the unique physicochemical properties of HA, the elevation of HA concentration in local tissue might cause increased turgidity and hydration, which in turn facilitates cell migration. The disruption of cell-to-cell junctions in Has2-overexpressing tumors may also imply that HA matrix provides microenvironments amenable to easy penetration by fibroblasts and endothelial cells. Furthermore, HA seems to promote cell motility by acting on intracellular signaling pathways through interaction with cell surface receptors.9 On the other hand, because tumor cells often exhibit elevated levels of hyaluronidase activity, HA oligosaccharides might be responsible for the enhanced intratumoral infiltration of host cells. Indeed, the Matrigel plug assay confirms the positive effect of HA oligosaccharides on bFGF-induced angiogenesis. HA-rich ECM therefore may serve a crucial role in tumor angiogenesis by providing a suitable microenvironment for cell growth and migration and by acting as a functional pool for angiogenic HA oligosaccharides, which can be generated by hyaluronidase. For a long time, it has been believed that HA oligosaccharides were angiogenic whereas high molecular mass forms were antiangiogenic. Currently, the mechanism for the antiangiogenic effect of high molecular mass HA is unknown, but our observation that its function was oppositely modulated Figure 8. Infiltration of stroma cells into Matrigel plugs supplemented with or without native HA-versican aggregates (HA/V). A: Matrigel plugs were analyzed by hematoxylin and eosin staining or by immunostaining of CD31 of paraffin sections. Scale bars ⫽ 100 ␮m. B: Sections from the center of each embedded Matrigel plugs (three animals per group) were quantified for cellular invasion. Data represent the means ⫾ SE (**P ⬍ 0.01). C: The sections of Matrigel plugs were immunostained with anti-human versican (2B1), ␣-SMA, or vimentin antibody. All specimens were counterstained with b-HABP. Scale bar ⫽ 20 ␮m.

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with versican at least in part accounts for the HA-mediated enhancement of tumor angiogenesis. Because CD44 has been implicated in proliferation, migration, and gene expression of endothelial cells as a major receptor for HA,38 – 40 the question arose as to whether CD44 contributes to the promotion of angiogenesis by HAversican aggregates. Recently, Cao et al41 demonstrated the involvement of endothelial CD44 during in vivo angiogenesis. We therefore used the Matrigel plug assay to evaluate in vivo angiogenesis in CD44-null mice and found that the angiogenic response to HA-versican aggregates persisted even in the absence of CD44 (data not shown). This discrepancy may be attributed to differences in mechanisms between angiogenic responses induced by HA oligosaccharides and HA-versican aggregates. This also suggests either the involvement of other HA receptors or an alternative receptor-independent mechanism for aggregate-induced angiogenesis; instead of through HA receptors, HA synthase can retain growing HA at cell surface, because newly synthesized HA is thought to be extruded through enzymes on the plasma membrane.42 This cell surface HA not only activates intracellular signals through receptors but also functions to localize other bioactive molecules as a constituent of ECM. Our current observations indicate that exogenous versican is predominantly incorporated and accumulated into the HA matrix surrounding stromal cells. Thus, HA produced endogenously by tumor and stromal cells may support compartmentalization of versican on the cell surface. Our data suggest that versican acts as a key player in HA-mediated angiogenesis by enhancing recruitment of host stromal cells. Versican has been reported to be highly expressed in tissue compartments undergoing active cell proliferation and migration under physiological and pathological conditions43 and is abundantly produced by vascular smooth muscle cells and/or myofibroblasts in atherosclerotic lesions where the cells actively proliferate in an autocrine and paracrine manner.44,45 Increased immunostaining of versican is often associated with vascular and perivascular elastic structures in malignant tumors.33 Because of the strong coincidence of HA and versican expression, versican is thought to modulate HA function and vice versa; blocking the formation of the HA pericellular coat, either by using antibodies to the HA receptors or by using short HA oligosaccharides, prevents cell proliferation and migration.16,27,45,46 Thus, it would seem that HA-versicanrich ECM allows cells to prepare for proliferation and migration. Versican may promote cell proliferation and migration by enhancing cell detachment from ECM or by participating in the assembly of intracellular machinery and transmitting signals in concert with HA.47,48 Although the requirement of HA for the role of versican is not fully resolved, the above results suggest that versican acts as an angiogenic modulator via interaction with HA. Genetic manipulation of HA biosynthesis has led to a better understanding of the role of HA in tumorigenesis and progression.11–15 The Has2 cTg mice presented here demonstrated the important role of HA in stimulating the early event of tumor formation. In our previous work, an abnormal production and HA accumulation by forced expression of HA synthases led to a diminution of contact

growth inhibition in a nontransformed cells.49 The induction of uncontrolled cell growth by abnormal HA production may therefore account for the higher incidence of mammary tumors observed in Has2⌬Neo mice. Without the neu oncogene, however, Has2 cTg did not develop any visible tumors over a period of 1 year (data not shown). Thus, overproduced HA is likely to enhance synergistically tumor development initiated by other oncogenic alterations. The occurrence of EMT during tumor progression allows tumor cells to acquire the capacity to infiltrate surrounding tissue and to metastasize ultimately to distant sites.50 Although our data suggested that HA overproduction elicited mesenchymal transition of mammary carcinoma cells, no obvious sign of invasion or metastasis was found in the Has2-overexpressing mice, suggesting that additional genetic alterations are required for the sufficient promotion of tumor malignancy. Detailed pathological examination and prolonged follow-up studies are needed to clarify this issue. Our overall understanding of the mechanism underlying tumor stromal reactions has, until now, been limited by a paucity of in vivo experimental data. In this experiment, however, Has2 transgenic tumors grew with formation of abundant stroma compared with those elicited by xenografting Has-overexpressing tumor cell lines. Thus, the transgenic mouse model provided the opportunity to investigate the HA-dependent stromal reactions rather than investigating tumor cell lines. Furthermore, this mouse model may also prove useful for the preclinical study of anti-cancer drugs targeting the host stromal reaction and tumor angiogenesis.

Acknowledgments We thank Dr. Yumi Kanegae and Dr. Izumu Saito (University of Tokyo) for providing pCALNL5 expression vector, Dr. Jun-ichi Miyazaki (Osaka University) for CAG promoter, and Seikagaku Corp. (Tokyo, Japan) for HA oligosaccharides.

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