Glioma-Derived Platelet-Derived Growth Factor-BB Recruits Oligodendrocyte Progenitor Cells via Platelet-Derived Growth Factor Receptor-α and Remodels Cancer Stroma

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SHORT COMMUNICATION Glioma-Derived Platelet-Derived Growth Factor-BB Recruits Oligodendrocyte Progenitor Cells via Platelet-Derived Growth Factor Receptor-a and Remodels Cancer Stroma Q15

Yang Zheng,* Seiji Yamamoto,* Yoko Ishii,* Yang Sang,* Takeru Hamashima,* Nguyen Van De,* Hirofumi Nishizono,y Ran Inoue,z Hisashi Mori,z and Masakiyo Sasahara* From the Departments of Pathology* and Molecular Neuroscience,z Graduate School of Medicine and Pharmaceutical Sciences, and the Division of Animal Experimental Laboratory,y Life Science Research Center, University of Toyama, Toyama, Japan Accepted for publication December 21, 2015. Address correspondence to Yoko Ishii, M.D., Ph.D., Department of Pathology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0152, Japan. E-mail: [email protected].

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Glioma is an aggressive and incurable disease, and is frequently accompanied by augmented platelet-derived growth factor (PDGF) signaling. Overexpression of PDGF-B ligand characterizes a specific subclass of glioblastoma multiforme, but the significance of the ligand remains to be elucidated. For this end, we implanted a glioma cell line transfected with PDGF-BBeoverexpressing vector (GL261-PDGF-BB) or control vector (GL261-vector) into wild-type mouse brain, and examined the effect of glioma-derived PDGF on the tumor microenvironment. The volume of GL261-PDGFBB rapidly increased compared with GL261-vector. Recruitment of many PDGF receptor (PDGFR)-a and Olig2-positive oligodendrocyte precursor cells and frequent hemorrhages were observed in GL261-PDGF-BB but not in GL261-vector glioma. We then implanted GL261-PDGF-BB into the mouse brain with and without Pdgfra gene inactivation, corresponding to PDGFRa-knockout (KO) and Flox mice, respectively. The recruitment of oligodendrocyte precursor cells was largely suppressed in PDGFRa-KO than in Flox mice, whereas the volume of GL261-PDGF-BB was comparable between the two genotypes. Frequent hemorrhage and increased IgG leakage were associated with aberrant vascular structures within the area where many recruited oligodendrocyte precursor cells accumulated in Flox mice. In contrast, these vascular phenotypes were largely normalized in PDGFRa-KO mice. Increased matrix metalloproteinase-9 in recruited oligodendrocyte precursor cells and decreased claudin-5 in vasculature may underlie the vascular abnormality. Glioma-derived PDGF-B signal induces cancer stroma characteristically seen in high-grade glioma, and should be therapeutically targeted to improve cancer microenvironment. (Am J Pathol 2016, -: 1e11; http:// dx.doi.org/10.1016/j.ajpath.2015.12.020)

Glioblastoma multiforme (GBM), World Health Organization grade IV astrocytoma, is the most common malignant brain tumor of adults. GBM is characterized by highly invasive features and prominent vascular involvement.1 In addition to the aggressive growth of tumor cells, the associating secondary events, like intracranial hemorrhage, are critical prognostic factors as well.2 Recently, tumor cells per se as well as microenvironmentddefined by extracellular matrix and tumor stromal cells, including vascular endothelial cells and glial cellsdgained attention as a therapeutic target.3,4 Typically, a

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treatment targeting tumor angiogenesis using an antibody against vascular endothelial growth factor, bevacizumab, is used at tumor recurrence, and achieves significant improvement of the disease.5 Furthermore, the current efforts are developing novel and potent agents that target multiple angiogenic pathways, including platelet-derived growth factor (PDGF) and Supported by Ministry of Education, Culture, Sports, Science, and Technology grants-in-aid for scientific research 25293093 (M.S.) and 15K08396 (Y.I.). Q1 Disclosures: None declared.

Copyright ª 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2015.12.020

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Zheng et al fibroblast growth factor, for the treatment of advanced solid tumors.6 PDGF family members include PDGF-A, PDGF-B, PDGF-C, and PDGF-D, which are assembled as disulfidelinked homodimers or heterodimers. The PDGF signal is mediated by two types of PDGF receptors, PDGF receptor a (PDGFRa) and PDGFRb.7 PDGF ligands and receptors were expressed in the brain; among them, PDGFRa is most abundantly expressed in oligodendrocyte progenitor cells (OPCs), and is implicated in the regulation of the proliferation, migration, and differentiation of OPCs in developing brain.8 Furthermore, in the recent studies, PDGFRa signaling is well documented in the disruption of the blood-brain barrier in different pathological conditions.9,10 PDGF autocrine mechanisms are assumed to be most important in glioma,11 and proteomic analyses demonstrated that the overexpression of PDGF-B with increased PDGFRb phosphorylation characterized a specific subclass of GBM that comprises nearly 30% of human GBM.12 Because PDGF-B activates PDGFR-a and PDGFR-b and is a chemoattractant of glial and mesenchymal cells,13 PDGF-B overexpressed in GBM may chemoattract a range of intermingling nonneoplastic parenchymal cells that were characteristically seen in high-grade brain tumors.14 Along this line, many PDGFRapositive OPC-like cells were recruited toward PDGFBeinduced rodent glioma.15 However, the mechanism of GBM-derived PDGF-B to induce cancer stroma, and the functional implication of tumor microenvironment defined by cancer stroma, remains to be elucidated for the establishment of a novel therapeutic target of GBM. In the present study, we hypothesized that GBM-derived PDGF-B induces tumor microenvironment and may contribute to the aggressive behavior of GBM. For this end, we developed a PDGF-BBeoverexpressing glioma cell line (GL261-PDGF-BB) and implanted them into the mouse brain. Furthermore, the same transplantation was conducted after the inactivation of host PDGFRa signaling by conditionally induced Pdgfra gene inactivation. Paracrine PDGF signal from glioma cells was shown to recruit many OPCs, and the recruited OPCs were implicated in the regulation of vascular leakage and hyperplasia with remodeling of extracellular matrix.

Materials and Methods All experimental animal procedures were approved by the Committee for Institutional Animal Care and Use at the University of Toyama (Toyama, Japan). All of our study protocols were approved by the Ethics Committee of the University of Toyama.

The Generation of Pdgfra Conditionally Inactive Mice Mutant mouse harboring a genetically mutated Pdgfra gene, in which exons 4 and 5 of Pdgfra were flanked by

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two loxP sequences (Pdgfraflox/flox), was generated as follows. Briefly, a BAC genomic clone (RP24-148N4), Q4 originating from the DNA of a C57BL/6 mouse and containing Pdgfra, was obtained from the BACPAC Resource Center CHORI. The constructed targeting vector included a DNA fragment containing a loxP sequence and pgk-Neo cassette flanked by two Flp recognition target (frt) sites16 and pMC1DTABGHA with a bovine growth hormoneederived polyadenylation signal sequence.17 The embryonic stem cell line RENKA, derived from the C57BL/6N strain,18 was used and the resulting targeting vector was electroporated, as described previously.19 The obtained male chimeric mouse was crossed with a female CAG-FLPe deleter mouse20 to remove the pgk-Neo selection cassette and obtain the heterozygous Pdgfraflox/þ strain. Genotyping was performed by PCR using the following primers: 50 -ATGCCAAACTCTGCCT- Q5 GATTGA-30 and 50 -CTCACGGAACCCCCACAAC-30 . Flox mice were crossbred with chicken b-actin-promoter/CMVenhanceredriven Cre-transgenic mice (CAGG-CreERþ/; The Jackson Laboratory, Bar Harbor, ME; CreER mouse)21 that harbor the fusion gene Cre recombinase and estrogen receptor (CreER). The resulting offspring were mice harboring CAGGCreERþ//Pdgfraflox/flox (CreER-Flox mice) or Pdgfraflox/flox (Flox mice). Mice with a systemic Pdgfra inactivation [PDGFRa-knockout (KO) mice] were obtained by tamoxifen (Sigma-Aldrich, St. Louis, MO) administration to CreER-Flox mice. Flox mice were treated identically and used as controls.

Generation of PDGF-BB Overexpression Glioma Cell Line To monitor the cells expressing the mouse PDGF-BB, we introduced IRES-EGFP DNA fragment from pIRES2EGFP (Clontech, Mountain View, CA) after the coding region of mouse Pdgfb expression plasmid pBLAST49mPDGFB (full) v24 (InvivoGen, San Diego, CA). The 0.12-Kbp DNA fragment containing mouse Pdgfb was amplified with primers PIE-U1 (50 -GGGGAATTCGGTGACCATTCGGACGGTGAG-30 ) and PIE-L1 (50 -GGGGGATCCCTAGGCTCCGAGGGTCTCC-30 ) using pBLAST49-mPDGFB (full) v24 as a template. The amplified DNA fragment was subcloned into the EcoRV site of the pLITMUS28 (New England Biolabs, Herts, UK) to yield the plasmid pLITPIE. The 0.12-Kbp EcoRI-BglII DNA fragment from pLITPIE was cloned between EcoRI and BamHI sites of the pIRES2-EGFP to yield the plasmid pIRES-PIE. The 1.4-kb BstEII-NotI (blunt) fragment from pIRES-PIE was ligated with the 3.6-kb BstEII-NheI (blunt) DNA fragment from pBLAST49-mPDGFB (full) v24 to yield the plasmid pPIE. Plasmid pPIE and empty vector were transfected to the GL261 glioma cell line (kindly provided by Dr. Atsushi Natsume, Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya, Japan) by electroporation (NEPA21 Electroporator; Nepa Gene, Chiba, Japan), according to the manufacturer’s procedure. Transfected cells were single cloned, and passaged several times to obtain

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Cancer Stroma by PDGF Signal in Glioma stable expression clones. Resulting PDGF-BB overexpression and vector cell lines were termed GL261-PDGF-BB and GL261-vector, respectively.

Immunoblotting

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Sample preparations and all other procedures for Western blot analysis have been described elsewhere.22 Briefly, cultured cells were lysed using radioimmunoprecipitation assay buffer (Thermo Fisher, Waltham, MA) on ice. Whole cell lysates were separated by SDS-PAGE and then electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 hour at room temperature in a 5% milk containing 50 mmol/L Tris buffer with 150 mmol/L NaCl and 0.05% Tween 20. The membranes were then probed with rabbit anti-PDGFRa (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDGFRb (1:500; Merck Millipore, Billerica, MA), and mouse antieb-actin (1:20,000; Sigma-Aldrich, St. Louis, MO) antibodies. The membranes were washed in a 50 mmol/L Tris buffer containing 150 mmol/L NaCl and 0.05% Tween 20, and were incubated with the appropriate horseradish peroxidaseeconjugated secondary antibodies. Immunoreactive bands were detected using enhanced chemiluminescence reagents (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), according to the manufacturer’s instructions.

RNA Extraction, cDNA Synthesis, and Real-Time PCR

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Sample preparations and all other procedures for real-time PCR have been described elsewhere.23 Briefly, total RNA was isolated from cultured cells using miRNA easy Mini Kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. Total RNA (150 mg) was reverse transcribed, as detailed in the PrimeScript RT reagent kit protocol (Takara Bio Inc., Shiga, Japan). For real-time PCR performed with a Takara Thermal Cycler Dice Real Time System TP800 (Takara Bio Inc.), cDNAs were diluted 1:25 in the reaction mixture consisting of SYBR Premix EX Taq II (Takara Bio Inc.). The real-time PCR program consisted of hot start enzyme activation at 95 C for 10 seconds and 45 cycles of amplification at 95 C for 10 seconds and 60 C for 40 seconds. Finally, to obtain the dissociation curve, a final cycle was performed at 95 C for 1 minute, 60 C for 30 seconds, and then 95 C for 10 seconds. Primers were as follows: Pdgfb, 50 -TGCTGAGCGACCACTCCATC-30 and 50 -CTCGGGTCATGTTCAAGTCCA-30 ; Pdgfra, 50 -AGCAAACATCTTGACTTGGGAACA-30 and 50 -ACTTGCATCATTCCCGGACAC-30 ; Pdgfrb, 50 -GAACGACCATGGCGATGAGA-30 and 50 -GCATCGGATAAGCCTCGAACA-30 ; and Actb, 50 -CATCCGTAAAGACCTCTATGCCAAC-30 and 50 -ATGGAGCCACCGATCCACA-30 . For data analysis, mouse b-actin (Actb) housekeeping gene was used as an internal control. Induction values were calculated using analysis software (Takara Bio Inc.).

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Colony Formation Assay GL261-PDGF-BB cells and GL261-vector cells were cultured in a soft agarebased three-dimensional culture system, CytoSelect 96-Well in Vitro Tumor Sensitivity Assay (Cell Biolabs, San Diego, CA). Tumor cells were seeded at 5  103 cells per well in the 96-well plate, and were photographed under the conventional microscopy on day 1 after starting of culture. Six days later, tumor cellederived colonies were microphotographed, and the growth rate was measured by OD 570 nm after MTT reagent reaction, according to the manufacturer’s procedure.

Tumor Implantation and Tamoxifen Administration Tamoxifen (Sigma-Aldrich) was orally administered once a day (225 mg/kg body weight) for 5 consecutive days, starting on 2 days before tumor implantation. Eight-monthold male mice were used for the experiments. All surgical procedures were conducted under anesthesia with sodium pentobarbital (i.p. injection, 100 mg$kg1). Using a small Q8 dental drill, a hole was made in the skull at the correct stereotaxic coordinates. After this, the dura matter was removed using small curved forceps, and exposure of the meninges was confirmed by observation of a small amount of cerebrospinal fluid at the exposed position. A needle (internal diameter, 0.13 mm) was set in the stereotaxic device (David Kopf Instruments, Tujunga, CA), connected to a Hamilton microsyringe via polyethylene tubes filled with phosphate-buffered saline, and set on a microsyringe pump (CMA 400; Harvard Apparatus, Holliston, MA). The needle was inserted to the targeted location in the striatum at 1.0, 2.0, and 3.0 mm at anteroposterior relative to bregma, mediolateral, and dorsoventral from surface of the brain, respectively. Tumor cell suspension (1 mL; 1.0  105 cells$mL1) was infused at a flow rate of 0.1 mL$minute1. After the infusion, the needle was retained at the same position for 10 minutes to ensure no backflow. Under deep anesthesia with sodium pentobarbital (i.p. injection, 50 mg/kg body weight; Dainippon Sumitomo, Osaka, Japan), mice were transcardially perfused with phosphate-buffered saline, followed by perfusion and immersion in 4% paraformaldehyde at day 20 after tumor implantation.

Immunohistochemistry and Immunofluorescence Staining of Paraffin-Embedded Sections For paraffin section preparation, brains were immediately fixed with 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4), dehydrated with a graded series of ethanol solutions, and embedded in paraffin. The sections (5 mm thick) were subjected to either hematoxylin and eosin staining or immunostaining. Immunohistochemistry and immunofluorescence were performed, as described previously.24,25 Briefly, the sections were incubated at 4 C overnight with the following primary

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Figure 1 In vitro characterization of the platelet-derived growth factor (PDGF)-BB overexpression glioma cell lines. AeC: Real-time PCR analyses for the Pdgfb and Pdgfrs in glioma cell lines transfected with PDGF-BBeoverexpressing vector (GL261-PDGF-BB) and with control vector (GL261-vector). Four clones were examined from GL261-PDGF-BB and Gl261-vector, respectively. A: Pdgfb mRNA expression is much higher in GL261-PDGF-BB clones than in GL261-vector clones. B: Pdgf receptor a (Pdgfra) mRNA expression is lower in Gl26L-PDGF-BB than in GL261vector. C: Pdgfrb mRNA expression is comparable in all clones, except for one GL261-vector clone. D: Western blot analysis shows that PDGFRa is abundant in GL261-vector than in GL261-PDGFBB. PDGFRb was undetected in all examined cell lines. The lysates from cultured mouse skin fibroblasts were used as positive controls. Equal sample loading was confirmed by b-actin. E: Representative photographs of GL261-vector and GL261PDGF-BB cell lines in colony formation assay. F: Colony formation assay of four clones of GL261PDGF-BB and Gl261-vector. n Z 6 from each clone (AeC). Scale bar Z 200 mm (E). AU, arbitrary unit.

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antibodies: goat polyclonal anti-PDGFRa (1:1000; Neuromics, Edina, MN), goat polyclonal anti-PDGFRb antibody (1:100; R&D, Minneapolis, MN), rabbit polyclonal anti-Olig2 (1:500; IBL, Hamburg, Germany), goat polyclonal anti-Sox10 (1:100; Santa Cruz Biotechnology), rat monoclonal anti-CD31 (1:100; Dianova, Hamburg, Germany), rabbit polyclonal anti-collagen type IV (1:500; Millipore, Darmstadt, Germany), rabbit polyclonal antiematrix metalloproteinase (MMP)-9 (1:100; Abcam, Cambridge, UK), and rabbit polyclonal antieclaudin-5 (1:100; Invitrogen, Carlsbad, CA). Colorimetric immunostaining was conducted using the appropriate Histofine Simple Stain Mice System (Nichirei Biosciences, Tokyo, Japan) and 3,30 diaminobenzidine tetrahydrochloride (Dako, Glostrup, Denmark) reaction. The leakage of serum protein was detected by immunohistochemistry for IgG using an anti-IgG detection system from Histofine Simple Stain Mice System (Nichirei Biosciences), as per the supplier’s protocol. For the fluorometric immunostaining, the positive immunoreactions were visualized using the following fluorescent dyeeconjugated secondary antibodies that were applied at room temperature for 3 hours: donkey anti-goat IgG conjugated with Alexa Fluor 488, donkey anti-rabbit IgG conjugated with Alexa Fluor 488, 594, or 633,

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and donkey anti-rat IgG conjugated with Alexa Fluor 594. These secondary antibodies were used at 1:500 dilutions and were obtained from Life Technologies Corporation (Carlsbad, CA). Images were captured by a microscopy system (BX 51; Olympus, Tokyo, Japan) connected to a digital camera (DP70; Olympus) and TCS SP5 confocal system (Leica, Heidelberg, Germany), and were processed using Photoshop software version 7.0 (Adobe, San Jose, CA).

Zymography Taken out brains under deep anesthesia were immediately Q9 snap frozen into liquid nitrogen. Frozen brains were serially cut with 30 mm intervals in the coronal plane to obtain a representative cut surface of the tumor using a Leica CM3000 cryostat (Leica Microsystems, Wetzlar, Germany). Then, the surrounding nonneoplastic tissues were manually trimmed off the frozen brain; at this step, we saved OPC-rich area in tumor side. Then, six of the frozen sections (30 mm thick) were cut from each mouse, and lysed in the sample buffer of Gelatin-Zymography Kit (Cosmo Bio, Tokyo, Japan). Protein concentration was measured by OD 280 nm

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Figure 2 Histological characterization of platelet-derived growth factor (PDGF)-BBeoverexpressing glioma and control glioma after implantation in the brain of wild-type mouse. A and B: Hematoxylin and eosin (H&E) staining (A) and immunohistochemistry (B) for Olig2 of wild-type mouse brain at day 20 after the implantation of GL261-PDGF-BB and GL261-vector. C: Tumor volume (mm3) of GL261-PDGF-BB and GL261-vector at day 20 after implantation. D: Numbers of Olig2-positive cells per field in the marginal region of tumor. E: Immunohistochemistry for PDGF receptor a (PDGFRa) in the marginal region of tumors of GL261-PDGF-BB and GL261-vector. The images are from adjacent sections of boxed area in A and B. F: Percentage of PDGFRa-positive area per field in the marginal region of tumor of GL261-PDGF-BB and GL261vector. G: Numbers of extravasated red blood cells (RBCs) along the entire circle of tumor of GL261-PDGF-BB and GL261-vector. Counted numbers of RBCs were divided by the circumferential distance of each tumor for the comparison. H: Higher magnification of boxed area of H&E staining in the marginal region of tumors of GL261-PDGF-BB and GL261-vector. Dotted lines in E and H indicate the edge of tumor. Error bars represent SEM (C, D, F, and G). n Z 16 from four clones (C, GL261-PDGF-BB); n Z 14 from four clones (C, GL261-vector); n Z 8, two fields from four mice (D and F); n Z 4 mice (G). *P < 0.05, ***P < 0.001. Scale bar: 500 mm (A); 100 mm (E and H).

for ensuring equal protein volume loading. Samples were sonicated to reduce viscosity, and electrophoresis and complete blood cell count stainings were performed according to the manufacturer’s procedure. Translucent MMP enzymatic

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reaction bans were measured by ImageJ software (NIH, Q10 Bethesda, MD; http://imagej.nih.gov/ij), and normalized to the major protein bands of complete blood cell count staining.

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Cancer Stroma by PDGF Signal in Glioma 807 808 809 Images were obtained by a microscopy system (BX 51; 810 Olympus) connected to a digital camera (DP70; Olympus). PDGF-BBeOverexpressing Glioma Induces Massive 811 Morphological and morphometric analyses were performed Recruitment of OPCs and Exacerbates Glioma-Related 812 using a BioRevo BZ-9000 microscope (Keyence, Osaka, Hemorrhage 813 Q11 Japan), BZ-II Analyzer software (Keyence), and ImageJ soft814 ware. The tumor volume (mm3) was estimated on the basis of First, we implanted four clones of glioma cells from lines 815 the tissue section prepared from largest cut surface of each with and without PDGF-BB overexpression into the 816 817 tumor using the following equation: volume Z 4/3  p  striatum of wild-type mouse. The tumor volumes after 818 long axis  short axis2.26 transplantation were significantly larger in GL261-PDGF-BB than in GL261-vector (Figure 2, AeC). Olig2 is a marker ½F2 819 820 of oligodendrocyte lineage, in which lineage, PDGFRa821 Statistical Analysis 8 positive cells correspond to OPCs. At higher magnifi822 cation, we found that many Olig2 and PDGFRa-positive 823 Statistical significance was determined using a t-test. P < 0.05 OPCs recruited and surrounded the tumor mass in all of 824 was considered statistically significant. Quantified data are GL261-PDGF-BB glioma, but none of GL261-vector 825 presented as means  SEM. Graphs were drawn using glioma (Figure 2, A, B, and DeF). Furthermore, we 826 GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA). found frequent hemorrhages around the tumor mass of 827 GL261-PDGF-BB, but not GL261-vector (Figure 2H). 828 829 The hemorrhagic foci were closely distributed to the Results 830 recruited OPCs. The number of extravasated red blood 831 cells around tumor was substantially larger in GL261Characterization of PDGF-BBeOverexpressing Glioma 832 PDGF-BB than in GL261-vector (Figure 2G). Cell Line 833 834 Glioma cell lines transfected with either PDGF-BBe Pdgfra Inactivation Suppresses OPC Recruitment, 835 overexpressing vector (GL261-PDGF-BB) or control vector Hemorrhage, and Vascular Permeability 836 (GL261-vector) were characterized in vitro before using in 837 transplantation into mouse brain. In real-time PCR analyses, To understand the relevance of PDGFRa signaling in the 838 four of the GL261-PDGF-BB cell lines expressed higher levels induction of cancer stroma, we next implanted GL261839 of Pdgfb mRNA compared with four of the GL261-vector cell PDGF-BB and GL261-vector clones into the striatum of 840 PDGFRa-KO and Flox mice. The tumor volumes after im½F1 lines (Figure 1A). The levels of Pdgfra mRNA expression were 841 842 plantation of GL261-PDGF-BB were similar between two lower in GL261-PDGF-BB cell lines than in GL261-vector cell 843 genotypes, and Pdgfra inactivation in host tissue did not lines (Figure 1B). Pdgfrb expression was low at comparable 844 significantly affect the growth of implants (P Z 0.146) levels in all examined clones, except that one GL261-vector cell (Figure 3, A, B, and G). The tumor volumes of GL261-vector ½F3 845 line showed Pdgfrb mRNA at an exceptionally high level 846 (Figure 1C). GL261-PDGF-BB clones released significant were also equivalent between the two genotypes of mice 847 3 3 amounts of PDGF-BB into culture media in enzyme-linked (Flox, 5.06  1.60 mm ; PDGFRa-KO, 8.02  1.91 mm ; 848 immunosorbent assay (data not shown). In Western blot anaP Z 0.276, data not shown). After GL261-PDGF-BB im849 lyses, PDGFRa protein expression levels were lower in plantation, we detected many recruited OPCs and hemor850 GL261-PDGF-BB than in GL261-vector cell lines (Figure 1D). rhagic foci around the tumor of Flox mice (Figure 3, A, C, D, 851 The expression of PDGFRb was undetectable in all cell lines and HeJ), as previously observed after implantation in 852 (Figure 1D). The colony-forming activity in GL261-vector cell wild-type mice (Figure 2, A and DeH). In contrast, the 853 854 lines was significantly higher than that in GL261-PDGF-BB recruitment of either Olig2-positive cells of oligoden855 cell lines (P < 0.001, four to five wells from each of the four drocyte lineage or PDGFRa-positive OPCs was largely 856 857 858 Figure 3 Histological characterization of platelet-derived growth factor (PDGF)-BBeoverexpressing glioma (GL261-PDGF-BB) 20 days after implantation in the 859 brain of PDGF receptor a (PDGFRa) knockout mouse (PDGFRa-KO) and PDGFRa-preserving control (Flox) mice. A and B: Hematoxylin and eosin (H&E) staining and immunohistochemistry for Olig2 of GL261-PDGF-BB in PDGFRa-KO and Flox mice. C: Immunohistochemistry for PDGFRa in the marginal region of tumor in Flox and 860 PDGFRa-KO mice. The images are from adjacent sections of boxed area in A and B. D: H&E staining in the marginal region of tumors in Flox and PDGFRa-KO. The 861 images are enlarged boxed area in A and B. Inset: Higher magnification of hemorrhage. E: Immunohistochemistry for IgG in Flox and PDGFRa-KO mice. F: Higher- Q14 862 magnification images of immunohistochemistry for IgG and Olig2 in the marginal region of tumor in Flox and PDGFRa-KO mice. Images correspond to the boxed 863 3 areas in E, respectively. G: Tumor volume (mm ) of GL261-PDGF-BB in Flox and PDGFRa-KO (a-KO). H: Number of Olig2-positive cells per field in the marginal region 864 of tumor. I: Percentage of PDGFRa-positive area per field in the marginal region of tumor. J: Numbers of extravasated red blood cells (RBCs) along the entire circle of 865 each tumor. Counted numbers of RBCs were divided by the circumferential distance of each tumor for the comparison. K: Percentage of PDGFRa-positive area per field 866 in the magical region of tumor. Dotted lines in C, D, and F indicate the edge of tumor. Error bars represent SEM (GeK). n Z 4 mice (G and J); n Z 8, two fields 867 from four mice (H, I, and K). *P < 0.05, ***P < 0.001. Scale bar: 500 mm (A, B, and E); 100 mm (C, D, and F). 868

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clones in GL261-PDGF-BB and GL261-vector cell lines, respectively; t-test) (Figure 1, E and F).

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Cancer Stroma by PDGF Signal in Glioma 993 recruited, and the staining was more intense in Flox than with PDGFRa-KO in zymography (Figure 4M), whereas the 994 in PDGFRa-KO (Figure 3, E, F, and K). difference was not significant (P Z 0.3349, n Z 4 in Flox and 995 n Z 3 in PDGFRa-KO mice). The proeMMP-2 activity was 996 similar between Flox and PDGFRa-KO. Glioma-Associated OPC Induces Vessel Dilatation and 997 Permeability and Down-Regulates Claudin-5 998 Discussion 999 The diameter of blood vessel within GL261-PDGF-BB glioma 1000 1001 appeared to be larger in Flox than in PDGFRa-KO mice Herein, we implanted GL261-vector and GL261-PDGF-BB 1002 (Figure 3, A and B). Morphometrically, the diameter of CD31glioma cells into the brain of wild-type mouse, and found that 1003 positive blood vessels was significantly decreased in the volume of GL261-PDGF-BB glioma was extensively 1004 ½F4 PDGFRa-KO compared with Flox (Figure 4, A and B). The larger than that of GL261-vector glioma. The large tumor in 1005 density of the blood vessel was equivalent between Flox and GL261-PDGF-BB was accompanied by many Olig2/ 1006 PDGFRa-KO (Figure 4, A and C). When we focus on the PDGFRa-positive OPCs, and showed increased hemorrhage. 1007 blood vessels in the margin but not inside of the tumor, the Therefore, PDGF-BBeoverexpressing glioma cells showed 1008 immunofluorescence of claudin-5, a tight junction complex more high-grade malignant features, and these findings were 1009 protein that maintains the blood tumor barrier,27 was less in accordance with a previous report showing that the 1010 strong in Flox than in PDGFRa-KO (Figure 4, A and D). strength of PDGFR signaling is well correlated with high1011 1012 grade characteristics of glioma.30 Previously, the host 1013 tissueederived PDGF-CC was shown to recruit OPCs toward Glioma-Associated OPC Induces Remodeling of 1014 orthotopic GL261 glioma by inactivating host Pdgfc or Collagen 1015 Pdgfra genes.31 In our present study, an inactivation of host 1016 Pdgfra resulted in the substantial decrease of OPC recruitCollagen type IV is one of the major components of the 1017 ment in GL261-PDGF-BB glioma, and it was clearly shown extracellular membrane in glioma, and is mainly synthesized 1018 28,29 that glioma-derived paracrine signal of PDGF-BB recruited by vascular endothelial cells. Collagen type IV expression 1019 OPCs via PDGFRa activation in OPCs. Intriguingly, the was increased in the marginal region of GL261-PDGF-BB 1020 inactivation of host Pdgfra was accompanied by the decrease glioma, where many OPCs were recruited, but was low inside 1021 of hemorrhage and vascular leakage in PDGFRa-KO mouse, of the glioma in Flox mouse (some data not shown) (Figure 4, 1022 even though the glioma volume was comparable between E and G). Collagen type IV expression was at low level both 1023 1024 Flox and PDGFRa-KO mice. It was suggested that the outside and inside of glioma in PDGFRa-KO (Figure 4, E and 1025 paracrine PDGF signal of glioma could be causally related G). In the tumor border of Flox, but not PDGFRa-KO, triple 1026 with hemorrhage and brain edema that contribute to the staining for PDGFRb/collagen type IV/CD31 revealed that the 1027 serious prognostic factors of GBM, independently of the collagen type IVepositive basement membrane was aberrantly 1028 tumor volume.2 thick and multilayered (Figure 4, F and H), and that the 1029 PDGFRb-positive pericytes were detached from endothelial Activated PDGFRa after intracerebral hemorrhage 1030 cells and were interspersed within multilayer basement meminduced blood-brain barrier (BBB) dysfunction in 1031 brane (Figure 4, F and I). Next, we examined the expression of ischemic stroke and cerebral hemorrhage, in which MMP1032 4 9,32 glioma invasion-related protein, MMP-9. 9 was critically involved. Many recruited Similarly, OPCs were shown 1033 OPCs were highly immunoreactive for MMP-9 around tumor to release MMP-9 under acute phase of white matter 1034 of Flox, but not in PDGFRa-KO, whereas glioma cells were injury to initiate the early onset of BBB breakdown.33 We 1035 1036 similarly immunoreactive to MMP-9 in both genotypes of found frequent hemorrhage and increased IgG leakage in 1037 mice (Figure 4, J and K). We confirmed that most of the the marginal region of GL261-PDGF-BB glioma, where 1038 Sox10-positive OPCs expressed MMP-9 (Figure 4L). The MMP-9eexpressing OPCs were recruited. Specific to this 1039 proeMMP-9 activity tended to be higher in Flox compared area, aberrant vasculatures were found that were 1040 1041 1042 Figure 4 Morphological analyses of the parameters related with blood vessel formation and extracellular matrix in platelet-derived growth factor (PDGF)1043 BBeoverexpressing glioma. A: Immunohistochemistry for CD31 and immunofluorescence for claudin-5 (red) in the marginal region of tumor of Flox and PDGF 1044 receptor a knockout (PDGFRa-KO). B and C: Blood vessel diameter and numbers measured from the images of CD31 staining, respectively. D: Claudin-5 1045 immunofluorescence intensity per blood vessel was converted into gray color values, and mean gray value per blood vessel was calculated. E: Immunohistochemistry for collagen type IV in the border part of tumor of Flox and PDGFRa-KO mice. F: Triple immunofluorescence staining for PDGFRa (green), 1046 collagen type IV (magenta), and CD31 (red) in the border part of tumor of Flox and PDGFRa-KO. G: Percentage of collagen type IVepositive area per field. H: 1047 Thickness of the collagen type IVepositive basement membrane of blood vessel. I: Measurement of stratification of the PDGFRb-positive cells around the blood 1048 vessels. J: Immunohistochemistry for matrix metalloproteinase (MMP)-9 in the marginal region of the tumor of PDGFRa-KO and Flox mouse. K: Number of MMP1049 9epositive cells in the marginal region per field. L: Double-immunofluorescence staining for SOX10 (green) and MMP-9 (red) in the marginal region of the 1050 tumor of PDGFRa-KO and Flox mice. M: A representative image of the gelatin zymography gel. Dotted lines in A, J, and L indicate the edge of tumor. Error bars 1051 represent SEM (BeD, GeI, and K). n Z 16, each four fields from four mice (B and C); n Z 77 (D, blood vessels from nine Flox mice); n Z 83 (D, blood vessels 1052 from eight PDGFRa-KO mice); n Z 8, two fields each from four mice (G and K). ***P < 0.001. Scale bars: 100 mm (A, CD31); 50 mm [A, claudin-5, E, F (left 1053 panels), J, and L); 20 mm [F (right panels)]. 1054

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Zheng et al accompanied by a multilayered thick collagen; they contain interspersed pericytes, whose vascular phenotypes were similar to those reported in previous experimentally induced glioma.34,35 These vascular abnormalities in the tumor border of GL261-PDGF-BB in Flox mice were mostly normalized in PDGFRa KO. Taken altogether into consideration, recruited OPCs by paracrine PDGF signal were indicated to be involved in the BBB dysfunction to cause hemorrhage and vascular leakage. Decreased expression of claudin-5, a tight junction complex protein, that maintains the blood-tumor barrier,27 could be an underlying cause of BBB dysfunction; however, the mechanism of decrease remains unknown. Glioma cellederived MMP-9 was not sufficient to induce hemorrhage within tumor, suggesting that OPC-derived additional factors were required for the vascular phenotype. MMPs strongly correlate with malignant progression of glioma,36,37 among which MMP-9 mediates glioma cell adhesion to the extracellular membrane and invasive growth along collagen type IV.38 In our study, a cancer stroma was established with abundant collagen type IV and MMP-9 expression around GL261-PDGF-BB glioma in wild-type and Flox mice, but not in that of PDGFRa-KO mice; however, the tumor volume was similar between Pdgfra-preserving mice and PDGFRa-KO mice. Different PDGF signal in glioma may differentially regulate invasive tumor growth and associating events, like hemorrhage and edema; further studies are needed. Colony-forming activities were significantly lower in GL261-PDGF-BB than in GL261-vector in vitro, which was compatible with low expression levels of PDGFRs in GL261-PDGF-BB glioma cells. The volume of either GL261-PDGF-BB or GL261-vector glioma was comparable between Flox and PDGFRa-KO mice. Accordingly, either of autocrine PDGF-BB signal in glioma or host PDGFRa signaling was not a major determinant of GL261 glioma volume in our study. These observations implied that activated host PDGFRb signaling might have been involved in the larger GL261-PDGF-BB than GL261-vector in wildtype mice. One of the subclasses of GBM was characterized by high levels of PDGF-B ligand expression and PDGFRb phosphorylation.12 The PDGFRb was insignificant in our GL261 cells. Further studies are required to explore the contribution of the two types of PDGFRs in PDGF-BBeoverexpressing glioma. Our study showed that glioma-derived paracrine signal of PDGF-B extensively recruits OPCs via the activation of PDGFRa, and that the recruited OPCs were implicated in the formation of cancer stroma that defines microenvironment, such as BBB dysfunction, hemorrhages, and vascular abnormality with extracellular membrane remodeling. PDGFRa inactivation could normalize vascular phenotype induced by PDGF-BB secreted from glioma, and vascular normalization is crucial for radiation therapy of GBM.34 Our findings may open prospects to develop a new

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Acknowledgments We thank Dr. Atsushi Natsume (Graduate School of Medicine, Nagoya University) for the GL261 glioma cell line; Prof. Yoshimichi Ueda (Kanazawa Medical University) for the valuable scientific discussion; and members of the Department of Pathology and Life Science Research Center (University of Toyama) for thoughtful discussion and careful animal care. Y.I., S.Y., and M.S. conceived and designed the project; Y.Z., Y.I., S.Y., Y.S., N.V.D., H.N., H.M., and R.I. performed experiments; and Y.I., S.Y., and M.S. wrote the manuscript with significant contributions from Y.Z. and T.H.

References 1. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, Scheithauer BW, Kleihues P: The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 2007, 114: 97e109 2. Velander AJ, DeAngelis LM, Navi BB: Intracranial hemorrhage in patients with cancer. Curr Atheroscler Rep 2012, 14:373e381 3. Jones TS, Holland EC: Standard of care therapy for malignant glioma and its effect on tumor and stromal cells. Oncogene 2012, 31: 1995e2006 4. Mammoto T, Jiang A, Jiang E, Panigrahy D, Kieran MW, Mammoto A: Role of collagen matrix in tumor angiogenesis and glioblastoma multiforme progression. Am J Pathol 2013, 183: 1293e1305 5. Curry RC, Dahiya S, Alva Venur V, Raizer JJ, Ahluwalia MS: Bevacizumab in high-grade gliomas: past, present, and future. Expert Rev Anticancer Ther 2015, 15:387e397 6. Zhao Y, Adjei AA: Targeting angiogenesis in cancer therapy: moving beyond vascular endothelial growth factor. Oncologist 2015, 20: 660e673 7. Shim AH, Liu H, Focia PJ, Chen X, Lin PC, He X: Structures of a platelet-derived growth factor/propeptide complex and a plateletderived growth factor/receptor complex. Proc Natl Acad Sci U S A 2010, 107:11307e11312 8. Nishiyama A, Komitova M, Suzuki R, Zhu X: Polydendrocytes (NG2 cells): multifunctional cells with lineage plasticity. Nat Rev Neurosci 2009, 10:9e22 9. Su EJ, Fredriksson L, Geyer M, Folestad E, Cale J, Andrae J, Gao Y, Pietras K, Mann K, Yepes M, Strickland DK, Betsholtz C, Eriksson U, Lawrence DA: Activation of PDGF-CC by tissue plasminogen activator impairs blood-brain barrier integrity during ischemic stroke. Nat Med 2008, 14:731e737 10. Yao H, Duan M, Buch S: Cocaine-mediated induction of plateletderived growth factor: implication for increased vascular permeability. Blood 2011, 117:2538e2547 11. Heldin CH: Autocrine PDGF stimulation in malignancies. Ups J Med Sci 2012, 117:83e91 12. Brennan C, Momota H, Hambardzumyan D, Ozawa T, Tandon A, Pedraza A, Holland E: Glioblastoma subclasses can be defined by activity among signal transduction pathways and associated genomic alterations. PLoS One 2009, 4:e7752 13. Heldin CH, Westermark B: Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 1999, 79:1283e1316

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1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288

Cancer Stroma by PDGF Signal in Glioma

Q12

14. Charles NA, Holland EC, Gilbertson R, Glass R, Kettenmann H: The brain tumor microenvironment. Glia 2012, 60:502e514 15. Appolloni I, Calzolari F, Tutucci E, Caviglia S, Terrile M, Corte G, Malatesta P: PDGF-B induces a homogeneous class of oligodendrogliomas from embryonic neural progenitors. Int J Cancer 2009, 124:2251e2259 16. Takeuchi T, Nomura T, Tsujita M, Suzuki M, Fuse T, Mori H, Mishina M: Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting. Biochem Biophys Res Commun 2002, 293: 953e957 17. Kitayama K, Abe M, Kakizaki T, Honma D, Natsume R, Fukaya M, Watanabe M, Miyazaki J, Mishina M, Sakimura K: Purkinje cellspecific and inducible gene recombination system generated from C57BL/6 mouse ES cells. Biochem Biophys Res Commun 2001, 281: 1134e1140 18. Fukaya M, Tsujita M, Yamazaki M, Kushiya E, Abe M, Akashi K, Natsume R, Kano M, Kamiya H, Watanabe M, Sakimura K: Abundant distribution of TARP gamma-8 in synaptic and extrasynaptic surface of hippocampal neurons and its major role in AMPA receptor expression on spines and dendrites. Eur J Neurosci 2006, 24:2177e2190 19. Miya K, Inoue R, Takata Y, Abe M, Natsume R, Sakimura K, Hongou K, Miyawaki T, Mori H: Serine racemase is predominantly localized in neurons in mouse brain. J Comp Neurol 2008, 510: 641e654 20. Kanki H, Suzuki H, Itohara S: High-efficiency CAG-FLPe deleter mice in C57BL/6J background. Exp Anim 2006, 55:137e141 21. Hayashi S, McMahon AP: Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol 2002, 244:305e318 22. Yamamoto S, Yoshino I, Shimazaki T, Murohashi M, Hevner RF, Lax I, Okano H, Shibuya M, Schlessinger J, Gotoh N: Essential role of Shp2-binding sites on FRS2alpha for corticogenesis and for FGF2dependent proliferation of neural progenitor cells. Proc Natl Acad Sci U S A 2005, 102:15983e15988 23. Yamamoto S, Niida S, Azuma E, Yanagibashi T, Muramatsu M, Huang TT, Sagara H, Higaki S, Ikutani M, Nagai Y, Takatsu K, Miyazaki K, Hamashima T, Mori H, Matsuda N, Ishii Y, Sasahara M: Inflammation-induced endothelial cell-derived extracellular vesicles modulate the cellular status of pericytes. Sci Rep 2015, 5:8505 24. Shen J, Ishii Y, Xu G, Dang TC, Hamashima T, Matsushima T, Yamamoto S, Hattori Y, Takatsuru Y, Nabekura J, Sasahara M: PDGFR-beta as a positive regulator of tissue repair in a mouse model of focal cerebral ischemia. J Cereb Blood Flow Metab 2012, 32: 353e367 25. Sato H, Ishii Y, Yamamoto S, Azuma E, Takahashi Y, Hamashima T, Umezawa A, Mori H, Kuroda S, Endo S, Sasahara M: PDGFR-beta plays a key role in the ectopic migration of neuroblasts in cerebral stroke. Stem Cells 2015. doi:10.1002/stem.2212

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26. Xue Y, Lim S, Yang Y, Wang Z, Jensen LD, Hedlund EM, Andersson P, Sasahara M, Larsson O, Galter D, Cao R, Hosaka K, Cao Y: PDGF-BB modulates hematopoiesis and tumor angiogenesis by inducing erythropoietin production in stromal cells. Nat Med 2012, 18:100e110 27. Musch MW, Walsh-Reitz MM, Chang EB: Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption. Am J Physiol Gastrointest Liver Physiol 2006, 290:G222eG231 28. Huijbers IJ, Iravani M, Popov S, Robertson D, Al-Sarraj S, Jones C, Isacke CM: A role for fibrillar collagen deposition and the collagen internalization receptor endo180 in glioma invasion. PLoS One 2010, 5:e9808 29. Zamecnik J: The extracellular space and matrix of gliomas. Acta Neuropathol 2005, 110:435e442 30. Shih AH, Dai C, Hu X, Rosenblum MK, Koutcher JA, Holland EC: Dose-dependent effects of platelet-derived growth factor-B on glial tumorigenesis. Cancer Res 2004, 64:4783e4789 31. Huang Y, Hoffman C, Rajappa P, Kim JH, Hu W, Huse J, Tang Z, Li X, Weksler B, Bromberg J, Lyden DC, Greenfield JP: Oligodendrocyte progenitor cells promote neovascularization in glioma by disrupting the blood-brain barrier. Cancer Res 2014, 74:1011e1021 32. Ma Q, Huang B, Khatibi N, Rolland W 2nd, Suzuki H, Zhang JH, Tang J: PDGFR-alpha inhibition preserves blood-brain barrier after intracerebral hemorrhage. Ann Neurol 2011, 70:920e931 33. Seo JH, Miyamoto N, Hayakawa K, Pham LD, Maki T, Ayata C, Kim KW, Lo EH, Arai K: Oligodendrocyte precursors induce early blood-brain barrier opening after white matter injury. J Clin Invest 2013, 123:782e786 34. Winkler F, Kozin SV, Tong RT, Chae SS, Booth MF, Garkavtsev I, Xu L, Hicklin DJ, Fukumura D, di Tomaso E, Munn LL, Jain RK: Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases. Cancer Cell 2004, 6:553e563 35. Kamoun WS, Ley CD, Farrar CT, Duyverman AM, Lahdenranta J, Lacorre DA, Batchelor TT, di Tomaso E, Duda DG, Munn LL, Fukumura D, Sorensen AG, Jain RK: Edema control by cediranib, a vascular endothelial growth factor receptor-targeted kinase inhibitor, prolongs survival despite persistent brain tumor growth in mice. J Clin Oncol 2009, 27:2542e2552 36. Wang M, Wang T, Liu S, Yoshida D, Teramoto A: The expression of matrix metalloproteinase-2 and -9 in human gliomas of different pathological grades. Brain Tumor Pathol 2003, 20:65e72 37. Levicar N, Nuttall RK, Lah TT: Proteases in brain tumour progression. Acta Neurochir (Wien) 2003, 145:825e838 38. Zhao Y, Xiao A, diPierro CG, Carpenter JE, Abdel-Fattah R, Redpath GT, Lopes MB, Hussaini IM: An extensive invasive intracranial human glioblastoma xenograft model: role of high level matrix metalloproteinase 9. Am J Pathol 2010, 176:3032e3049

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