Laminin isoforms and their integrin receptors in glioma cell migration and invasiveness: Evidence for a role of α5-laminin(s) and α3β1 integrin

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Laminin isoforms and their integrin receptors in glioma cell migration and invasiveness: Evidence for a role of α5-laminin(s) and α3β1 integrin Tomoyuki Kawataki a,b , Tetsu Yamane c , Hirofumi Naganuma b , Patricia Rousselle d , Ingegerd Andurén a , Karl Tryggvason e , Manuel Patarroyo a,⁎ a

Department of Odontology and Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, S 141 04 Stockholm, Sweden Department of Neurosurgery, Yamanashi University, Japan c Department of Pathology, Yamanashi University, Japan d Institut de Biologie et Chimie des Protéines, Université Lyon I, Lyon, France e Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

Glioma cell infiltration of brain tissue often occurs along the basement membrane (BM) of blood

Received 16 March 2007

vessels. In the present study we have investigated the role of laminins, major structural

Revised version received

components of BMs and strong promoters of cell migration. Immunohistochemical studies of

12 July 2007

glioma tumor tissue demonstrated expression of α2-, α3-, α4- and α5-, but not α1-, laminins by

Accepted 27 July 2007

the tumor vasculature. In functional assays, α3 (Lm-332/laminin-5)- and α5 (Lm-511/laminin-

Available online 16 August 2007

10)-laminins strongly promoted migration of all glioma cell lines tested. α1-Laminin (Lm-111/

Keywords:

laminins were practically inactive. Global integrin phenotyping identified α3β1 as the most

Glioma

abundant integrin in all the glioma cell lines, and this laminin-binding integrin exclusively or

Laminin

largely mediate the cell migration. Moreover, pretreatment of U251 glioma cells with blocking

Integrin

antibodies to α3β1 integrin followed by intracerebral injection into nude mice inhibited invasion

Migration

of the tumor cells into the brain tissue. The cell lines secreted Lm-211, Lm-411 and Lm-511, at

Invasion

different ratios. The results indicate that glioma cells secrete α2-, α4- and α5-laminins and that

laminin-1) displayed lower activity, whereas α2 (Lm-211/laminin-2)- and α4 (Lm-411/laminin-8)-

α3- and α5-laminins, found in brain vasculature, selectively promote glioma cell migration. They identify α3β1 as the predominant integrin and laminin receptor in glioma cells, and as a brain invasion-mediating integrin. © 2007 Elsevier Inc. All rights reserved.

Introduction Gliomas are the most common primary brain tumors and are often highly malignant. Glioblastoma multiforme, the most aggressive glioma, has a poor prognosis and most patients die

of their disease in less than a year, independently of treatment [1]. Malignant glioma diffusely infiltrates the normal brain tissue as single cells, and this infiltration makes complete surgical resection practically impossible. The invasive cells often migrate along myelinated fiber tracts

⁎ Corresponding author. Fax: +46 8 7467915. E-mail address: [email protected] (M. Patarroyo). 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.07.038

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and in the perivascular space along blood vessels [2–5]. In the latter case, the glioma cells interact with the abluminal side of the vessels, adopting a pericytic-like location, without intravasating. This pattern of migration may be due to specific interactions between extracellular matrix (ECM) components of the vascular basement membrane (BM) and tumor cell surface receptors. Alternatively, glioma tumors may create a permissive migratory substrate by synthesizing and depositing autologous ECM components. Laminins are a growing family of large heterotrimeric αβγ proteins which promote cell adhesion and migration via integrin receptors [6–8]. Together with type IV collagen, perlecan and nidogen, these ECM proteins are major components of all BMs, including the vascular BM [9]. Five α, three β

Table 1 – Monoclonal antibodies to laminin and integrin chains a Name

Specificity

Source

Reference

EB7 5H2 4H8 P3H9-2 3H2 6C3 4C7 15H5 DG10 C4 17 22 2E8 D4B5 FB12 P1E6 P1B5 HP2/1 P1D6 BQ16 GoH3 9.1 AMF7 69.6.5 SZ.22 13 6S6 P4C10 IB-4 SZ.21 ASC-9 B5-IVF2 FIB27 Y9A2 LM609 P1F6 G46-2.6

Lmα1 Lmα2 Lmα2 Lmα3 Lmα4 Lmα4 Lmα5 Lmα5 Lmβ1 Lmβ2 Lmβ3 Lmγ1 Lmγ1 Lmγ2 Intα1 Intα2 Intα3 Intα4 Intα5 Intα6 Intα6 Intα7 IntαV IntαV IntαIIb Intβ1 Intβ1 Intβ1 Intβ2 Intβ3 Intβ4 Intβ5 Intβ7 Intα9β1 IntαVβ3 IntαVβ5 HLA-ABC

I.V. Chemicon Sigma Chemicon M.P. M.P. Dako I.V./K.S. I.V. Neomarkers BD Biosciences BD Biosciences E.E Chemicon Chemicon Chemicon Chemicon Immunotech Chemicon Ancell Immunotech R.K. Immunotech Immunotech Immunotech BD Biosciences Chemicon Chemicon S.W. Immunotech Chemicon Sigma BD Biosciences Serotec Chemicon Chemicon BD Biosciences

[26]

a

[31] [26] [27] [25]

[25] [25]

[55]

[31]

All mAbs are mouse IgG. I.V., Ismo Virtanen; Chemicon, Chemicon International, Temecula, CA; Sigma, Sigma Chemical Co., St. Louis, MO; M.P., Manuel Patarroyo; Dako, Dako, Copenhagen, Denmark; K.S., Kiyotoshi Sekiguchi; Neomarkers, Neomarker, Fremont, CA, USA; BD Biosciences, BD Biosciences, Erembodegem, Belgium; E.E., Eva Engvall; Ancell, Ancell Corporation, Bayport, MN, USA; Immunotech, Immunotech, Marseille, France; R.K., Randall Kramer; S.W., Samuel Wright; Lm, laminin; Int, integrin.

Fig. 1 – Expression of laminin chains in glioma tissue vasculature. Expression of α2-, α3-, α4- and α5-laminins in tumor blood vessels. Frozen sections of tissue from human glioblastoma multiforme were immunostained with mAb EB7 (Lmα1), mAb 5H2 (Lmα2), mAb P3H9-2 (Lmα3), mAb 3H2 (Lmα4), mAb 4C7 (Lmα5), mAb DG10 (Lmβ1), mAb C4 (Lmβ2) and mAb 2E8 (Lmγ1). Scale bar, applicable to all fields, represents 50 μm.

and three γ chains constitute by combination over 15 different laminin isoforms, and the α chains, which display a cell- and tissue-specific expression, are differentially recognized by nearly 10 out of 24 different integrins [6,10]. In a new simplified nomenclature, laminin isoforms are designated according to their chain composition (e.g., laminin-8 or α4β1γ1 is now called Lm-411) [8]. Though the first laminin, Lm-111 or laminin-1, was reported over 25 years ago, a large number of additional laminin isoforms with wider tissue distribution, different integrins receptors and different biological activities have been identified since then. Several studies concerning gliomas and laminins have been reported in the literature. By immunohistochemistry, laminin

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has been localized mainly to the tumor vasculature, but also within the tumor as punctate deposits and at the brain/tumor confrontation zone, particularly in glioma models [11–14]. Glioma cells are known to secrete several laminins, but their isoforms are poorly characterized [15,16]. Moreover, among several BM components and other ECM proteins, laminins appeared to be most permissive for adhesion and migration of glioma cells in vitro, and these interactions were mediated by integrins, including α3β1 and α6β1 [16–23]. Compared to normal brain and astrocytes, astrocytomas and glioblastomas showed increased expression of the former integrin both in vivo and in vitro [24]. These studies have provided most valuable information. However, the anti-laminin antibodies used were often cross-reactive with most laminin isoforms, or

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their chain specificity was unknown. Moreover, the laminin preparations were often poorly characterized molecularly, differing from one another and consisting of highly fragmented proteins, and/or a mixture of laminin isoforms [25,26]. In addition, only a limited number of laminin isoforms, predominantly Lm-111, or their integrin receptors were tested, and no systematic analysis of α5-laminin(s) was reported. More recently, the chain specificity of many laminin antibodies has been established, and several recombinant laminin isoforms have been produced [25–29]. In the present study, a panel of antibodies to all laminin chains, except γ3, was used to identify the laminins of glioma tumor tissue, particularly of the vasculature. Moreover, the glioma cell migration-promoting activity of laminins covering all known

Fig. 2 – Migration-promoting activity of laminin isoforms in glioma cells. (A, B) α1 (Lm-111, laminin-1)-, α3 (Lm-332, laminin-5)- and α5 (Lm-511, laminin-10)-laminins, but not α2 (Lm-211, laminin-2)- and α4 (Lm-411, laminin-8)-laminins, specifically promote glioma cell migration. (A) Photomicrographs of KG1C cells in the cell migration assay. (B) Cell migration of A172, KG1C, T98G and U251 glioma cell lines on different laminin isoforms compared to human serum albumin (HSA). Both placenta (p) and recombinant (r) Lm-511 were tested on KG1C cells. Glioma cell migration through 8-μm pore filters precoated with proteins at 20 μg/ml was measured after 16-h incubation at 37 °C. The number of transmigrated glioma cells were counted at 400× magnification in nine microscopic fields randomly selected and calculated as mean cell number per field. Scale bar in panel A, applicable to all fields, represents 50 μm. Statistical analyses (Student's t test) including mean and SD were calculated, as well as level of significance comparing all Lm isoforms to HSA control (*p b 0.05; **p b 0.01; ***p b 0.001).

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α chains was established by using fully characterized natural and/or recombinant proteins, and their major integrin receptor was identified with a panel of antibodies to all known integrins. Finally, the effect of blocking antibodies to this integrin in the invasiveness of human glioma cells transplanted into the brain of nude mice was determined, and the laminin isoforms secreted by glioma cells were identified.

Material and methods Cells, purified proteins and mAbs Human malignant glioma cell lines (A172, KG1C, T98G, U251) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, and antibiotics. All cell lines were obtained from the Health Science Research Resources Bank, Tokyo, Japan. α1-Laminin (Lm-111, laminin1) was isolated from mouse Engelbreth–Holm–Swarm (EHS) tumor and obtained from Chemicon (Chemicon International, Temecula, CA). α2-Laminins were used either as merosin isolated from human placenta (Lm-211 (laminin-2)) with some Lm-221 (laminin-4) purchased from Chemicon, or as recombinant human laminin-2 (Lm-211) produced in a mammalian expression system and purified by affinity chromatography [29]. α3-Laminin (Lm-332, laminin-5) was purified from the culture medium of human oral squamous carcinoma SCC25 cells as previously described [30]. α4 (Lm-411, laminin-8)- and α5 (Lm-511, laminin-10)-laminins were produced in a mammalian expression system as full-length recombinant molecules and purified by affinity chromatography as previously reported [27,28]. Another α5-laminin preparation (Lm-511, laminin-10) purchased from Sigma (Sigma, St. Louis, MO) was also used. This preparation, isolated from human placenta by mild pepsin digestion and purified by immunoaffinity chromatography, was used only when reactive with mAb 4C7 to Lmα5 chain globular domain [26]. Monoclonal antibodies (mAbs) used in the present study are listed in Table 1, together with their source and reported specificity.

Immunohistochemistry Glioma tissues were obtained from three patients who underwent tumor resection. Glioblastoma was diagnosed according to the criteria of the World Health Organization. Six-μm tissue sections were fixed in acetone at −20 °C for 10 min and incubated for 15 min with 3% hydrogen peroxide to block endogenous peroxidase. After addition of mAbs to laminin chains, tissues were immunostained with the avidin biotin complex (ABC) technique using Dakopatts (Glostrup, Denmark) and Amersham (Amersham, UK) reagents. Colors were developed with 3,3′-diaminobenzidine (DAB) tetrahy-

drochloride. Sections were dehydrated, coverslipped with Eukitt (O. Kindler, RFG) and observed using light microscopy.

Cell migration assay To investigate the effect of exogenous laminin isoforms on glioma cell migration, transmigration of cells through protein-coated filters which separate upper and lower chambers was determined microscopically. The filters had been previously coated for 3 h with 20 μg/ml of laminin isoforms or HSA, and blocked with 0.5% HSA for 1 h. A single cell suspension (2 × 105 cells in 100 μl RPMI1640 medium) from glioma cell cultures was seeded in the upper chamber of a 6.5-mm diameter, 8-μm pore polycarbonate Transwell culture insert (Costar, Cambridge, MA), and then incubated at 37 °C for 16 h in a 5% CO2 humidified incubator. After incubation, the chamber was disassembled, the upper side of the filter was wiped off to remove non-migrating cells, and cells attached to the lower side were fixed in 3% glutaraldehyde in PBS for 15 min and stained with hematoxylin. Migrated cells were counted at 400× magnification in 9 randomly selected microscopic fields. Cell migration inhibition assay was performed by pretreating the cell suspension with mAbs (20 μg/ml) for 15 min before adding the cells to the upper chamber. Cell migration in presence of control IgG was defined as 100% migration. In statistical analysis (Student's t test), mean and SD were calculated as well as well as level of significance (⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001), comparing the blocking mAbs to the IgG control.

Immunofluorescence flow cytometry Flow cytometric analysis was performed as described previously [31]. Briefly, single suspended cells were obtained by trypsinization of cultured glioma cell lines. After blocking with 1 mg/ml of heat aggregated human IgG (Sigma), cells were incubated for 30 min at 4 °C with saturating amount of mAbs, followed by fluorescein-conjugated F(ab)2 fragments of rabbit anti-mouse Ig (Dakopatts) at 1:20 dilution. After staining, the cells were gated (according to forward and side scatter) and analyzed in a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).

Glioma transplantation model Human U251 glioma cells were transplanted into the brain of nude mice as previously reported [32]. Briefly, rhodaminelabeled U251 cells (5 × 104) were injected into the basal ganglion of anesthetized mice, 3 mm deep from the dura mater, using a Hamilton syringe with a stereotactic frame. Six- to eight-weekold male KSN Slc nude mice obtained from Japan SLC, Inc. (Shizuoka, Japan) were used. A midline skin incision had been

Fig. 3 – Laminin-binding α3β1 integrin is the predominant integrin expressed by glioma cells. Detection of integrin chains in glioma cells lines (A172, KG1C, T98G, U251) by immunofluorescence flow cytometry. Reactivity of monoclonal antibodies with each glioma cell line is shown. Mouse IgG control (white profiles) and mAbs (black profiles) P1B5 (Intα3), BQ16 (Intα6), 9.1 (Intα7), AMF-7 (IntαV), P4C7 (Intβ1), SZ21 (Intβ3), LM609 (IntαVβ3), ASC-9 (Intβ4), PIF6 (IntαVβ5). All integrin staining, but Intα7, was performed simultaneously for each cell line.

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made in the head, before drilling a hole in the skull 1 mm anterior to the coronal suture and 1.5 mm lateral to the midline to expose the dura. Glioma cells, treated with 100 μg/ml of

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mIgG control, mAb G46-2.6 to HLA-ABC or mAb P1B5 to Intα3, were injected in 3 μl of PBS. All antibodies were mouse IgG1 and used in purified form free of NaN3. Five days after transplan-

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tation of the glioma cells, mice were decapitated. Thereafter, the whole brain was removed and, after immersion in OCT compound for 30 min at −80 °C, sectioned into 4-μm coronal slices. The invasive activity of rhodamine-labeled U251 cells was observed under a fluorescence microscope and quantified by counting the migration distance of the twenty most far migrated cells from the inoculation area center, first in millimeters in images and then converted to micrometers. All protocols for animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at Yamanashi University.

RT-PCR Total RNA was extracted from cultured glioma cells by using Total RNA Extraction kit (Amersham Bioscience Sweden). Briefly, after oligo(dT)18 priming, mRNA was reverse-transcribed into cDNA by incubating for 60 min at 42 °C with 200 U

of M-MLV reverse transcriptase in 20 μl of reaction buffer containing 20 U of recombinant RNase inhibitor, and 10 μM of each dNTP. The reactions were terminated by heating the samples at 94 °C for 5 min and the mixture was diluted to 100 μl with DEPC-treated water. Single-stranded cDNAs in 20 μl of reaction buffer containing 1.5 mM MgCl2, 10 μM of each primer and dNTP mix (10 μM each) were amplified by 30 cycles of PCR using 1.25 U of Ampli Taq DNA polymerase (Perkin Elmer/ Roche Molecular Systems, Inc., Branchburg, NJ, USA). The conditions for PCR were 94 °C, 20 s; 60 °C, 30 s; 72 °C, 2 min. For PCR of human Lmα2 cDNA, paired primers 5′-GTCAAGAATTCAAAAACAAG-3′ (sense strand) and 5′-TATCTTCTGTTTTCATCCAC-3′ (antisense strand) (nucleotides 7330-7983) were used to direct synthesis of a 654-bp product. For human Lmα4 cDNA, paired primers 5′-AATGCGTCAGGGATTTATGC-3′ (sense strand) and 5′-GAGACTTGGATCTTGCTGGC-3′ (antisense strand) (nucleotides 1837–2672) were used to direct synthesis of an 836-bp product. For human Lmα5 cDNA, paired

Fig. 4 – Laminin-promoted glioma cell migration is largely mediated by α3β1 integrin. Cell migration inhibition assays were performed to assess the effect of function-blocking mAbs to integrins in the migration of KG1C cells (A) and U251 cells (B) on α1-, α3- and α5-laminins. Both placenta (p) and recombinant (r) Lm-511 were tested on KG1C cells. Glioma cell migration through 8-μm pore filters precoated with proteins at 20 μg/ml was measured after 16-h incubation at 37 °C. mAbs P1B5 (INTα3), GoH3 (INTα6) and 13 (INTβ1), and mIgG (negative control), were used at 20 μg/ml. Cell migration in presence of control IgG was defined as 100% migration. Statistical analyses (Student's t test) including mean and SD were calculated, as well as level of significance comparing the blocking mAbs to the IgG control (*p b 0.05; **p b 0.01; ***p b 0.001).

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primers 5′-ATGAGCATCACATTCCTGGA-3′ (sense strand) and 5′-AGTGGGTTCCCAAAGAATCC-3′ (antisense strand) (nucleotides 5291–5946) were used to direct synthesis of a 656-bp product. For human Lmβ1 cDNA, paired primers 5′-TTGGACCAAGATGTCCTGAG-3′ (sense strand) and 5′-CAATATATTCTGCCTCCCCG-3′ (antisense strand) (nucleotides 955– 1633) were used and a 679-bp product was expected. For human Lmβ2 cDNA, paired primers 5′-TTGTACGTGGCAACTGCTTC-3′ (sense strand) and 5′-ACTGTTGGGGTCACAAGGAG-3′ (antisense strand) (nucleotides 998–1629) were used and a 632-bp product was expected. For human Lmγ1 cDNA, paired primers 5′-GCAAGACTGAACAGCAGACC-3′ (sense strand) and 5′-TCCTATCAAGATCGCTGACC-3′ (antisense strand) (nucleotides 4241–4927) were used and a 687-bp product was expected. A reaction without reverse transcriptase product was performed as negative control to exclude the possibility of contaminating genomic DNA. PCR products were analyzed by electrophoresis using 2% agarose gels.

Immunoprecipitation, gel electrophoresis and immunoblotting

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in the tumor vessels. Lm-221 (α2β2γ1, laminin-4), Lm-421 (α4β2γ1, laminin-9) and Lm-521 (α5β2γ1, laminin-11), as well as Lm-311 (α3β1γ1, laminin-6) and/or Lm-321 (α3β2γ1, laminin-7), may also be expressed, but at lower levels. Staining of the tumor cells in situ for Lm chains was faint or negative (Fig. 1 and data not shown).

α3 (Lm-332, laminin-5)- and α5 (Lm-511, laminin-10)-laminins strongly and specifically promote glioma cell migration To determine the effect of vascular laminin isoforms on glioma cell migration, the migration-promoting activity of α1-, α2-, α3-, α4- and α5-laminins was examined on four glioma cell lines (A172, KG1C, T98G and U251). Human serum albumin (HSA) was used as negative control. In this in vitro assay, glioma cells were allowed to migrate for 16 h through Transwell filters precoated with the proteins. α3- and α5-laminins, and to a lesser extent α1-laminin, efficiently promoted glioma cell migration when compared to HSA, and this effect was highly significant (Figs. 2A and B). When compared to placenta Lm511, which was used in most studies, recombinant Lm-511

Laminin secretion was determined in 100 ml of conditioned medium from glioma cell lines that had reached confluence. Following clearing by centrifugation, the medium was precipitated with 80% ammonium sulfate and 1 ml of lysis buffer (1% Triton X-100 in 500 mM NaCl with 20 mM Tris–HCl) was added to the pellet. Thereafter, the precipitate was dialyzed against H2O for 3 h and against PBS overnight. After removal of smaller molecules by filtration (Macrosep 50K, Pall Life Sciences, Ann Arbor, MI), the material was precleared with protein G-Sepharose beads (Amersham Pharmacia AB), and immunoprecipitation was performed by adding 20 μg of mAb to 200 μl of the material, followed by the addition of 100 μl of protein G-Sepharose beads previously coated with 10 μl of rabbit anti-mouse Ig (Dakopatts). Following separation by SDS-PAGE, immunoprecipitates were analyzed by Western blotting. In Western blots, filters were blocked with 0.1% Tween 20/5% dry milk in PBS. Peroxidase-linked anti-mouse or anti-rabbit Ig (Dakopatts) was used as secondary Ab, and ECL (Amersham, UK) was used as developer.

Results Glioma tissue vasculature expresses α2-, α3-, α4- and α5-laminins To identify the laminin isoforms in the vasculature of gliomas, immunohistochemistry of glioblastoma tissues was first performed with mAbs to laminin chains (Fig. 1). The tumor vessels were strongly stained with mAbs to Lmα2, Lmα4, Lmα5, Lmβ1 and Lmγ1, whereas staining for Lmα3 and Lmβ2 was less intense. Faint or no staining was observed with mAbs to Lmα1, Lmβ3 and Lmγ2, though the mAb to Lmα1 clearly reacted with the vasculature of normal brain tissue (Fig. 1 and data not shown). Under the present experimental conditions, no double BMs could be visualized in the brain vasculature with any of the mAbs (Fig. 1 and data not shown). These results are consistent with the presence of Lm-211 (α2β1γ1, laminin-2), Lm-411 (α4β1γ1, laminin-8) and Lm-511 (α5β1γ1, laminin-10)

Fig. 5 – Effect of blocking antibody to α3β1 integrin on the invasive activity of glioma cells transplanted into nude mouse brain. Rhodamine-labeled U251 glioma cells were stereotactically injected into the brain of nude mice and cell invasion was observed 5 days after. Microphotographs showing invasive activity of U251 cells pretreated with 100 μg/ml of mouse IgG (A) and mAb P1B5 to Intα3 (B). Scale bar in panel A, also applicable to panel B, 20 μm. (C) Measurement of invasion activity of U251 glioma cells pretreated with either mIgG or P1B5. Values represent cell migration distance in μm from the inoculation site (n = 6 in each group). Statistical analyses (Student's t test), mean and SD were calculated, as well as level of significance comparing mAb P1B5 to the IgG control (*p b 0.05).

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behaved similarly. Due to shortage of protein, the latter preparation was tested only on KG1C cells. Lm-211, either as natural or recombinant protein, and Lm-411 exerted a minimal

effect, if any (Figs. 2A and B and data not shown). Though T89G was less migratory than the other cell lines, the pattern of laminin recognition by all cell lines was similar.

Fig. 6 – Synthesis and secretion of heterotrimeric α2-, α4- and α5-laminins by glioma cells. (A) RT-PCR of laminin chains in glioma cell lines. Amplification of laminin α2, α4, α5, β1, β2, γ1 cDNA fragments by RT-PCR of total RNA from each glioma cell line (A172, KG1C, T98G, U251). (B) Western blot analysis of immunoprecipitates from conditioned medium of glioma cells obtained with mAbs to laminin α2, α4 and α5 chains. Immunoprecipitated proteins were separated by SDS-PAGE using 5% (left and middle panel) or 6% (right panel) polyacrylamide gels under reducing conditions. mAb 4H8 (Lmα2), 3H2 (Lmα4) and 4C7 (Lmα5) were used for immunoprecipitation. mIgG (negative control), mAb DG10 (Lmβ1), mAb 22 (Lmγ1), mAb C4 (Lmβ2), mAb 6C3 (Lmα4) and mAb 15H5 (Lmα5) were used for Western blotting. Bands of 230, 220, 190, 220/180 and 350 kDa, corresponding to Lmβ1, Lmγ1, Lmβ2, Lmα4 and Lmα5 were detected. The marker line shows 211 kDa.

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Glioma cells express laminin-binding α3β1 as the predominant integrin

Glioma cells synthesize and secrete heterotrimeric α2-, α4and α5-laminins

To identify laminin-binding integrins, the glioma cell lines were analyzed by immunofluorescence flow cytometry (Fig. 3). All four cell lines were positive for Intα3, Intα6, IntαV, Intβ1 and IntαVβ5. Three cell lines (A172, KG1C and U251) expressed Intβ4, and two cell lines (T98G and U251) were positive for Intβ3 and IntαVβ3. Only U251 cells expressed Intα7, but at low levels. Further analysis with a library of mAbs that covers all 24 integrin heterodimers (Table 1) indicated that expression of Intα3 and Intβ1 was about 5- to 10-fold higher than that of any other integrin (Fig. 3 and data not shown). Altogether, the results indicated that glioma cell lines express several laminin-binding integrins, and that α3β1 is by far the most abundant integrin.

To identify laminin isoforms produced by glioma cells, synthesis, expression and secretion of heterotrimeric laminin molecules were investigated in the glioma cell lines. Expression of mRNAs encoding for various laminin chains was first investigated by RTPCR (Fig. 6A). Amplified products with the expected size were detected with primers for Lmα2 (654 bp), Lmα4 (836 bp), Lmα5 (637 bp), Lmβ1 (679 bp), Lmβ2 (632 bp) and Lmγ1 (687 bp) after 30 cycles of PCR with reverse transcribed mRNA in all four glioma cell lines. Transcripts for the remaining laminin chains were either weakly expressed or absent, or the data were inconclusive (data not shown). Thereafter, laminins secretion was investigated in the cell conditioned medium by immunoprecipitation with mAb against each laminin α chain, followed by Western blotting of the immunoprecipitates with mAbs to the corresponding laminin α chain, and to laminin β and γ chains to confirm physical association of the components (Fig. 6B). Under these experimental condition, Lmβ1 (230 kDa) and Lmγ1 (220 kDa) chains were detected from all cell lines when mAbs to Lmα2, Lmα4 or Lmα5 were used for immunoprecipitation, whereas Lmβ2 chain (190 kDa) was observed only in some cases. No immunoprecipitates were obtained by using mAbs to either Lmα1 or Lmα3 chains, and Lmβ3 and Lmγ2 chains were not detected (data not shown). The Lmα2 chain could not be directly visualized by Western blotting, but Lmα4 and Lmα5 chains consisted of 180/220 and 320/350 kDa polypeptides, respectively. No components were detected when mouse IgG control was used for either immunoprecipitation or Western blotting (Fig. 6B and data not shown). Among the laminin isoforms, α5-laminins appeared to be the most abundant, followed by α4- and α2laminins (data not shown). Altogether, the results indicated that glioma cells are able to synthesize, assemble and secrete complete laminin molecules, including Lm-211, Lm-411 and Lm-511, and smaller amounts of Lm-221, Lm-421 and Lm-521.

α3β1 integrin mediates promotion of glioma cell migration by laminins To identify integrins mediating glioma cell migration on α1-, α3- and α5-laminins, the effect of function-blocking mAbs against laminin-binding integrins was examined on two glioma lines, KG1C and U251 (Figs. 4A and B). mAb to Intα3 chain inhibited almost completely the migration of both cell lines on α3- and α5-laminins. Inhibition was similar on placenta and recombinant α5-laminins, but slightly more pronounced on the former preparation. On α1-laminin-promoted migration, the antibody was highly inhibitory on KG1C cells, but exerted only moderate inhibition on U251 cells. In contrast, mAb to Intα6 was practically inactive on α3- and α5laminin-promoted cell migration, but exerted a partial inhibitory effect on α1-laminin. mAb to Intβ1 chain blocked migration of both cell lines on the three laminin isoforms. No significant inhibition was observed with mAbs to IntαV (data not shown). Thus, α3β1 integrin predominantly mediates glioma cell migration on α3- and α5-laminins and, depending on the cell line, on α1-laminin. α6β1 integrin contributes to a lower extent to the cell migration, and preferentially on α1laminin.

A blocking antibody to α3β1 integrin inhibits glioma cell invasion of brain tissue Fluorescence microscopy studies indicated that, 5 days after injection, rhodamine-labeled U251 cells treated with mIgG invaded diffusely into the nude mouse brain tissue around the site of injection (Fig 5A). In contrast, cells treated with mAb P1B5 against Intα3 remained mainly in the inoculation site and infiltrated to a much lower extent (Fig. 5B). When quantified microscopically in 12 animals, the migrated cell distance in the mIgG control group (n= 6) was about 240 μm, compared to 60 μm in the mAb P1B5-treated group (n= 6) (Fig. 5C). This difference was statistically significant (p b 0.05) and indicated that pretreatment of glioma cells with a blocking mAb to Intα3 inhibits their ability to invade brain tissue. Immediately after injection the tumor cells showed either a very focal localization or a linear distribution (data not shown). Moreover, mAb G46-2.6 to HLAABC, which was expressed at the same density as Intα3, behaved similarly to mIgG control (data not shown).

Discussion In the present study, the migration-promoting activity of laminins covering all known α chains was systematically compared on glioma cells for the first time, and a laminin isoformspecific effect was observed. α3- and α5-laminins, which were found in the tumor vasculature, strongly promoted migration, whereas α2- and α4-laminins exerted a minimal effect, if any. α1-Laminin displayed an intermediate activity. Among all integrins, α3β1 was identified as the most abundant in glioma cells, and as the receptor mediating the laminin-promoted cell migration. Moreover, a blocking antibody to this integrin inhibited invasion of the tumor cells into the brain tissue of nude mice, and glioma cells were found to secrete heterotrimeric α2-, α4- and α5-laminins. Our immunohistochemical studies of tumor tissue are consistent with the expression of Lm-211, Lm-411 and Lm511, and, to a lower extent, Lm-311, Lm-321, Lm-221, Lm-421 and Lm-521 in the tumor vasculature. Normal brain blood vessels are known to express several laminin isoforms and the larger vessels can display double BMs. The endothelial (inner) BM contains α4- and α5-laminins, whereas the parenchymal

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(outer) BM expresses α1- and α2-laminins, which are derived from epithelial meningeal cells and astroglia, respectively [33]. Though early studies were unable to detect α3-laminins in normal brain vasculature [33,34], positive reactivity for Lmα3 chain was recently found by two groups [35,36]. In glial tumors, blood vessels have been reported to express α1-, α2-, α4- and α5-, but not α3-laminins [34]. Moreover, a higher Lmβ1:Lmβ2 ratio, when compared to normal brain vasculature, has been also found, indicating that during tumor angiogenesis the laminin composition of vascular BM may shift. Gene microarray analysis has similarly demonstrated upregulation of α2-, α4-, α5, β1- and γ1-laminin gene expression in malignant gliomas [34,37,38]. When our results are compared to others, the apparent discrepancies concerning α1- and α3-expression in the tumor vasculature may be explained by the use of different antibodies. Concerning Lmα1, the faint/negative staining described by us (present study) was obtained with a mAb (EB7), whereas the positive staining reported by another group was obtained with polyclonal antibodies [34], which may have a stronger reactivity. On the other hand, the unambiguous positive staining of blood vessels in normal brain tissue with mAb EB7, observed in the present study, suggests significantly lower expression of α1-laminin(s) in the tumor vessels. Though we and others were unable to visualized double BMs in the vasculature of normal and tumor tissues by single immunohistochemistry and conventional microscopy (present study) [34], Farin et al. [5] have demonstrated by using triple fluorescence confocal microscopy that transplanted glioma cells migrate between the endothelial cells and the astrocytic end feet, which constitute two different BMs [33], of the host brain vasculature. Interestingly, these glioma cells displaced the astrocytes and interacted physically with the abluminal surface of the endothelial cells. This finding supports a role of α3- and α5-laminins, which are expressed by the endothelial BM [33,36], as promoters of glioma cell migration along blood vessels. Moreover, co-culture studies have demonstrated that glioma cells, as well as melanoma cells, localize and migrate along the external surfaces of vascular tubes generated from endothelial cells [2]. Lm-511, which strongly promoted glioma cell migration (present study), is a major endothelial laminin isoform [6,7]. In the in vitro cell migration assay, laminins covering all known α chains were selected, as these subunits are differentially bound by integrin receptors, mainly at the globular domain. Moreover, serum-free conditions were used to avoid contribution of other adhesive proteins such as fibronectin and vitronectin. Our present results confirm the migration-promoting activity of Lm-111 (laminin-1) and Lm-332 (laminin-5) described for several glioma cell lines, and of Lm-511 (laminin10) reported for a single glioma cell line, T98G, in a different context [16,23]. The negligible activity of Lm-211 (laminin-2) and Lm-411 (laminin-8) found by us is not due to an artifact because these preparation were biologically active for other cell types under similar conditions [29,39]. However, these results are in clear contrast with data obtained by other groups who reported strong migration-promoting activities of commercial preparations containing Lm-211, also known as merosin, and of a preparation of Lm-411 isolated from conditioned medium of T98G glioma cells by using immunoaffinity chromatography with a Lmβ1-specific mAb [16,21].

It is possible that the latter preparation was contaminated with small amounts of Lm-511, which is also found in T98G cells (our present results). This concept is supported by the different integrin usage and adhesive activity of Lm-411 preparations isolated from the glioma cells by either Lmβ1 or Lmα4 mAbs [40]. The recently reported reduction of glioma cell invasion both in vivo and in vitro by antisense inhibition of α4-laminins is intriguing and, in the context of our results, unexplained [32,41]. Concerning the commercial merosin preparations, we recently described contamination of the early preparations with other laminin isoforms, including small amounts of α5laminins [26]. Thus, purity of the preparations appears critical. In contrast to commercial mouse laminin which consists of Lm-111, the poorly characterized commercial human laminin preparations reported to promote glioma cell migration in early studies probably contained α5-laminins, though with variable degree of proteolytic degradation [26]. Moreover, a similar human laminin preparation was found to bind α3β1 integrin from glioma cells [42]. In the present study, reproducible results were obtained with five different preparation of recombinant Lm-411, and both molecularly characterized commercial Lm-211 and Lm-511 isolated from human placenta behaved similarly to their recombinant counterparts. Global integrin screening of glioma cell lines by flow cytometry identified α3β1 as the most abundant integrin of all. In a partial phenotyping, high expression of this lamininbinding integrin has been reported by others [12,22–24,43]. These high levels are not due to an artifact introduced by cell culture conditions since, by a similar screening, melanoma cell lines display αVβ3 as the predominant integrin (Oikawa et al., unpublished data). In fact, α3β1 integrin appears to be upregulated in malignant glioma tumor tissue as demonstrated by both immunohistochemistry and gene expression profiling [24,37,44]. In a glioma model, this integrin was mainly expressed in areas where the tumor cells invade the host tissue, compared to within the tumor parenchyma [14]. Though originally considered as a promiscuous receptor for a large variety of ligands, including collagens and fibronectin, recent studies have demonstrated that α3β1 integrin has a more restricted set of ligands [45]. In experiments with isolated integrins and purified laminin isoforms, α3β1 integrin showed clear specificity for α3- and α5-laminins, and no or minimal binding to α1-, α2- and α4-laminins [46]. This ligand specificity agrees very well with the laminin-isoform-specific promotion of glioma cell migration described in the present study. Moreover, the inhibition of cell migration by blocking mAbs to Intα3 and Intβ1 chains confirms the results obtained by two other groups which used Lm-332 and a human laminin preparation, presumably containing α5-laminins, as substrata [22,23]. In both α3- and α5-laminins, the LG3 module of their globular domain appears to be essential for α3β1 integrin binding [47–49]. Thus, independently of whether α3-laminins in brain vasculature exist as Lm-332, Lm-311, Lm-321, or any other β/γ chain combination, α3β1 integrin appears to be their common receptor, and the present results obtained with Lm332 may still apply to other α3-laminins. Likewise, the results obtained with Lm-511 may be relevant to other α5-laminins. Though α3- and α5-laminins are also recognized by α6β1 and α6β4 integrins, low expression of these receptors by glioma cells may explain the dominant role of α3β1 integrin. Moreover, low

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expression, if any, of α7β1 in the cells precludes any major role of this integrin. Though the Lmα5 chain contains RGD sequences in the short arm, and glioma cells expressed αVβ3 and αVβ5 integrins, we were unable to demonstrate participation of these integrins under the present experimental conditions [50,51]. Since inhibition of glioma cell migration on Lm-511 with mAbs to Intα3, Intα6 and/or Intβ1 was not 100%, minor participation of other adhesive receptors could be envisaged. On α1-laminin-promoted cell migration, the contrasting inhibitory effect of mAb to Intα3 on KG1C and U251 cells indicates a higher contribution of α3β1 integrin on the former cells. Since mAb to Intβ1 was equally inhibitory in both cell lines, the participation of β1 integrins other than α3β1 and α6β1 in U251, such as α7β1 integrin, is likely. In the present study, inhibition of glioma cell invasion into mouse brain tissue by a blocking antibody to Intα3 suggested interference of tumor cell migration. Since treatment with either mIgG control or mAb to HLA-ABC exerted minimal effects, antagonism of α3β1 integrin function appears to be the mechanism responsible for the antibody-mediated inhibition in vivo. Whether laminins participate in this model is presently unknown. However, these results point α3β1 integrin as a feasible target for anti-tumor therapy by inhibiting tumor cell migration. The same blocking antibody has been previously found to inhibit invasion of human glioma cells into rat brain aggregates, which apparently lack vasculature [52]. Thus, in addition to vascular laminins, glioma cells may utilize endogenous laminins, or exogenous laminin-related molecules or other permissive ligands during invasion into the brain tissue, for example along myelinated fiber tracts. Still, α3β1 integrin may be used by the glioma cells as a functional receptor. Thrombospondin-1, which is upregulated in malignant gliomas, is also a ligand for this integrin [45,53]. The present results do not exclude additional contribution of other integrins in glioma invasion. Excluding the vasculature, staining of the tumor tissue with the anti-laminin antibodies was faint or negative in the present study. Other groups have previously reported diffuse or punctate staining, immunoreactivity at the brain/tumor confrontation zone and/or lack of staining, depending on different conditions [12–14]. In glioma models, the brain tissue appears to react to the growing tumor by synthesizing laminins and other ECM proteins [12,14]. Moreover, astrocytes are also known to synthesize laminins [54]. Thus, both glioma cells and reactive astrocytes may secrete these molecules. A decade ago, LeMosy et al. [15] reported secretion of Y- and cross-shaped laminins by U251 glioma cells. These laminins are now identified as Lm-411/Lm-421 and Lm-511/Lm-521, respectively. The presently used methodology, namely immunoprecipitation followed by Western blotting, allows not only identification of the Lm chains but also demonstration of their physical association as heterotrimers. Our inability to detect Lmα2 in the Western blots may be due to the relative low amounts of α2-laminins, and the faint Lmα5 bands, when compared to the Lmβ1 and Lmγ1 bands, could be explained by the weaker reactivity of Lmα5-specific mAb 15H5 by Western blotting. Though all four glioma cell lines synthesized α2-, α4and α5-laminins, the ratio varied among the cell lines, being in general α5-laminins the most abundant and α2-laminins the least. In accordance with our data, Lm-411 was recently

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isolated from T98G glioma cells [16]. Faint or negative reactivity of tumor parenchyma with anti-laminin antibodies, in contrast to glioma cell lines, in the present study may be due to masking of the laminin epitopes in the tumor tissue and/or lower sensitivity of immunohistochemistry. Altogether the results suggest that vascular α3- and/or α5laminins contribute to glioma cell migration along the brain blood vessels and spreading of the tumor. On the other hand, glioma-derived laminins could be used by the tumor cells themselves, other glial cells or vascular endothelial cells during tumorigenesis. Moreover, the predominant expression of α3β1 integrin by glioma cells and its pivotal role in migration and invasiveness of the tumor cells makes antagonists of this integrin most promising agents for novel and more effective therapies of malignant gliomas.

Note added in proof During revision of the present paper, Chia et al. (Am J Pathol 170:2135 2007) reported evidence for a role of tumor-derived laminin-511 in the metastatic progression of breast cancer. Moreover, additional studies from our laboratory have recently demonstrated expression, recognition and use of α5-laminin(s) by human malignant melanoma cells (Oikawa and Patarroyo, unpublished data), similarly to glioma cells. These findings indicate participation of α5-laminin(s) and α3β1 integrin in several malignancies.

Acknowledgments The authors thank Drs. Ismo Virtanen, Eva Engvall, Kiyotoshi Sekiguchi, Randall Kramer and Samuel Wright for providing mAbs, Drs. Sergei Smirnov and Peter Yurchenco for providing rLm-211, and Drs. Monica Nistér and Bengt Westermark for their comments on the manuscript. This work was supported by the Swedish Cancer Society and Karolinska Institutet.

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