Elsevier

Experimental Cell Research

Volume 313, Issue 18, 1 November 2007, Pages 3819-3831
Experimental Cell Research

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

https://doi.org/10.1016/j.yexcr.2007.07.038Get rights and content

Abstract

Glioma cell infiltration of brain tissue often occurs along the basement membrane (BM) of blood vessels. In the present study we have investigated the role of laminins, major structural components of BMs and strong promoters of cell migration. Immunohistochemical studies of glioma tumor tissue demonstrated expression of α2-, α3-, α4- and α5-, but not α1-, laminins by the tumor vasculature. In functional assays, α3 (Lm-332/laminin-5)- and α5 (Lm-511/laminin-10)-laminins strongly promoted migration of all glioma cell lines tested. α1-Laminin (Lm-111/laminin-1) displayed lower activity, whereas α2 (Lm-211/laminin-2)- and α4 (Lm-411/laminin-8)-laminins were practically inactive. Global integrin phenotyping identified α3β1 as the most abundant integrin in all the glioma cell lines, and this laminin-binding integrin exclusively or largely mediate the cell migration. Moreover, pretreatment of U251 glioma cells with blocking antibodies to α3β1 integrin followed by intracerebral injection into nude mice inhibited invasion of the tumor cells into the brain tissue. The cell lines secreted Lm-211, Lm-411 and Lm-511, at different ratios. The results indicate that glioma cells secrete α2-, α4- and α5-laminins and that 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.

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 and in the perivascular space along blood vessels [2], [3], [4], [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], [7], [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 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 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], [12], [13], [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], [17], [18], [19], [20], [21], [22], [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 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], [26], [27], [28], [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 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.

Section snippets

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, laminin-1) 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

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

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 isoform-specific 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

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

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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