Cell. Mol. Life Sci. (2013) 70:4431–4448 DOI 10.1007/s00018-013-1339-8
Cellular and Molecular Life Sciences
Review
Positive and negative influence of the matrix architecture on antitumor immune surveillance Elisa Peranzoni · Ana Rivas‑Caicedo · Houcine Bougherara · Hélène Salmon · Emmanuel Donnadieu
Received: 12 November 2012 / Revised: 18 March 2013 / Accepted: 8 April 2013 / Published online: 7 May 2013 © Springer Basel 2013
Abstract The migration of T cells and access to tumor antigens is of utmost importance for the induction of protective anti-tumor immunity. Once having entered a malignant site, T cells encounter a complex environment composed of nontumor cells along with the extracellular matrix (ECM). It is now well accepted that a deregulated ECM favors tumor progression and metastasis. Recent progress in imaging technologies has also highlighted the impact of the matrix architecture found in solid tumor on immune cells and especially T cells. In this review, we argue that the ability of T cells to mount an antitumor response is dependent on the matrix structure, more precisely on the balance between pro-migratory reticular fiber networks and unfavorable migration zones composed of dense and aligned ECM structures. Thus, the matrix architecture, that has long been considered to merely provide the structural framework of connective tissues, can play a key role
E. Peranzoni · H. Bougherara · E. Donnadieu Inserm, U1016, Institut Cochin, Paris, France E. Peranzoni · H. Bougherara · E. Donnadieu Cnrs UMR8104, Paris, France
in facilitating or suppressing the antitumor immune surveillance. A new challenge in cancer therapy will be to develop approaches aimed at altering the architecture of the tumor stroma, rendering it more permissive to antitumor T cells. Keywords Tumor · T cells · Stroma · Extracellular matrix · Motility · Imaging Abbreviations 3D Three-dimensional ECM Extracellular matrix EMT Epithelial-mesenchymal transition FRC Fibroblastic reticular cells LOX Lysyl oxidase LTi Lymphoid tissue inducer MMP Metalloproteinases SHG Second-harmonic generation SLO Secondary lymphoid organs TACS Tumor-associated collagen signature TIL Tumor-infiltrating lymphocytes TLO Tertiary lymphoid organs
E. Peranzoni · H. Bougherara · E. Donnadieu Université Paris Descartes, Sorbonne Paris Cité, France
Introduction
A. Rivas‑Caicedo Alta Tecnología en Laboratorios SA de CV, Comoporis #45, El Caracol, Mexico, Mexico
A large majority of human solid tumors are of epithelial origin. However, carcinomas cannot be simply considered as a group of proliferating epithelial cells but rather as a complex ecosystem composed of a variety of cells organized in a specialized microenvironment, referred to as stroma. Indeed, the tumor stroma contains non-cancer cells, including endothelial cells and fibroblasts, along with the ECM. Although epithelial neoplastic cells and stromal cells are found in distinct areas (Fig. 1a), the growth of a tumor depends on dynamic and complex interactions between these cell populations.
H. Salmon Department of Oncological Sciences, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA E. Donnadieu (*) Département d’Immunologie et d’Hématologie, Institut Cochin, 22 Rue Méchain, 75014 Paris, France e-mail:
[email protected]
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4432 Fig. 1 Spatial organization of tumor cells, T cells and ECM components in human lung and ovarian carcinomas. a In a human lung tumor, tumor cells (brown, stained for cytokeratin) form well-delineated tumor islets surrounded by a stroma (blue). b In a human lung tumor, T cells (brown, stained for CD3) are preferentially distributed within the tumor stroma and very rarely in contact with tumor cells. Dotted white lines denote tumor islets. c In a human lung tumor, thick and linear fibronectin fibers (brown, stained for fibronectin) surround tumor islets denoted by dotted white lines. d In a human ovarian tumor, tumor cells (red, stained for EpCAM) are surrounded by straight and parallel collagen fibers (green) detected by SHG on a two-photon microscope. Bar 100 μm
E. Peranzoni et al.
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An increasing body of evidence suggests that several cells of the immune system are able to control the growth of a tumor. For instance, the presence of high numbers of memory Th1 T cells and CD8+ cytotoxic T cells has been reported as an indicator of good prognosis in many human cancers, such as melanoma or lung cancer [1]. However, to mount an effective antitumor response, T cells must pass through several distinct steps. First, T lymphocytes need to be fully activated by mature dendritic cells in the tumor-draining lymph node. Second, cancer-specific effector T cells must enter the tumor after they have left the blood vessels. Finally, tumor-infiltrating lymphocytes (TIL) need to accomplish their functional activities which eventually lead to tumor regression. However, experiments performed over the last decade indicate that the tumor environment has the ability to compromise antitumor immunity, and several escape mechanisms have been identified [2]. For example, the entry of lymphocytes into the tumor does not always occur normally, mostly due to abnormal vessel formation and reduced expression of adhesion molecules [3]. In addition, T cells found within a progressive tumor are usually unable to respond normally to antigen stimulation and show signs of anergy [4, 5]. Finally, T lymphocytes, as well as other immune cells, are not randomly distributed within tumors and are
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Fibronectin
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Tum. / Collagen (SHG)
usually more concentrated in the stroma than in tumor islets [6–9] (Fig. 1b). In recent years, functional alterations of lymphocytes in the tumor microenvironment have been accurately reviewed [10]. However, defects in T cell infiltration into tumor islets might well constitute an important obstacle for T cell-mediated antitumor activity, and understanding the mechanisms underlying the distribution of T cells would be of major interest. Given the pivotal role of chemokines in controlling the positioning of immune cells in various tissues, the paucity of T cells in carcinoma regions has been envisaged to result from a lack or an inactivation of chemoattractants within tumor islets [11, 12]. Another plausible mechanism by which T cells are prevented from entering tumor islets could be due to the expression of molecules by tumor cells that will act as a chemical barrier for T cells. These include proteins with chemorepulsive capacities like neuropilin-1, originally described in the nervous system and, more recently, in the immune system [13] but also chemokines that, under certain conditions, e.g., high doses or chronic exposure, can provoke active movements of lymphocytes away from chemokinetic agents [14]. Apart from features that depend on tumor cells, the recent development of improved imaging technologies, enabling the dynamic visualization of cells within intact
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Matrix architecture and antitumor immune surveillance
tissues, has identified additional environmental cues that regulate the distribution and migration of T cells [15]. Indeed, evidence is accumulating that the ECM, by its physical and biochemical features, is essential for regulating T cell behavior. Along with signals transmitted by ECM proteins, leading to T cell activation and differentiation, the architecture of a tissue can both facilitate and create environmental obstacles to T cell traffic. In this review, we will examine the diverse matrix structures found in normal, chronically inflamed, and tumor tissues, focusing our attention on the recent advances regarding the role of the ECM in the control of immune cell migration. We will also discuss some potential therapeutic approaches that, by modifying the ECM architecture, could favor T cell ability to contact and eliminate tumor cells.
Matrix architecture: from normal to tumor situation Components and structures of the ECM In this review, we will focus on the interstitial matrix that leukocytes encounter once they have entered a tissue. The structure and function of the basement membrane, which is a specialized form of sheet-like ECM interacting with the epithelium and endothelium, will not be addressed here. The ECM is the non-cellular component of any tissue and organ [16]. It can be functionally divided into two main classes of macromolecules: the fibrillar fraction, that consists of arrays of fibrillar collagen bundles, elastin and fibronectin fibers, and the non-fibrillar fraction, which mainly consists of glycosaminoglycans and proteoglycans. These large and negatively charged sugars efficiently bind water and fill the space between the fibers. The ECM primarily fulfils a structural role by maintaining an insoluble scaffold, which ultimately defines the shape and stiffness of organs. Most ECM components are produced by fibroblasts that also play a role in their assembly into fibers and their spatial disposition. Collagen cross-linking is almost exclusively mediated by an enzyme, the lysyl oxydase (LOX) [17]. The synthesis and cross-linking of ECM fibers is balanced by the action of metalloproteinases (MMPs) that degrade collagen and other ECM proteins [18]. Depending on the origin of the tissue and on the context (injury, inflammation, tumor), the three-dimensional (3D) matrix architecture can adopt a variety of conformations [19]. In some tissues, the ECM can be dense and stiff, while, in others, it is soft and more porous with gap size of different diameters. ECM fibers also display highly variable thickness, straightness, and spatial arrangements. They can be relaxed and non-oriented or, in contrast, linearized and oriented in a specific direction. These physical
characteristics determine the architecture of the tissue but, as we will see later, they are also essential for regulating immune cell migration and behavior. Our understanding of the matrix structure mainly comes from histological techniques, which include immunostaining, as well as the use of Masson’s trichrome and picrosirius red to stain collagens and examine their linearization and orientation. However, these standard procedures require tissue processing such as fixation, paraffin embedding, and cutting and therefore give only a static snapshot of the ECM and cannot document its complex dynamics. More specialized techniques, such as second harmonic generation (SHG) microscopy, have addressed these limitations [20]. SHG is an intrinsic signal highly sensitive to collagen that therefore rules out the use of external dyes. In addition, the non-linear excitation of a multi-photon microscope permits deep penetration in freshly biopsied tissues. Thus, a 4D spatio-temporal resolution of fibrillar collagen in thick samples, and even in intact organs, can be assessed with this non-invasive technique. However, SHG cannot be a routine technique designed to replace standard histological techniques, because of the expense of multiphoton microscopes and the amount of time required for the analysis, but it can still be envisaged as a complement to semi-quantitative histopathology of collagen deposition in tissues. Matrix architecture in a normal epithelium Epithelial tissues usually consist of a single layer of epithelial cells surrounded by a stroma composed primarily of non-activated fibroblasts, rare immune cells, and endothelial cells forming blood and lymph vessels. Even if each tissue has a unique matrix composition and structure, the stroma of a normal epithelium contains a relaxed meshwork of collagen fibers and ECM components, such as elastic fibers and fibronectin, embedded in a hydrogel of proteoglycans that fills most of the interstitial space. As an example, the density and physical organization of the collagen network found in normal mammary tissue have been relatively well characterized. By using SHG, several studies have shown the presence of collagen fibrils with a rod-like structure, measuring 67 nm in diameter in stromal regions surrounding epithelial cells [19, 21–24]. These works highlight the heterogeneous texture of the collagen networks in normal breast. Thereby, the presence of wavy, as well as straight, collagen fibers have been observed wrapping around the epithelial duct but also radiating away from the duct. Additionally, the connective tissue displays dense collagen bundles interspersed with loosely organized networks of variable widths, ranging from below 1 to 20 μm. As we will see later, these ECM determinants have profound impact on the way immune cells migrate within a tissue.
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The structure of a tissue is tightly controlled by multiple regulatory mechanisms that ensure organ homeostasis, so that any deviation from the steady state induces a response aimed at rapidly restoring the integrity of the tissue. Notably, disruption of such control mechanisms must be transient in order to avoid organ dysfunction. Alterations of matrix architecture in wound healing, fibrosis and chronic inflammation Alterations of the matrix architecture are observed in several physiological and pathological conditions. Acute tissue injury triggers a reparation program during which the architecture of the stroma is strongly modified [25]. Multiple events are involved in repair, one of them being the recruitment to the wounded tissue of fibroblasts that become activated into myofibroblasts. These cells are actively involved in the tissue remodeling required for healing [26]. First, they exhibit increased capacities to produce and cross-link ECM proteins into fibers compared to non-activated fibroblasts [27]. Second, due to their contractile activity, myofibroblasts are able to organize matrix fibrils into linear and thick cables. This rich collagen network is beneficial to the host, as it provides a scaffold onto which other cells can migrate during tissue repair. In normal conditions, this activation phase is transient and, once the tissue has regained its integrity, myofibroblasts die by apoptosis. However, under extreme conditions, such as prolonged tissue injury, myofibroblasts remain within the tissue leading to continuous ECM deposition, organ fibrosis, and, finally, organ dysfunction [26]. Chronic inflammation that accompanies an infection or an autoimmune disease gives rise to tissue modifications that can adopt different textures. The formation and development of fibrous or connective tissue (defined as desmoplasia) is a major pathological feature of many chronic inflammatory diseases, including autoimmune diseases
such as scleroderma, rheumatoid arthritis, and Crohn’s disease [28]. Apart from regions harboring extensive collagen deposition, a variety of inflamed tissues, both in human and mice, are often heavily infiltrated with immune cells that form aggregates showing various degrees of internal organization [29–32]. In some, but not all, cases, these inflammatory infiltrates closely resemble secondary lymphoid organs (SLO), especially lymph nodes, with regard to cellular composition, organization, chemokines, and vasculature. For this reason, such organized structures induced at sites of chronic inflammation are usually referred to as tertiary lymphoid organs (TLO) [29]. According to pathologists, chronic infiltrates must fulfill a series of criteria to be named TLO, including distinct yet adjacent T and B cell compartments, the existence of functional germinal centers, and the presence, in the T cell area, of high endothelial venules (HEV), as well as a specialized subtype of fibroblasts. In both secondary and tertiary lymphoid organs, fibroblasts present within the T cell zone express contractile molecules that are normally restricted to smooth muscle cells (desmin, smooth muscle actin, etc.) and myofibroblasts of wounded and fibrotic tissues [33] (Table 1). These fibroblasts, named fibroblastic reticular cells (FRC), have been relatively well studied in secondary lymphoid organs, but less so in chronically inflamed tissues. These cells are responsible for building a 3D network that is highly interconnected via cell–cell contacts (Fig. 2, left panel). In SLO, FRC produce multiple ECM components that are specifically assembled around a core of collagen fibers and disposed in microvessels, called conduits, capable of transporting small molecules [34, 35]. Remarkably, unlike other tissues where the ECM produced by fibroblasts surrounds the cells, matrix fibers of the lymph nodes are ensheathed by FRC. As a result, most of the conduit system is shielded from lymphocytes within the T cell zone. By analogy, this 3D network, composed of FRC wrapping ECM fibers, structurally resembles
Table 1 Key features of reticular fiber and fibrotic networks
Physical characteristics Tissue gaps Disposition of matrix fibers Phenotype of fibroblasts
Reticular fiber network
Fibrotic network
15–20 μm [7, 36] In conduits, ensheathed by FRC [35] Among many genes, FRC express αSMA, gp38, CCL19, CCL21, IL-7 [33]
