The extracellular matrix (ECM)1
is a fundamental and important component of metazoan organisms providing architectural support and anchorage for the cells. The ECM consists of a complex meshwork of highly cross-linked proteins and exists as interstitial forms within organs and as specialized forms, such as basement membranes underlying epithelia, vascular endothelium, and surrounding certain other tissues and cell types (e.g.
neurons, muscles). Cells adhere to the ECM via transmembrane receptors, among which integrins are the most prominent (1
). These cell-matrix interactions result in the stimulation of various signaling pathways controlling proliferation and survival, differentiation, migration, etc. The composition of the ECM and the repertoire of ECM receptors determine the responses of the cells. The biophysical properties of the ECM (deformability or stiffness) have also been shown to modulate these cellular functions (3
). In addition to core ECM components (fibronectins, collagens, laminins, proteoglycans, etc.), the ECM serves as a reservoir for growth factors and cytokines and ECM-remodeling enzymes that collaborate with ECM proteins to signal to the cells (5
). Hence, the ECM provides not only biophysical cues but also biochemical cues that regulate cell behavior. In addition to being important for normal development, alterations of the ECM have been associated with various pathologies such as fibrosis, skeletal diseases, and cancer (7
) and it has been emphasized recently that the ECM proteome needs better characterization (10
The role of the ECM in cancer is of particular interest. Long-standing as well as recent data implicate tumor ECM as a significant contributor to tumor progression. Indeed, the ECM is a major component of the tumor microenvironment (11
) and classical pathology has shown that excessive deposition of ECM is a common feature of tumors with poor prognosis. More recently, gene expression screens have revealed that many genes encoding ECM components and ECM receptors are dysregulated during tumor progression (13
). Finally, modifications of the extracellular matrix architecture and biophysical properties have been shown to influence tumor progression (6
). Despite these clear indications that tumor ECM and the interactions of cells with it are very likely to play important roles in tumor progression, we do not have a good picture of ECM composition, origins and functions in tumors. One reason for this lies in the biochemical properties of ECM proteins (large size, insolubility, cross-linking, etc.) that have rendered very challenging attempts to characterize systematically the composition of the ECM from tissues and tumors.
Thanks to the completion of the genomes of many species and to previous studies (19
), it is now clear that vertebrate genomes contain hundreds of genes encoding ECM proteins. Specific features of ECM proteins have emerged from these studies, in particular their distinctive structures based on the repetition of conserved domains (22
). During the last few years, several attempts have been made at in silico
predictions of the complement of ECM proteins (24
). Furthermore, recent studies have begun to characterize experimentally the composition of the extracellular matrix of specific model systems such as retinal and vascular basement membranes (27
), mammary gland (30
), and cartilage (32
). However, there remains a pressing need for a better definition of the number and diversity of ECM proteins and even of what should be included in that definition. Limitations arise also from the lack of experimental reagents and approaches because of the biochemical intractability of ECM and the lack of an adequate library of antibodies or other probes to characterize ECM proteins in situ
. Thus, deciphering the complexity of the extracellular matrix in vivo
represents an important scientific challenge.
We describe here the development of proteomics-based methods coupled with a bioinformatic definition of the “matrisome” (ECM and ECM-associated proteins) to analyze the protein composition of the tissue extracellular matrix. We have successfully applied this strategy to characterize in detail the extracellular matrices both of normal murine tissues (lung and colon) and of melanoma tumors (nonmetastatic and metastatic), which each comprise well over 100 proteins. Moreover, we have applied this approach to understand the origins of tumor ECM proteins and have been able to show, using human into mouse xenograft models, that both tumor cells and stromal cells contribute in characteristic ways to the ECM of the tumor microenvironment. Furthermore, we show that both tumor and stromal cells contribute to significant changes in the extracellular matrices of tumors of differing metastatic potential. The strategy we describe and illustrate here can be broadly applied and we provide protocols and inventories of ECM domains and proteins to facilitate application of these methods by others.