**2.1. Glycosaminoglycans**

(fibroblasts, Schwann cells), tumor‐associated immune cells, and vascular (blood and lymph) endothelial cells are found, lying among a network of various reticulin, collagen, and elastic fibers merged within the interstitial fluids (glycosaminoglycans, proteoglycans, and glycopro‐ teins) and gradients of several chemical species, which constantly interplay with cells and provide much of the structural support available to parenchymal cells in tissues, providing

Numerous studies have demonstrated that the tumor ECM not only responds to and supports carcinogenesis, but actively contributes to tumor initiation, progression, and metastasis, and must not only be understood as a reactive neighboring, but also as an active contributor [4]. In fact, it has been published that chronic growth stimulation, ECM remodeling, alteration of cell mechanics, and disruption of tissue architecture are a non‐genetic basis, influencing cancer progression [5–7]. Given the complexity both within and outside the cancer cell, and the interactions between cancer cells and the surrounding stroma, it is not surprising that a single perturbation within a tumor can create a cascade of changes in multiple pathways and

Tumor malignancy is driven, among other factors, by the remodeling of contiguous stromal tissue to foster growth, metastasis, and therapy resistance. Tumor cells alter the mechanical properties of the microenvironment to create favorable conditions for their proliferation and/ or dissemination [9]. They promote stiffening of their environment, which feeds back to increase malignant behaviors such as loss of tissue architecture and invasion [10]. Matrix remodeling by tumor‐associated stromal cells entails the assembly of a highly dense ECM, whose physicochemical attributes enhance malignancy through morphogenic deregulation, tumor cell proliferation, vascular recruitment, and stromal cell differentiation [11–13]. Both tumor and stromal cells produce and assemble a matrix of collagens, proteoglycans, and other molecules such as cytokines that hinder the transport of macromolecules and stimulate the otherwise quiescent host cells to initiate a variety of processes, including desmoplasia and angiogenesis [14, 15]. Mediated by increased deposition, unfolding, and crosslinking of fibrilar adhesion proteins, stiffening increases cell contractility which, in turn, can directly and indirectly modify gene expression via altering transcription factor activity and the release of matrix‐bound pro‐tumorigenic growth factors [16–19]. Similarly, changes in the microstruc‐ ture, elasticity, distribution of pore sizes, chemical composition, and fiber arrangement due to ECM remodeling can control aspects of tumor cell phenotype such as adhesion, mechanics, and motility [20–24]. The speed of malignant cells *in vitro* is also affected by the geometry of the ECM. Human glioma cells move faster through narrow channels than through wide channels or in non‐stretched 2D surfaces. This is thought to be triggered by an increase in the polarity of the traction forces between cell and ECM [25]. Recent publications describe that not only neoplastic ECM stiffness, but also the firmness of tumor cells play a significant role in tumor progression. Steadiness of tumor cells, especially the metastatic cells, is lower than that of the normal cells of the same sample, and is currently caused by the loss of actin filaments

and/or microtubules, and the subsequent lower density of the scaffold [26, 27].

The study of ECM elements composing the architectural scaffolding and the blood vessels can be performed by means of automated or semi‐automated quantification. This technique can

tensile strength and flexibility [2, 3].

262 Composition and Function of the Extracellular Matrix in the Human Body

networks, some of which may have lethal repercussions [8].

Since glycosaminoglycans (GAGs) have been recognized as essential players in critical biological processes regulating cellular properties, owing to their particular biofiltering, scaffolding, and cell anchoring properties, they have been related to diverse malignancies and they are now understood as key elements involved in cancer cell biology and novel thera‐ peutical agents [28, 29].

GAGs are long, non‐branched polymers of several disaccharides (up to 200 repeated saccha‐ rides), consisting of one uronic acid (almost always glucuronic acid and sometimes iduronic acid) and one hexosamine (glucosamine or galactosamine), presenting variable degrees of sulfation, and constitute the main components of the fundamental substance of the ECM [2]. According to their chemical composition, GAGs can be divided in acid and sulfated: chon‐ droitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate or heparin (HS, Hep); acid and non‐sulfated: hyaluronan (HA); and non‐acid and sulfated: keratan sulfate (KS). GAGs can form proteoglycans by means of a linkage tetrasaccharide, linking GAGs to a central protein (core protein) through a serine residue and catalyzed by four specific enzymes. The formation of the linkage tetrasaccharide is key for GAG synthesis to start in *GAGosomes* [30–32]. Proteo‐ glycans can be located at the cell surface (syndecan, glypican), in the intracellular compartment (serglycin), secreted to the extracellular medium (small leucine‐rich proteoglycans, hyalec‐ tans), or within the basement membrane (agrin, collagen XVIII, perlecan) [33, 34].

Diagnostic methods have typically centered on the analysis of GAG structure and concentra‐ tion. Hyaluronan has the capacity to bind large amounts of water to form viscous gels with special filtering properties and it is capable of forming polyvalent connections with other ECM proteins. This GAG, as well as the rest, has the capacity of modulating host‐tumor interaction, lymphangiogenesis, angiogenesis, and multidrug resistance. It can therefore be used as a drug carrier [35]. Hyaluronan accumulates in the stroma of various human tumors and modulates intracellular signaling pathways, cell proliferation, motility, and invasive properties of malignant cells: high stromal hyaluronic acid content is associated with poor differentiation and aggressive clinical behavior in human adenocarcinomas. On the contrary, squamous cell carcinomas and malignant melanomas tend to have a reduced HA content [35]. When tumors produce hyaluronan, this fact is associated with invasion, host‐tumor interactions, lymphan‐ giogenesis/angiogenesis, epithelial‐mesenchymal transition and with local and distant metastases in glioma, pancreatic adenocarcinoma, breast cancer and prostate cancer, respec‐ tively [35]. Other studies in prostate carcinoma have found a link between poor prognosis and under‐sulfation or overexpression of chondroitin sulphate. Aggressive breast cancer shows an increase of approximately 15% on GAG content with longer chains compared to non‐lethal breast cancer tissue [36].

## **2.2. Fibrous component**

The architectural role of the fibrous component of the ECM is clear and central for tissue homeostasis. In fact, scaffold architecture has been found to have a significant impact on cell growth [37].

The primary proteins present in the ECM are the collagens. They are secreted by connective tissue cells, as well as by a variety of other cell types. As a major component of skin and bone, they represent the most abundant proteins in mammals, constituting 25% of the total protein mass in these animals. A typical collagen molecule is extremely rich in proline and glycine, long, stiff, and presents a triple‐stranded helical structure, in which three collagen polypeptide chains called α chain are wound around one another in a ropelike superhelix [38]. Reticulin fibers, or type III collagen, are fine fibers forming an extensive branching network in certain organs. Their distribution is rather restricted: they are usually found mainly in the basement of epithelial tissues, the surface of adipose cells, muscle cells and Schwann cells, outside the endothelium of the hepatic sinusoids, and in the fibrous reticulum of lymphoid tissues. These fibers have a diameter of less than 2 μm and support not only the physical structure of the cell, but also various biological functions, largely through their ability to bind multiple interacting partners such as other ECM proteins, growth factors, signal receptors, and adhesion molecules [3, 39]. Collagen type I fibers account for 90% of the body collagen and usually form thick bundles in bone, skin, tendons, ligaments, cornea, and internal organs. These collagen strands measure up to 100 μm thick and usually follow a wavy course without branching in normal tissues.

The architecture of the collagen scaffolds in tumors is severely altered [18, 40]. It has been found that an aligned fiber matrix enhances differentiation of human neural crest stem cells towards the Schwann cell lineage [41] and evidence has pointed to collagen crosslinking as a significant contributor to the changes in cellular mechanical microenvironment that accom‐ panies tumor progression [42]. Reticulin fibers are considered to play an important role in the adherence of cells and constitute a skeletal framework suitable for individual cells and tissues [43]. They are known to increase in amount and disorganize with aging and stress in physio‐ logical conditions [44]. Desmoplasia (collagen type I and reticulin fibers accumulation) is associated with several malignancies. The deregulation and disorganization of the tumor stroma alter the composition, structure, and stiffness of the ECM, leading to the creation of niches within tissues and organs that offer a proper environment for tumors to successfully establish metastasis and activate therapy resistance programs [40, 45, 46]. In primary breast tumors, collagen type I density is associated with breast cancer metastasis, and may serve as an imaging biomarker of metastasis. The expression of COL11A1 gene continuously increases during ovarian cancer disease progression, with the highest expression in recurrent metasta‐ ses. Knockdown of COL11A1 decreases in vitro cell migration and invasion and tumor progression in mice [47]. The tumor‐stromal interface of breast primary tumors in 3D culture was studied with second harmonic generation, showing that randomly organized matrix realigned the collagen fibers allowing individual cells to migrate out along radially aligned fibers [48].

Elastic fibers are generally twisted or straight strands of 0.2–1.5 μm which sometimes branch to form a coarse network in loose connective tissue or form flattened sheets in dense elastic tissues such as the aorta [39]. As basic structural elements, elastic fibers aberrations trigger severe pathologies such as Marfan's syndrome, emphysema, hypertension, actinic elastosis, and aortic aneurysms [49]. Nevertheless, little evidence has been reported about its role in neoplastic processes. It has been found that elastic fiber increase, together with a decrease in collagen fibers is associated with oral squamous cell carcinoma and lymph node metastasis [50]. Elastin‐rich lung ECM is largely remodeled during tumor invasion. The degradation of elastin produces peptides displaying a wide range of biological activities and increase invasive capacities of lung cancer cells by post‐transcriptional regulation of metalloproteinase‐2 [51]. This mechanism has also been found to act in melanoma progression, another cancer associated with rich elastin microenvironment [52].
