**2.3. Vascular component**

increase of approximately 15% on GAG content with longer chains compared to non‐lethal

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

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

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

breast cancer tissue [36].

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

**2.2. Fibrous component**

growth [37].

tissues.

Depending on their size and composition, blood vessels can be divided in capillaries (5–15μm), post‐capillaries/metarterioles (15–20μm), sinusoids (20–50μm), venules/ arterioles (50– 200μm), and veins/ arteries (>200μm). In order to grow over the limit of oxygen and nutrients diffusion, tumors have developed different strategies to provide blood supply. These mecha‐ nisms are sprouting angiogenesis: the growth of new capillary vessels out of preexisting ones; intussusceptive angiogenesis: the division of preexisting vessels in two new vessels by the formation of transvascular tissue in the lumen of the vessels; recruitment of endothelial progenitor cells or angioblasts; vessel co‐option: tumor cells can grow along existing vessels without evoking an angiogenic response; vasculogenic mimicry: tumor cells dedifferentiate to an endothelial phenotype and make tube‐like structures, providing tumors with a secondary circulation system; mosaic vessels: both endothelial cells and tumor cells form the luminal surface of the vessels [53–55].

In many aspects, tumor vessels are different from normal vessels. They are dilated, tortuous, and poorly covered by pericytes [56, 57]. The fact that tumor growth is dependent on angio‐ genesis has given rise to anti‐angiogenic therapies targeting different pro‐angiogenic mole‐ cules [58]. The tumor microenvironment comprises numerous signaling molecules and pathways that influence the angiogenic response. Understanding how these components functionally interact as angiogenic stimuli or as repressors and how mechanisms of resistance arise is required for the identification of new therapeutic strategies [59].

In some malignancies, different studies have shown conflicting results, some indicating a prognostic value of angiogenesis and others rejecting such conclusions [60–64]. This may arise from the fact that tissue vascularization has been quantified following different methods [62, 63], all based on the detection of the differential staining of the vessels with more or less specific immunostaining (anti‐factor VIII, von Willebrand factor, CD34, CD31, caveolin or CD105) on image sections [65], hot spots [60], whole sections [62], or in tissue microarrays sections [64], thereby indicating the need for robust morphometric techniques which may standardize the measurement of angiogenesis.

#### **2.4. Immune system**

The immune system is composed of different lineages of immune cells with different functions that protect every organism against infection. Among its tasks, immunosurveillance impedes transformed cells to become neoplastic cells. Indeed, increasing literature support the hypoth‐ esis that cancer development is influenced by the host immune system [66]. In fact, specialized blood vessels, the high endothelial venules (HEV), allowing the extravasation of lymphocytes, are present in primary human solid cancers [67, 68]. Therefore, it has been postulated that analyzing the composition, distribution, and architecture of the immune infiltrate for each tumor type, will offer new prognostic or predictive biomarkers [68, 69].

Immune infiltrates are heterogeneous between tumor types, and are diverse from patient to patient. All immune cell types may be found in a tumor, including macrophages, dendritic cells (DC), mast cells, natural killer (NK) cells, naïve and memory lymphocytes, B cells, and T lymphocytes (which include various subsets of T cell: TH1, TH2, TH17, regulatory T cells (TREGS), T follicular helper cells (TFH), and cytotoxic T cells). The analysis of the location, density, and functional orientation of different immune cell populations (termed the immune contexture) in large collections of annotated human tumors has allowed the identification of components that are beneficial for patients and those that are deleterious [70–72]. The prog‐ nostic impact of immune cells such as B cells, NK cells, myeloid derived suppressor cells (MDSC), macrophages, and subset of Thelper populations (TH2, TH17, TREG cells) may differ depending on the type of cancer, and on the cancer stage [72]. In contrast, cytotoxic T cells, TH1 cells, and memory T cells were strongly associated with good clinical outcome for all cancer types [66, 72]. Notably, two large studies have shown that tumor immune infiltrate patterns and subsets in colorectal cancer are significant prognostic biomarkers [73, 74]. A potential clinical translation of these observations is the establishment of an Immunoscore, based on the numeration of two lymphocyte populations (CD3/CD45RO, CD3/CD8 or CD8/ CD45RO), both in the core and in the invasive margin of tumors, as a clinically useful prog‐ nostic marker [75]. This immunoscore sheds light into the prognostic role of the tumoral immune infiltrate, but still needs to be validated in colorectal cancer. Its utility in other malignancies should be tested as well.
