A tumor can be considered as a highly heterogeneous functional tissue, connected and dependent on the microenvironment, which sends and receives signals to and from the tumor tissue itself. Tumor cells alter the mechanical properties of the microenvironment in order to create favorable conditions for their proliferation. Stromal cells and non‐cellular elements of the extracellular matrix (ECM), including the host immune system, the fibrous scaffolding, the fundamental substance, and blood vascularization can determine tumoral cell morphologies, functions, aggressiveness, and response to treatment, as well as an accurate assessment of prognosis of the patients. Robust morphometric digital pathology techniques that are able to standardize measurements and analyse whole sets of immunohistochemical images are called for to identify, describe, and quantify the elements of the ECM. The computer‐automated segmentation algorithms are therefore required to increase the knowledge on the tumor microenvironment heterogeneity and to provide new therapeutic targets.
- Extracellular matrix
- digital pathology
- tumor microenvironment
- reticulin fibers
Many processes relevant to cancer, such as differentiation, maturation, and the malignant potential of tumor cells have all been shown to be influenced by extracellular matrix (ECM) stiffness . In the general context of cancer, within the tumor ECM, various support cells (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 glycoproteins) 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 tensile strength and flexibility [2, 3].
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 . 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 networks, some of which may have lethal repercussions .
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 . They promote stiffening of their environment, which feeds back to increase malignant behaviors such as loss of tissue architecture and invasion . 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 microstructure, 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 . 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 be used on histologic sections of all malignancies, providing important tumor characteristics such as the morphometric features and the spatial context of tumor and stromal cells at single‐cell resolution. Differences in the ECM of different cancer subtypes can contribute to a more comprehensive understanding of the highly heterogeneous tumor microenvironment, and can provide abundant and novel targets for therapy.
2. Masterpiece of biology
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 therapeutical agents [28, 29].
GAGs are long, non‐branched polymers of several disaccharides (up to 200 repeated saccharides), 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 . According to their chemical composition, GAGs can be divided in acid and sulfated: chondroitin 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]. Proteoglycans can be located at the cell surface (syndecan, glypican), in the intracellular compartment (serglycin), secreted to the extracellular medium (small leucine‐rich proteoglycans, hyalectans), or within the basement membrane (agrin, collagen XVIII, perlecan) [33, 34].
Diagnostic methods have typically centered on the analysis of GAG structure and concentration. 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 . 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 . When tumors produce hyaluronan, this fact is associated with invasion, host‐tumor interactions, lymphangiogenesis/angiogenesis, epithelial‐mesenchymal transition and with local and distant metastases in glioma, pancreatic adenocarcinoma, breast cancer and prostate cancer, respectively . 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 .
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 .
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 . 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  and evidence has pointed to collagen crosslinking as a significant contributor to the changes in cellular mechanical microenvironment that accompanies tumor progression . 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 . They are known to increase in amount and disorganize with aging and stress in physiological conditions . 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 metastases. Knockdown of COL11A1 decreases in vitro cell migration and invasion and tumor progression in mice . 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 .
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 . As basic structural elements, elastic fibers aberrations trigger severe pathologies such as Marfan’s syndrome, emphysema, hypertension, actinic elastosis, and aortic aneurysms . 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 . 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 . This mechanism has also been found to act in melanoma progression, another cancer associated with rich elastin microenvironment .
2.3. Vascular component
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 mechanisms 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 angiogenesis has given rise to anti‐angiogenic therapies targeting different pro‐angiogenic molecules . 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 .
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 , hot spots , whole sections , or in tissue microarrays sections , 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 hypothesis that cancer development is influenced by the host immune system . 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 prognostic 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 . 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 prognostic marker . 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.
3. Methods for the quantification of ECM elements above mentioned
The methods explained are based on the experience of the group in the study of the ECM in neuroblastic tumor samples.
The study of tissue microarrays (TMAs) have emerged as a tool for rapid analysis for diagnostic and prognostic studies because several markers can be tested in huge amounts of samples [76–79]. The main advantage of TMAs consists in the standardization of the methods, given that all samples contained in a single TMA are subject to the same protocols. TMA are mainly used to validate biologic markers with potential diagnostic value, which can be used to develop screening programs or enhance a subclassification of a disease . The steps to follow are summarized in Figure 1. Alternatively, whole slides for individual samples can be used.
Several serial sections of 3μm can be made and stained with histochemistry (HC) for GAGs, reticulin fibers and collagen type I fibers, and with immunohistochemistry (IHC) anti‐CD31 for blood vessels.
Alcian blue pH 2.5 Stain Kit (Artisan™, Dako) stains acid GAGs (HS, CS, DS, and HA), sulfomucins and sialomucins in blue, the nucleus in red and the cytoplasm in pale pink (Figure 2).
Reticulin stains are silver stains based on the argyrophilic properties of reticulin fibers and a slightly modified Gomori can be used, which stains reticulin fibers in black (Figure 3). The first step in the staining procedure consists of oxidation of the hexose sugars in reticulin fibers to yield aldehydes. The second step is called sensitization in which a metallic compound such as ammonium sulfate is deposited around the reticulin fibers, followed by silver impregnation in which an ammonical or diamine silver solution is reduced by the exposed aldehyde groups to metallic silver. Further reduction of the diamine silver is achieved by transferring the sections to formaldehyde; this step is called revealing. The last step consists of toning by gold chloride in which the silver is replaced by metallic gold and the color of the reticulin fibers changes from brown to black. Masson's trichrome stain consists in the sequential staining with Harris hematoxylin which stains nuclei dark red, aniline blue which stains collagen and reticulin blue, and molybdic orange G which stains erythrocytes dark orange (Figure 4). Orcein is a natural dye obtained from lichens which are found to stain elastic fibers dark brown (Figure 5).
CD31 is a single chain type 1 transmembrane protein with a molecular mass of approximately 135 kDa, belonging to the immunoglobulin superfamily. CD34 can also be applied to a subgroup, but this marker also stains cells other than endothelial. CD31 is expressed on endothelial cells of epithelial origin (all continuous endothelia, including those of arteries, arterioles, venules, veins, and capillaries, but it is not completely expressed on discontinuous endothelium in, for example, splenic red pulp). In addition, CD31 is expressed diffusely on the surfaces of megakaryocytes, platelets, myeloid cells, natural killer cells, and some subsets of T cells, as well as on B‐cell precursors. Cells labeled by the antibody predominantly display membrane staining with weaker cytoplasmic staining (Figure 6).
Finally, IHC is used to detect diverse subpopulations of lymphocytes in tumoral tissues. For example, CD45 is a transmembrane glycoprotein expressed on most nucleated cells of hematopoetic origin, i.e., all human leucocytes; CD20 reacts with an epitope located on the surface of B cells and appears early during B‐cell maturation; CD3 is a pan‐T cell marker for identification of T cells. It is well‐suited for labeling reactive T cells in tissue with lymphoid infiltrates, and for classification of T‐cell neoplasms; CD7 is expressed by the majority of peripheral blood T cells, NK cells, and all thymocytes. It is one of the earliest surface antigens on T and NK‐cell lineages; CD4 is a transmembrane glycoprotein, expressed on normal thymocytes, T‐helper cells, majority of mature peripheral T cells, and a subset of suppressor or cytotoxic T cells; CD8 is a 68 kDa transmembrane glycoprotein expressed as a heterodimer by a majority of thymocytes, and by class I major histocompatibility complex restricted, mature, suppressor/cytotoxic T cells; CD68 labels human monocytes and macrophages, but not myeloid cells; CD163 has been shown to mark cells of monocyte/macrophage lineage; CD11b is expressed on the surface of many leukocytes including monocytes, neutrophils, natural killer cells, granulocytes and macrophages, as well as on 8% of spleen cells and 44% of bone marrow cells; and CD11c is expressed prominently on the plasma membranes of monocytes, tissue macrophages, NK cells, and most dendritic cells (Figure 7).
3.3. Evaluation of the samples
3.3.1. Subjective assessment
The samples contained in TMAs or in whole slides can subjectively be analyzed by a pathologist to assess the amount of each ECM element as non‐informative: artefact, scant material, lost cylinder; negative: no expression or <5% of stained area is detected; positive 1+: mild staining, 5–10% of the area; positive 2+: moderate staining, 10–50% of the area.; and positive 3+: strong staining, >50% of the area.
3.3.2. Automated quantification
The growing size and number of medical diagnostic images requires the use of computer‐automated segmentation algorithms for the delineation of ECM structures of interest, which play a vital role in the research of new biomedical‐imaging markers . This is critical to analyzing whole sets of HC and IHC images to identify, describe, and quantify tissue alterations of the ECM, and require digitation of the samples. Morphometric techniques based on computer‐automated segmentation algorithms attempt to decrease human error, increase efficiency, assess large areas or huge amounts of tumor samples, create reproducible results, and help to standardize the measurements .
22.214.171.124. Digitization of the sections
The digitization of the samples is required to perform morphometric analysis. The method available in most laboratories is the capture of several images per sample with a photomicroscope. Sequential photos can be done at 20x or 40x magnifications with a photomicroscope and then carefully merged with Adobe Photoshop to reconstruct a single whole cylinder image (Figure 2). Following our experience, this method needs approximately two weeks to digitize 30 samples (2 cylinders per sample) for a single staining at 20x magnification, or approximately 6 to 7 weeks at 40x magnification.
The use of a slide scanner is the only method that permits the digitization of a high number of cases for a reasonable time when developing a routine image biobank [82, 83] or a research project. Additionally, the use of slide scanners provides the possibility to standardize the image quality using preserved light conditions.
In order to enhance the standardization of the image capture conditions and therefore, the quality of the measurements and to save time, whole slide scanning is advised, as described next. Our group used the ScanScope XT scanner, Aperio technologies, but increasing alternatives such as Panoramic Midi from3D Histech and the Ventana iScann Coreo Au from Roche, among others can also be considered [82, 84]. ScanScope XT scanner is a brightfield scanner that digitizes whole sections at 20x or 40x magnification providing high resolution images in approximately 15 to 30 minutes per slide, depending on the magnification and the size of the tissue. 40x magnification was used, originating images with a resolution of 0.25 μm/pixel. Given to the enormous amount of pixels scanned, the images were compressed in JPEG2000 to 100–200 megabytes for the average size of a TMA, and saved to a proprietary TIFF format (SVS).
The sections were placed in a mobile plate one by one. An option for TMAs digitization provided by the scanner driver was used. This option recognizes the tissue cylinders and places several points per sample where the objective adjusts the focus to obtain clear and focused images. The mobile plate with the section moves in consecutive stripes, until it sweeps the whole section while the objective scans. Individual scanned stripes are originated and stitched together automatically to reconstruct the whole image, which can be visualized up to 40x magnifications with the Image Scope viewer software (Aperio technologies). The process is briefly shown in Figure 3.
126.96.36.199. Design of automated image analysis algorithms
Depending on the staining to be measured and the availability of morphometric systems, different methods for image analysis can be used, all following a common workflow (Figure 4).
OPTION 1: To use commercial software for image analysis with established protocols which the user can adapt to the marker specific color.
Example 1: Algorithm for the fundamental substance (GAGs). Each cylinder was analyzed individually with Aperio Positive Pixel Count Algorithm on Aperio ScanScope software by selecting each cylinder outline in the whole section image. This algorithm was used with the default parameters and counted the number of pixels belonging to a given staining intensity, being the intensity of each pixel the average between the values of red, green, and blue (RGB) intensities. It recognizes Alcian blue stained pixels which have average intensities over 221. Nuclear fast red stained pixels were detected with weak, middle, and strong intensities painted in yellow, orange, and brown following default parameters (Figure 5). The remaining customizable parameters were set by default.
Example 2: Algorithm for the immune system cells. Each cylinder was analyzed individually with the Nuclear Quant algorithm on Panoramic Viewer software (3D Histech) by selecting each cylinder outline in the whole section image. This algorithm was used with the default parameters and counted the number of cells presenting a given staining intensity. Stained cells were detected with weak, middle, and strong intensities painted in yellow, orange, and maroon (Figure 6).
OPTION 2: To use commercial software for image analysis which allows the user to configure personal image analysis protocols or macros capable of recognizing and describing the specific color and shape of the element of interest.
Example: Algorithm for the reticulin fibers. The grading of fibrosis in general and namely of reticulin fibers is of main interest in bone marrow pathologies  and fibrous diseases of the liver . For this reason, different methods for the quantification of reticular fibrosis have been developed, some of them consisting in automated morphometry [88–93]. However, these methods quantify the percentage of stained area and we considered that not only the amount of fibers, but also the morphometric features were relevant, given that these are usually subjectively assessed. Image Pro‐Plus software (Media Cybernetics), which enables the design of specific algorithms, was used to analyze the fibrous component. An image of every cylinder was extracted in a separate JPEG‐quality 80 image from the whole section scan. An algorithm capable of specifically detecting the reticulin fibers and measure their amount, size, and shape was customized. Image J is a free software providing similar options. An example of the segmentation process in a control tissue (kidney) is provided by Figure 7.
OPTION 3: To design a personal application capable of solving commercial softwares lacks.
Example: Algorithm for the vascular component. A common feature of tumor vessels studies is that the researchers focus on microvessel density overlooking other parameters that might be significant, such as the size and shape of the microvessels . Studies have revealed the importance of the size and shape of blood vessels in, for instance, laryngeal tumors . The morphometric tool Angiopath closes blood vessels with discontinuous endothelial layer, recognizes all blood vessels and classifies them in six categories corresponding to different types of vessels, differentiated by their largest diameter or length. Density (density and occupied area), size (area, width, length, and perimeter), and shape (perimeter‐ratio, shape index, branching, aspect, roundness, and deformity) parameters are extracted [96, 97]. An example of the segmentation process is provided in Figure 8.
188.8.131.52. Spatial distribution of microenvironmental components
Topological network analysis and the graph theory in combination with Vonoroi tessellations  have recently been found to be useful in the diagnosis of muscular dystrophies and neurogenic atrophies, in the classification of neuromuscular disease or to model the progression of dementia [99–102]. All the generated information is subject to capture relevant information about the organization of different tissue markers.
184.108.40.206. Texture analysis
Another novel approach to cancer research is the texture analysis of different tissue images and machine‐learning methods to train automated algorithms which find different patterns in the tissue capable of discriminating between prognostic groups, among other variables, resulting in computer‐aided diagnosis tools [103–106].
4. Therapeutic implications of the study of ECM elements
The accumulation of non‐fluid elements in the tumor ECM, understood as an increased density of fibrous elements with huge crosslinking, carries the establishment of a solid pressure within the tumor tissue, which, together with other pressure sources, leads to an elevated tumor interstitial pressure . Elevated tumor pressure alters gene expression, cancer cell proliferation, apoptosis and invasiveness, stromal cell function, and ECM synthesis and organization [108–110]. Additionally, it has a negative impact on cancer treatment and is known to reduce the efficacy of cancer therapy through a reduced uptake and heterogeneous distribution of drugs [111, 112]. The direct intratumoral injection of anticancer agents has been evaluated extensively in the past decades, as one approach to counteract such intratumoral pressure . For example, is has been shown that intratumoral injection of immunostimulatory oligonucleotides induces differentiation and reduces immunosuppressive activity of myeloid‐derived suppressor cells in mice inoculated with colon cancer cell lines . Regarding ECM fibers, therapies that attack some of the major physical changes themselves, such as inhibiting matrix crosslinking, could be developed to revert pressure within the tumor tissue . Most of the therapeutic strategies under study are based on altering the fibroblastic pathways of collagen synthesis and inhibiting fibroblast growth factor [116–118]. Other strategies focusing on the destruction of existing fibrosis are being developed. In this sense, collagenase has been shown to reduce tumor interstitial pressure by cleaving collagen and consequently destroying the collagen crosslinked networks in human osteosarcoma xenografts . Moreover, this enzyme could indirectly affect tumor ECM by degrading the reticulin fibers associated with blood vessels. Another therapeutic approach not affecting the amount of fibers but the morphology consists in impeding an increased crosslinking. The reduction of lysyl‐oxydase‐like‐2, catalyzing the production of a crosslinked collagenous matrix, by the monoclonal antibody AB0023, was efficacious in reducing disease in different models of cancer and fibrosis . Incorporating biomechanical forces into our current picture of the tumor microenvironment, through the study of the interaction and the status of different ECM elements, fundamentally changes the context in which we study the basic cell and molecular biology of cancer. If there is one core message from the last two decades of research into the tumor ECM, it is that context matters .
Solid stresses affect tumor pathophysiology directly by compressing cancer and stromal cells but also indirectly by deforming blood and lymphatic vessels as described in pancreatic cancer, where compressed vessels are observable at the histological level . The compression of tumor vasculature also leads to a reduction in tumor blood flow and subsequently reduces the anticancer agent transport to tumor cells . Therapeutic chemicals are normally transported through the interstitial space by convection, a transport process that is dependent on pressure gradients, but increased tumor vessel permeability creates a less steep gradient along the vessels. This leads to a reduction in convective transport across the vessels in tumor bulk . Additionally, an increase in vessels permeability enables the extravasation of stem and immune cells, among others, and the entrance of malignant cells into the bloodstream to promote metastasis . Moreover, abnormal and tortuous tumor vasculature causes blood stasis, which leads to the reduction of oxygen and blood flow in tumors causing hypoxia and subsequently ischemia and necrosis . This effect increases anabolic metabolism which produce an acid ECM that degrades or deactivates some therapeutic drugs and renders them ineffective . Hypoxia also can cause resistance to radiotherapy and can help tumor cells to escape immunosurveillance . Because of its importance to the tumor, tumor vasculature represents an “Achilles’ heel” that can be used for cancer therapy . Indeed, multiple antiangiogenic therapeutic strategies have been developed in the last decades for many different malignancies . Several direct angiogenic inhibitors (angiostatin, endostatin, and thrombospondin) targeting endothelial cells and indirect anti‐antiangiogenic agents blocking the production or activity of pro‐angiogenic molecules, such as vascular endothelial growth factor (VEGF), have been developed . In this case, again intratumoral injections of adenoviral vectors encoding tumor‐targeted immunoconjugates induce a cytotoxic immune response against the neovasculature endothelial cells and tumor cells in mice xenografted with human melanoma cell lines . Recent studies have focused on the value of normalizing tumor vasculature to improve responses to conventional anticancer therapies rather than destroying tumor vessels to starve primary tumors from oxygen and induce tumor shrinkage . Vessel normalization results in reduced vessel diameter, increased pericyte coverage, and normalized basement membrane, accompanied by normalization of its function. This normalized tumor vasculature becomes less permeable and tortuous and leads to reduced fluid and protein extravasation into the interstitium, resulting in the decrease tumor interstitial pressure [129, 130]. For example, various preclinical studies have shown that the high levels of VEGF observed in tumors induce vessel abnormalities [131, 132]. Therefore, targeting VEGF using anti‐VEGF antibodies and inhibitors such as bevacizumab has been shown to reduce tumor interstitial pressure [133, 134]. Another protein playing an important role in tumor angiogenesis is PDGF. Imatinib, a PDGFR‐inhibitor has been shown to reduce tumor interstitial pressure and enhance therapeutic efficacy . Other drugs such as hydralazine and cachectin are vasodilators that cause a decrease in vascular resistance followed by an increase in tumor blood flow, which can potentially improve intratumoral transport of macromolecules and that have been shown to reduce intratumoral pressure [136, 137]. In addition to these therapeutic agents, some physical approaches have been tested to decrease tumor ECM pressure that can be summarized as follows: irradiation reduces the vascular wall permeability to fluid and leads to a reduction of intratumoral pressure ; induced hyperthermia or hypothermia have shown similar results regarding the reduction of intratumoral pressure as for irradiation ; ultrasounds due to mechanical (cavitation) and thermal pressure cause damage to tumor cells and ECM, which increase the interstitial hydraulic conductivity, reduces matrix tension and enhances tumor blood flow ; hyperbaric oxygen increases oxygen tension and delivery to tissues ; finally, photodynamic therapy impaired microcirculation in tumors . All these strategies are possible by the knowledge of the physiopathology of vascularity in tumors ECM. Most of them are in the experimental phases and others are already in clinical trials.
In the specific case of neuroblastic tumors, many relevant processes, such as stem cell differentiation, neuronal maturation, neurite extension, MYCN expression and the malignant potential and phenotype of tumor cells have all been shown to be influenced by ECM stiffness . In this respect, our group performed a preliminary study where some differences regarding ECM elements could be described between favorable and unfavorable neuroblastic tumors . This study was extended to a cohort of more than 500 samples where a stiffer, crosslinked and less porous ECM was mainly found in unfavorable tumors [143, 144]. Additionally, unfavorable tumors presented a highly vascularized ECM with mostly sinusoid vessels with abnormal morphologies . Some ECM elements morphometric features helped the description of an ultra‐high risk subgroup which could beneficiate of novel therapies .
Cancer cells often express a variety of abnormal proteins that can serve as targets for an immune response. Although spontaneous immune responses to these antigens can occur, these reactions are rarely sufficient to cause tumor regression; however, the local administration of immune‐activating agents can induce tumor‐associated inflammation and protective immunity. By binding to their targets, antibodies exercise their functions through several effector mechanisms, including steric inhibition and neutralization, complement activation, and activation of cell‐mediated cytotoxicity. Each of these mechanisms may play a role in the antitumor activity of monoclonal antibodies; however, at present the relative importance of these mechanisms is not completely clear. In general, immune adjuvant–based therapies have only proven effective against early stage tumors; yet in this context they can be remarkably effective with minimal risk of serious adverse reactions . Advances in immunotherapy have changed the management of several malignancies, being monoclonal antibodies the most specific targeted therapies currently in use . Therefore, understanding how tumor cells develop immune escape mechanisms and create microenvironments that improve their survival and dissemination and to reach assay normalization seems crucial to achieving optimal treatment [69, 149, 150]. Most notable has been the ability of the anti‐CTLA4 antibody, ipilimumab, to achieve a significant increase in survival for patients with metastatic melanoma, for which conventional therapies have failed. In the context of advances in the understanding of how tolerance, immunity and immunosuppression regulate antitumor immune responses together with the advent of targeted therapies, suggesting that active immunotherapy represents a path to obtain a durable and long‐lasting response in cancer patients . Five monoclonal antibodies are clinically approved for the treatment of hematologic tumors such as chronic lymphocytic leukemia (alemtuzumab), acute myelogenous leukemia (gemtuzumab), and non‐Hodgkin's lymphoma (rituximab, ibritumomab tiuxetan, and tositumomab) . Cetuximab and panitumumab increase progression‐free survival in patients with metastatic colorectal cancer who have previously failed standard chemotherapy and are associated with 10–20% and 10% response rates, respectively [152, 153]. Trastuzumab was the second monoclonal antibody approved for cancer therapy and is used, either alone or in combination with paclitaxel, for the treatment of invasive, HER2/neu‐positive breast cancer, which represents approximately 20–30% of invasive breast cancers with a response rate of 50% when used in combination with chemotherapy . Bevacizumab is the only monoclonal antibody with anticancer activity that does not directly target malignant cells. Instead, bevacizumab binds VEGF, a critical mediator of tumor angiogenesis. Inhibiting angiogenesis slows the delivery of nutrients and oxygen to tumors, inhibiting growth without severely compromising normal tissue function [155, 156].
In cancer biology, tumors are described as complex tissues comprised of heterogeneous neoplastic cells interwoven with tumor associated stroma. The characterization of the ECM elements associated with the tumoral and stromal cells presents opportunities for targeted therapeutic intervention. However, the heterogeneity of tumoral elements dictates that, in order to achieve successful clinical treatment, it is necessary to employ a combination of targeted therapies.
Novel and accurate image analysis algorithms must be developed and used to further investigate about tumor ECM composition and conformation and its relationship with different malignancies prognosis. Digital pathology approaches based on automated image analysis are necessary to obtain standardization, reproducibility, and eliminate observers’ biases.
The definition of favorable and unfavorable ECM characteristics, together with the understanding of the fact that biomechanical forces affect cell‐cell and cell‐matrix crosstalk and alter tumor cells fate, suggests the possibility of developing new therapies that can target the behaviors that arise from these complex interactions and from the heterogeneity of tumor tissues.
The authors want to thank Marcial García Rojo, Gloria Bueno and Jose Benavent for technical support on digital pathology. This study was supported by the FIS contracts PI10/00015 and PI14/01008 and RTICC contract RD12/0036/0020, grants from the ISCIII & FEDER (European Regional Development Fund), Spain. The authors declare no conflict of interest.