**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 [107]. Elevated tumor pressure alters gene expression, cancer cell prolif‐ eration, apoptosis and invasiveness, stromal cell function, and ECM synthesis and organiza‐ tion [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 [113]. 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 [114]. 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 [115]. 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 [119]. 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 [115]. Incorporating biomechanical forces into our current picture of the tumor microenvir‐ onment, 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 [120].

neurogenic atrophies, in the classification of neuromuscular disease or to model the progres‐ sion of dementia [99–102]. All the generated information is subject to capture relevant

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,

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 [107]. Elevated tumor pressure alters gene expression, cancer cell prolif‐ eration, apoptosis and invasiveness, stromal cell function, and ECM synthesis and organiza‐ tion [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 [113]. 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 [114]. 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 [115]. 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 [119]. 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 [115]. Incorporating biomechanical forces into our current picture of the tumor microenvir‐ onment, 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,

information about the organization of different tissue markers.

**4. Therapeutic implications of the study of ECM elements**

resulting in computer‐aided diagnosis tools [103–106].

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

*3.3.2.4. Texture analysis*

it is that context matters [120].

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 [111]. The compression of tumor vasculature also leads to a reduction in tumor blood flow and subsequently reduces the anticancer agent transport to tumor cells [121]. Therapeutic chemicals are normally transport‐ ed 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 [122]. 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 [123]. 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 [121]. This effect increases anabolic metabolism which produce an acid ECM that degrades or deactivates some therapeutic drugs and renders them ineffective [124]. Hypoxia also can cause resistance to radiotherapy and can help tumor cells to escape immunosurveillance [45]. Because of its importance to the tumor, tumor vasculature represents an "Achilles' heel" that can be used for cancer therapy [125]. Indeed, multiple antiangiogenic therapeutic strategies have been developed in the last decades for many different malignancies [57]. 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 [126]. 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 [127]. 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 [128]. 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 intersti‐ tial pressure and enhance therapeutic efficacy [135]. 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 [138]; induced hyperthermia or hypothermia have shown similar results regarding the reduction of intratumoral pressure as for irradiation [139]; 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 [140]; hyperbaric oxygen increases oxygen tension and delivery to tissues [141]; finally, photodynamic therapy impaired microcirculation in tumors [142]. 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 differ‐ entiation, 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 [1]. In this respect, our group performed a preliminary study where some differences regarding ECM elements could be described between favorable and unfavorable neuroblastic tumors [85]. 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 [145]. Some ECM elements morphometric features helped the description of an ultra‐high risk subgroup which could beneficiate of novel therapies [146].

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 immun‐ ity. 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 [147]. Advances in immunotherapy have changed the management of several malignancies, being monoclonal antibodies the most specific targeted therapies currently in use [148]. 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 [151]. Five monoclonal antibodies are clinically approved for the treatment of hematologic tumors such as chronic lymphocytic leukemia (alemtuzumab), acute myelogenous leukemia (gemtuzu‐ mab), and non‐Hodgkin's lymphoma (rituximab, ibritumomab tiuxetan, and tositumomab) [147]. 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 [154]. 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].
