3.2.2　Tumor Vasculature

Tumor vasculature arises from two different biological process: angiogenesis consisting of formation of new blood vessel from pre-existing vessels and vasculogenesis consisting of formation of new blood vessels by recruitment of circulating endothelial progenitor cells.Angiogenetic process is regulated by several molecules, signaling pathways and their related receptors such as VEGFs and VEGF receptors (VEGFRs), FGFs and their receptors (FGFRs), PDGF-β and PDGF-β receptor (PDGFRβ), angiopoietin, Notch ligand, semaphorine, neuropilin, robo proteins, and Ephrin A and B.One of the major signals regulating angiogenesis process is hypoxia-inducible factors (HIFs) because they induce transcription of genes responsible for activation of angiogenesis.HIFs consist of an oxygen-regulated HIF1α and HIF2α subunit and a constitutively expressed HIF1β subunit.In physiological condition α subunits are hydroxylated by oxygen-dependent prolyl hydroxylation, bind to the von Hippel-Lindau (VHL) tumour suppressor protein responsible for their ubiquitylation and proteasomal degradation.This hydroxylation reaction depends upon the presence of some cofactors such as oxygen and α-ketoglutarate.In hypoxia condition this reaction is inhibited and consequently HIF1α and HIF2α dimerize with HIF1β subunit and subsequently bind to hypoxia response elements in promoters of targeted genes.In response to hypoxia, HIF regulates the expression of angiogenic factors responsible for vascular permeability, endothelial cell proliferation, sprouting, migration, adhesion and tube formation.Growth factors, oncogenic activation and loss or inactivation of VHL genes are responsible for HIF genes activation other than oxygen tension.Oxygen tension also induces EMT by increasing the expression of Smad3, promoting the release of TGF-β2 activating TGF-β signaling and blocking HIF-1α transcription [32-34].

Bevacizumab (Avastin), an antibody that targets VEGF, was the first anti-angiogenic approved by US FDA that is already in the clinics.The fluorescent form of Avastin, Bevacizumab-IRDye800CW,has been showing promising results for its use in tumor imaging.Concerning the therapeutic outcome of such drugs, although they showed to improve patient survival, it has not been as great as expected, mostly related to the fact that patients stop responding or do not respond at all to these therapies, or even due to the side effects, for instance, the increased risk of arterial thromboembolic events, which is currently under clinical trials (NCT03709771).Moreover, while some studies evaluate the action of two anti-angiogenic agents, e.g., NCT01684397 that evaluates the effect of Pazopanib and Bevacizumab (Avastin) in treating patients with metastatic kidney cancer, other studies compare their efficacy.One such example is the phase 2 trial that compares the action of Bevacizumab (Avastin), targeting VEGF, versus the action of Apatinib, targeting VEGFR2, in colorectal, intestinal, gastrointestinal and digestive system neoplasms (NCT03271255).Another example is the phase 3 trial NCT03533127 comparing the action of two anti-VEGF antibodies, the novel LY1008 and the US FDA-approved Bevacizumab (Avastin), when combined with Paclitaxel and Carboplatin for non-small cell lung cancer (NSCLC) therapy [35, 36].

The infiltration of tumor by immune and inflammatory cells may support tumor progression in contrast with the primary theory of the role of immune system in recognizing and eradicating a large part of nascent tumor cells.All immune cell types may be found in tumor microenvironment,including macrophages, dendritic cells, mast cells, natural killer (NK), naïve and memory lymphocytes B cells, effector T helper (Th) cells including: Th1 cells, Th2 cells and Th17 cells,regulatory T (Treg) cells, T follicular helper (TFH) cells and Tc cells.These immune cells can be located in the core of the tumor, in the invasive margin or in the adjacent tertiary lymphoid structures (TLS).Immune and inflammatory cells release growth factors such as epidermal growth factor (EGF), VEGF-A/C, FGF2, several cytokines amplifying inflammatory condition, and enzymes degrading extracellular matrix such as MMPs, cathepsin, and heparinases.These factors can promote tumor angiogenesis, tumor cells proliferation, and cancer metastatisation.In addition,these cells releasing TGF-β and IL-10 are responsible for immunosuppression by activation of Treg recruited into the tumor.TGF-β pathway seems to play a role in immune regulation together with IL-10 by several mechanisms.First of all TGF-β1 drives Th1 and 2 balance towards Th2 phenotypes without cytotoxic activity against tumor, inhibits Th1 response and M1-type macrophages activity, suppress lymphocytes CD8, NK, and dendritic cells function, generates Treg with immunosuppressive function, and promotes M2 type macrophages with pro-tumoral activity.TGF-β1 increases the expression of IL-10 and chemoattractant protein (MCP-1) enhancing tumor infiltration and immune suppression [32,37, 38].(https://www.daowen.com)

Several clinical and experimental data showed that the activation of inflammatory pathways promote cancer development.Two different inflammatory pathways linked to cancer have been identified: intrinsic driven by activation of oncogenes or inactivation of oncosuppressor genes and extrinsic pathway driven by inflammatory conditions.Both of them are triggered by the activation of transcription factors such as nuclear factor kappa-B (NFkB), STAT3 and HIF-1α in tumor cells.These transcription factors are responsible for production of cytokines and chemokines,prostaglandins recruiting and activating inflammatory cells, stromal cells, and tumourous cells promoting cancer growth and dissemination.NF-κB is a protein complex activated by oxidative and pro-inflammatory stimuli with subsequent nuclear accumulation where it binds proximal promoter region of genes encoding proinflammatory mediators, such as cytokines, inducible nitric oxide synthase (INOS) and cyclooxygenase-2 (COX-2), involved in inflammation associated carcinogenesis.Other genes encoded by NF-κB are: anti-apoptotic proteins (BCL2,BCL-XL),inflammatory mediators (TNF-α, IL-6, IL-8, COX-2), effectors of invasion and metastasis such as adhesion molecules and MMPs, promoters of DNA damage such as ROS, reactive nitrogen species(RNS), inducers of cell proliferation (c-MYC and cyclin D1), and angiogenic factors (VEGF and angiopoietin).

In particular, TNF-α interacting with its receptor TNF receptor 1 (TNFR1) induces cell survival and proliferation, inflammatory response and antiapoptotic signal by NFkB activation.TNF-α is also an important regulator of cancer-related inflammation by modulation of T cells, B cells and tumor-associated macrophages (TAMS).TNF mediates IL-17 production by CD4+ cells leading to recruitment of myeloid cells in TME responsible for tumor progression.Additionally, TNF-α promotes differentiation of myeloid tumor cells into cells expressing endothelial markers promoting angiogenesis and subsequently tumor growth.STAT3 is activated by different cytokines and growth factors and activates oncogenesis by promoting transcription of genes responsible for proliferation,survival, and angiogenesis.STAT3 has emerged as a critical regulator of tumor-associated inflammation because it negatively regulates Th1 immune response and promotes expansion of myeloid-derived suppressor cells (MDSCs) having a role in immunosuppression, favoring their differentiation in macrophages and dendritic cells and angiogenesis.Thus, targeting STAT3 has dual role to inhibit tumor growth directly, and also to alter the immunological environment so as to favor control of tumor proliferation.A strategy to inhibit STAT3 is blocking JAK kinase, upstream activator of STAT3.Pro-inflammatory cytokines released in TME promote tumor growth and metastasis by inducing expression of MMPs, chemokines, growth factors and TGF-β genes.They also stimulate the expression of endothelial adhesion molecules such as ICAM-1 and VCAM-1, and induce expansion of immunosuppressive MDSCs cells into the TME and their differentiation in TAMs [32, 39, 40].

Tocilizumab, a monoclonal antibody against IL-6 receptor (IL-6R), has been tested in combination with carboplatin/pegylated liposomal doxorubicin (PLD) and interferon-α2b in ovarian cancer because carboplatin is known to stimulate IL-6 from cancer cells.This drug showed to have an acceptable toxicity profile and by blocking IL-6R, increased levels of serum IL-6 and soluble IL-6R was found potentially associated with a survival benefit.Currently this drug is under evaluation in combination with chemotherapy in pancreatic cancer (NCT02767557).Also,chemokines were tested in clinical trials in solid tumors.Plerixafor is a symmetrical bicyclam approved in haematological malignancies that specifically competes with and blocks SDF-1 from binding to the CXCR4 receptor.Trabectedin, a cytotoxic drug registered for treatment of softtissue sarcoma and ovarian cancer, has showed to have a cytotoxic mechanism of action by inhibition of inflammatory mediators such as CCL2, IL-6 and CXCL8 and by activation of caspase 8 activity on TAM.Infliximab is a monoclonal antibody against TNFα tested in a phase 2 trial in renal cancer in which it showed to induce a stabilization of disease in 61% of patients.However, these results were not confirmed in a subsequent study testing this drug in combination with sorafenib.Other anti-TNFα drugs were also tested in other solid tumors showing a good toxicity profile but without any antitumor activity [32, 41].