Endothelial cells in tumor microenvironment: insights and perspectives

Abstract

The tumor microenvironment is a highly complex and dynamic mixture of cell types, including tumor, immune and endothelial cells (ECs), soluble factors (cytokines, chemokines, and growth factors), blood vessels and extracellular matrix. Within this complex network, ECs are not only relevant for controlling blood fluidity and permeability, and orchestrating tumor angiogenesis but also for regulating the antitumor immune response. Lining the luminal side of vessels, ECs check the passage of molecules into the tumor compartment, regulate cellular transmigration, and interact with both circulating pathogens and innate and adaptive immune cells. Thus, they represent a first-line defense system that participates in immune responses. Tumor-associated ECs are involved in T cell priming, activation, and proliferation by acting as semi-professional antigen presenting cells. Thus, targeting ECs may assist in improving antitumor immune cell functions. Moreover, tumor-associated ECs contribute to the development at the tumor site of tertiary lymphoid structures, which have recently been associated with enhanced response to immune checkpoint inhibitors (ICI). When compared to normal ECs, tumor-associated ECs are abnormal in terms of phenotype, genetic expression profile, and functions. They are characterized by high proliferative potential and the ability to activate immunosuppressive mechanisms that support tumor progression and metastatic dissemination. A complete phenotypic and functional characterization of tumor-associated ECs could be helpful to clarify their complex role within the tumor microenvironment and to identify EC specific drug targets to improve cancer therapy. The emerging therapeutic strategies based on the combination of anti-angiogenic treatments with immunotherapy strategies, including ICI, CAR T cells and bispecific antibodies aim to impact both ECs and immune cells to block angiogenesis and at the same time to increase recruitment and activation of effector cells within the tumor.

Keywords: T cells; endothelial cells; microenvironment; tumor; tumor immune evasion.

Figure 1

Immunoregulatory properties of tumor endothelial cells (ECs) in the tumor microenvironment. (A) Activated ECs recruit effector immune cells guiding their infiltration into the tumor microenvironment through a multi-staged adhesion process which includes binding of the integrins lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) on T cells to the respective ligands intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion protein-1 (VCAM-1) on tumor-associated ECs. The tumor-deriving cytokines vascular endothelial growth factor (VEGF), endothelin-1 (ET1), EGF-like domain-containing protein 7 (EGFL7), and fibroblast growth factor 2 (FGF2) downregulate gene expression of adhesion molecules and chemoattractants (e.g., CCL2, IL6, CXCL10, and CXCL7) to inhibit immune cell infiltration. Also, nitric oxide (NO) in the tumor microenvironment inhibits immune cell recruitment. Tumor hypoxia results in changes in EC glycosylation which impair adhesion and cell-matrix interactions. Tumor-associated ECs represent a selective barrier that favors regulatory T (Treg) cell traffic by the common lymphatic endothelial and vascular endothelial receptor 1 (CLEVER 1), but represses effector T cells, dendritic cells, natural killer (NK) cells and neutrophil granulocytes promoting immunological tolerance. Tumor-associated ECs can cause desensitization of NK cells through the expression of NKG2D ligands that interact with the receptor NKG2D on NK cells. The intracellular cGAS-cGAMP-STING pathway can enhance adhesion molecules and promote T cell infiltration. Shb deprivation in tumor-associated ECs led to the recruitment of myeloid-derived suppressor cells (MDSCs). (B) IL-8 signaling promotes EC activation and the migration of tumor cells, ECs, and infiltrating neutrophils at the tumor site. Macrophage-derived WNT7B increases VEGFA expression on tumor-associated ECs. Macrophage-derived WNT5A stimulates EC proliferation and migration and upregulates TIE2 in macrophages (MAs) and ECs. Tumor-associated macrophages-derived exosomes suppress EC migration by the miR-146b-5p/TRAF6/NF-kB/MMP2 pathway, whereas M2 macrophage-derived exosomal miR-155-5p and miR-221-5p mediate macrophage-EC interactions. Podoplanin (PDPN) on tumor-associated MAs mediates the attachment of MAs to lymphatic ECs by interaction with Galectin-8 (Gal-8) and stimulates new lymph vessel formation and the migration of tumor cells through the lymphatic system. Tumor-associated MAs express thrombospondin-1 which interacts with CD47 receptor expressed by tumor cells and tumor-associated ECs. Downregulation of CD47 in tumor-associated ECs increases angiogenesis, vascular integrity and stability, VEGF-A and VEGFR2 expression. A bidirectional crosstalk between ECs and B cells is realized by CD40 on microvascular ECs and CD40L on tumor-associated B cells. This interaction increases the EC release of BAFF and APRIL which further enhance the expression of CD40L on B cells. Moreover, tumor-associated B cells stimulate ECs via the signal transducer and activator of transcription 3 (STAT3)-YAP/TAZ signaling. Tumor-associated ECs induce endothelial-like differentiation of immature dendritic cells (iDC) within the tumor microenvironment by the expression of serine/threonine kinase 11 (Stk11). Mast cells release tryptase which acts on the proteinase-activated receptor-2 (PAR-2) stimulating both vascular endothelial and tumor cell proliferation. (C) STING activates also T cells by inducing a strong IFN-β production. The pro-inflammatory tumor-derived cytokines IFN-γ and TNF-α increase the expression of the immune checkpoint ligands programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2) on the tumor-associated EC surface. These ligands bind to the receptor programmed death 1 (PD-1) on T cells inhibiting T cell functions. ECs also induce tumor-infiltrating T cell exhaustion through the glycoprotein nonmetastatic melanoma protein B (GPNMB). Interferon (IFN)-γ, IFN-α and IFN-β upregulate indoleamine 2,3-dioxygenase (IDO) production by tumor-associated ECs promoting the metabolism of tryptophan (Tryp) to kynurenine (Kyn). Tryp depletion inhibits T cell proliferation, whereas Kyn accumulation promotes Treg differentiation and activation. (D) T cell apoptosis is triggered by IDO-mediated Tryp degradation and by upregulation of Fas ligand (FasL) on tumor-associated EC by tumor-derived VEGFA, interleukin (IL)-10 and prostaglandin E2 (PGE2). Tumor-associated ECs acquired the ability to kill effector CD8⁺ T cells but not Treg cells (due to expression of FoxP3). (E) Tumor-associated ECs can act as semi-professional antigen presenting cells. They can present processed antigens to memory T cells via class I and class II major histocompatibility complex (MHC) molecules, yet they do not express the co-stimulatory molecules CD80 and CD86 required for naïve T cell activation. They express the inducible co-stimulator ligand (ICOSL) which interacts with the receptor ICOS on Treg cells sustaining their activation and proliferation. ICOS/ICOSL interaction also increases the expression of the antiapoptotic protein Bcl-2. ICOSL can bind osteopontin (OPN) promoting EC migration.

Figure 2

Tertiary lymphoid structures (TLS) formation and maturation. When immune responses fail to eradicate the triggering stimulus, the formation of TLS at sites with persistent inflammatory stimulation takes place. Lymphocytes and stromal cells release CXCL13 and IL-7 involved in the recruitment of lymphoid tissue inducer (LTi) cells which interact with local stromal cells via lymphotoxin-α1β2 (LTα1β2). Many immune cells such as B cells, M1 macrophages and Th17 cells can substitute LTi cells in the initiation of TLS genesis in various pathological conditions. Stromal cells secrete vascular endothelial growth factor C (VEGFC) which promotes the formation of the high endothelial venules (HEVs) and release adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM1), intercellular adhesion molecule 1 (ICAM1) and mucosal addressin cell adhesion molecule 1 (MADCAM1), and various chemokines including CXCL12, CXCL13, CCL19 and CCL21. Together, these molecules recruit lymphocytes from HEVs and form a central B cell zone and a peripheral T cell zone enriched with CD8 T cells. CD4 T cells, CD4 T follicular helper (T_FH) cells and Treg cells can also be present, as well as CD68⁺ macrophages and antibody-producing long-lived plasma cells.