Reprogramming endothelial cells to empower cancer immunotherapy

Abstract

Cancer immunity is subjected to spatiotemporal regulation by leukocyte interaction with the tumor microenvironment. Growing evidence suggests an emerging role for the vasculature in tumor immune evasion and immunotherapy resistance. Beyond the conventional functions of the tumor vasculature, such as providing oxygen and nutrients to support tumor progression, we propose multiplex mechanisms for vascular regulation of tumor immunity: immunosuppressive vascular niche locoregionally educates circulation-derived immune cells by angiocrines, aberrant endothelial metabolism induces T cell exclusion and inactivation, and topologically and biochemically abnormal vascularity forms a pathophysiological barrier that hampers lymphocyte infiltration. We postulate that genetic and metabolic reprogramming of endothelial cells may rewire the immunosuppressive vascular microenvironment to overcome immunotherapy resistance, serving as a next-generation vascular-targeting strategy for cancer treatment.

The tumor vasculature – a barrier for cancer immunotherapy

Immunotherapy, an innovative therapeutic strategy which harnesses the immune system to battle cancer, is an exciting breakthrough in cancer treatment in recent decades. However, tumors develop ever-evolving mechanisms to evade immune detection, limiting the success of cancer immunotherapy in many solid tumors. This is largely due to insufficient T cell recruitment into and activation at the immune-hostile tumor microenvironment (TME; see Glossary). A significant contributor towards these resistance mechanisms is the development of an aberrant tumor vasculature. For immune cells to enter the tumor stroma and reach cancer cells, they need to pass through a vascular barrier. Endothelial cells (ECs) lining vascular structures express an array of cytokines and chemokines to direct immune cells as well as surface proteins to allow for immune cell attachment and extravasation. While these mechanisms provide guidance and balance to immune cell surveillance in a healthy setting, tumors seem to evade the immune system by dysregulating vascularity and EC interaction with immune cells, leading to the development of an aberrant immunosuppressive vascular microenvironment that drives tumor resistance to T cell-based immunotherapies. [1,2]

Here, we provide a brief overview of tumor vasculature-mediated therapeutic obstacles in T cell-based immunotherapies, including immune checkpoint blockade (ICB) and chimeric antigen receptor (CAR) T cell therapy, in treating solid tumors and propose new strategies for genetic and metabolic reprograming of tumor ECs (TECs) to overcome immunotherapy resistance. For decades, traditional therapies targeting aberrant vascular growth, such as vascular-obliterating or vascular-normalizing levels of anti-angiogenic agents, have been explored as strategies to improve drug delivery or anti-tumor immunity. While these strategies show benefits in combination therapy for some cancer types, they fail to produce long-term effects in others and tumors ultimately develop resistance. [1] Here, we propose a recently-developed novel alternative therapeutic method of directly targeting mechanisms within TECs driving tumor-promoting behavior, through genetic and metabolic reprograming, to minimize the development of resistance and sensitize previously-resistant tumor types to T cell-based immunotherapies.

Therapeutic difficulties of traditional vascular-targeting therapy

A fundamental component of the TME is the interacting vasculature. Cancers take advantage of the overgrown vasculature, with tumor-triggered angiogenesis providing oxygen and nutrient delivery to the tumor. First proposed in 1971, anti-vascular therapy aims to eradicate the vasculature by targeting key growth factors that induce EC proliferation and tumor angiogenesis, such as vascular endothelial growth factor (VEGF). [2,3] Since 2004, the US Food and Drug Administration (FDA) has approved several anti-angiogenic drugs for cancer treatment, including colon and kidney cancers, but the overall efficacy of anti-angiogenic therapies are often small and fail to produce long-term benefits in patients with most other cancer types, such as glioblastoma. [3,4,5] This is largely due to intrinsic and acquired tumor resistance, driven by the existence of parallel and redundant angiogenic pathways and adaptive mechanisms for tumor cells to survive an avascular and hypoxic tumor microenvironment, respectively. [2,4] Considering the potential role of pro-angiogenic factors for pro-tumor immunity, anti-angiogenic agents may alleviate immunosuppression, supported by promising results from recent clinical trials combining anti-angiogenic agents with checkpoint blockade immunotherapy. [5]

Destroying the tumor vasculature can adversely enhance tumor hypoxia and reduce drug delivery, however, rendering tumors more resistant to radio/chemotherapy and targeted molecular therapy. [6] Moreover, anti-vascular therapy may destroy the routes by which anti-tumor lymphocytes can reach the tumor and invade. Considering tumor vasculature is structurally and functionally abnormal, i.e., tortuous, leaky due to dysfunctional EC sprouting and overgrowth, a different strategy, to not just obliterate the tumor vasculature but rather restore normal vessel function, was attempted. [7] This could be achieved by not only inhibiting pro-angiogenic factors, but precisely re-balancing the pro- and anti-angiogenic factors presented in the TME, with improved perfusion allowing for proper drug delivery and immune infiltration through a process deemed “vessel normalization”. [8] Some vessel normalization therapeutic strategies include: controlling excessive EC growth by treating with lower doses of anti-angiogenic drugs, decreasing leakiness and increasing perfusion through improving pericyte-supported vascular structural integrity, and adding angiostatic factors. While it holds promise, the benefits of vessel normalization monotherapy have often been small and transient. Vessel normalizing doses of anti-angiogenic treatment improves T cell infiltration and enhances immunotherapy [9,10], due to enhanced vessel delivery and reduced intratumoral hypoxia. However, timing and dosing of vessel normalization therapy combined with immunotherapies and other conventional cytotoxic therapies still needs to be optimized in a tumor type-specific manner, as tumor immunogenicity, vascularity, and therapeutic resistance change over development and treatment exposure. [2,8]

Therefore, obliterating tumor vasculature through traditional anti-angiogenic therapy or restoring vascular structure and function through vascular-normalizing therapy have shown promise and even improved chemotherapy or immunotherapy response in some cancers, however, both of these strategies have limitations, with other cancer types failing to respond or tumors developing resistance via alternative vasogenic or immunesuppressing mechanisms. [2,8] Below, we discuss several immunosuppressive characteristics of the tumor vascular niche that contribute toward this resistance and how targeting pro-tumor mechanisms within TECs may directly bypass tumor resistance, improving immunotherapy efficacy.

Mechanisms of vascular regulation of tumor immunity

Tumor vasculature can spatiotemporally modulate immune cell recruitment and activation through compromised vessel delivery and aberrant paracrine effects. As the barrier circulation-derived immune cells must cross to reach cancer cells, the tumor vascular niche plays an important role in regulating the ability of cells of the innate and adaptive immune systems to fight cancer. Dysfunctional expression patterns of TECs can suppress anti-tumor immunity through the production of immunosuppressive factors, altered expression of metabolic gene pathways, decreased lymphocyte binding to ECs and extravasation via downregulation of adhesion molecules, and physical barriers resulting from aberrant angiogenesis. Better understanding of vascular microenvironment-dependent tumor immunity regulation may lead to the development of new therapeutic strategies (Figure 1), particularly for combination with immunotherapy.

Figure 1. Mechanisms of vascular-mediated tumor immunosuppression.

Tumor vasculature drives immune suppression through immunosuppressive niche function and abnormal vascularity at metabolic and genetic levels. Endothelial cells (ECs) develop adaptive metabolic mechanisms to support their survival and proliferation in the hypoxic tumor microenvironment (TME), including heightened rates of glycolysis and serine biosynthesis. Tumor ECs (TECs) also undergo genetic alterations mediated through cell plasticity or induced by TME cues, including pro-angiogenic factors, hypoxia, and oxidative stress. Aberrant EC metabolism stimulates vessel sprouting, induces vascular abnormalities, and results in the production of immunosuppressive metabolites including lactate and 2-hydroxyglutarate (2-HG), contributing to immunosuppressive vascular niche function. The altered transcriptome in tumor ECs is characterized by dysfunctional expression of genes associated with cell proliferation, migration, adhesion, and extracellular matrix (ECM) production, leading to vascular abnormality that induces T cell exclusion and inactivation. Furthermore, the genetically altered TECs express immunosuppressive factors, including prostaglandin Interleukin 10 (IL-10), Interleukin 6 (IL-6), Transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF), and inhibitory ligands, including programmed death-ligand 1 and 2 (PD-L1/2) and Fas, restricting T cell activation directly, or inhibiting T cell activity indirectly through alternative macrophage polarization. The figure was generated using Adobe Illustrator.

Immunosuppressive vascular niche

The vascular niche fuels tumor growth, progression, and metastasis and maintains stemness activity in cancer stem cells by cell-cell communication and secretion of paracrine factors, i.e., angiocrines (Box 1). [11-18] The role of the tumor vasculature in immunity regulation has been studied extensively in recent years, but is not fully defined. [19–22] As all leukocytes need to pass through the vascular niche to enter the tumor, the vascular niche may serve as an “educational facility” for immune cells via interaction with ECs and locally enriched, EC-secreted immunosuppressive factors. First, dysfunctional communication with TECs may suppress lymphocyte activation. For example, upregulation of inhibitory immune checkpoints, e.g., programmed death-ligand (PD-L)-1/-2 and Fas ligand in TECs inhibits T cell activation and facilitates tumor immune evasion. [19–21] Second, secretion of soluble angiocrine factors by TECs acts as immunosuppressive signals, affecting recruitment of lymphocytes and their activity. For example, TECs produce interleukin-6 (IL-6) and colony-stimulating factor-1, which promote anti-tumor alternative macrophage polarization via triggering Akt1/mTOR pathway-mediated hypoxia-inducible factor 2α (HIF-2α) upregulation, driving tumor progression and immunosuppression. [23] In addition, EC-derived C-X-C motif chemokine ligand 2 (CXCL2) promotes monocyte recruitment and macrophage education by tumor cells [24], while EC-derived prostaglandin E2 and IL-10 may also directly restrict T cell activity. [25] These examples highlight a few of the mechanisms by which the TECs of the tumor vascular niche promotes myeloid cell-mediated immunosuppression and suppresses lymphocyte activation.

Box 1 – Communication within the vascular niche.

The vasculature is comprised of many cell types that interact to maintain proper vessel structure and function. For example, pericytes typically interact with endothelial cells in smaller vascular structures, such as capillaries, and help regulate vascular function, growth, and maturation, as well as immune cell recruitment. [83] Different cell types within the tumor vascular niche, including tumor cells, cancer stem cells, and immune cells that travel through blood vessels, can influence each other’s behavior through direct cell-cell communication or through indirect means, such as nutrient deprivation. The main modes of direct communication between cells include interactions between surface molecules (mechanosignaling), transfer of secondary messengers such as ions through gaps between cells (i.e., gap junctions), production of signaling molecules (paracrine signaling), and transfer of extracellular vesicles (i.e., exosomes). [83] Exosomes can carry nucleic acids, proteins, lipids, and metabolites with immunosuppressive functions, playing a critical role in cross talk between ECs and immune cells within the vascular niche. Communication between cells can also induce indirect changes in cell behavior. Signals triggering proliferation in endothelial cells, for example, cause them to take up more nutrients from the microenvironment, leaving fewer resources available to fuel immune cell activity. [45]

Aberrant endothelial cell metabolism

Endothelial metabolism is a key regulator of EC function, vascular homeostasis, and angiogenic sprouting. [26-34] Single-cell metabolomic and transcriptome analyses show TECs acquire an active and proliferative state to support constant vascular sprouting, demanding quick production of high levels of metabolic energy and sufficient biomass synthesis. [33,35] Endothelial metabolism is also adaptively modulated by the hypoxic, oxidative, and nutrient-deficient TME. TECs heavily rely on glycolytic metabolism to produce sufficient energy in low-oxygen environments, storing glucose reserves in the form of glycogen, and coordinating vascular branching through glycolytic-mediated compartmentalization of actin. [26,33,36] Fatty acid oxidation (FAO) within ECs fuels vascular growth through de novo nucleotide synthesis necessary for DNA replication [28] and a post-translational mechanism by which malonyl-CoA-mediated mTOR lysine malonylation regulates protein synthesis required for vessel growth. [29] VEGF-B stimulates EC uptake of lipids [37], suggesting a potential feedback loop linking fatty acid metabolism and angiogenesis. In the nutrient-low microenvironment, ECs rely on glutamine uptake to sustain asparagine synthesis necessary to support proliferation. [38] Although the role of EC metabolism in cancer remains largely unknown, endothelial glycolysis, FAO, and glutamine metabolism might regulate vessel outgrowth and integrity in tumors, potentially contributing to vascular structure- and function-associated tumor immunity. Recent work by us and others shows that serine biosynthesis and glycolysis drive EC overgrowth and aberrant vascularity in tumors, leading to T cell exclusion, suggesting a critical role of endothelial metabolism for tumor immune evasion and immunotherapy resistance. [39,40]

Endothelial metabolism may also regulate tumor immunity through the production and uptake of various immunomodulatory metabolites. Dysfunctional metabolism in TECs produce excessive lactate and 2-hydroxyglutarate (2-HG), two metabolites that inhibit cytotoxicity functions in T cells, contributing to the formation of an immunosuppressive vascular niche. [39,41-43] Uptake of nutrients from the microenvironment by proliferative TECs limits what is available for nearby immune cells to take in, indirectly affecting immune cell activation and the anti-tumor immune response. For example, expression of indoleamine 2,3-dioxygenase in TECs may suppress T cell activity through depletion of tryptophan. [44] As such, excessive consumption of glucose and glutamine by ECs and tumor cells leads to their deprivation due to metabolic competition in the TME [45], potentially enhancing immunosuppressive macrophage phenotypes and reducing T cell effector functions. [46-48]

Altered metabolic pathways within TECs promote immunosuppression through fueling aberrant vascular growth, nutrient depletion, and production of immunosuppressive metabolites, highlighting potential therapeutic targets to abrogate TEC-derived immunosuppression.

Abnormal tumor vasculature

Tumor vasculature is structurally and topologically abnormal, which compromises vessel delivery functions and directly impedes lymphocyte infiltration into tumors. Vascular abnormality can be driven extrinsically by angiogenic factor-mediated vessel sprouting and outgrowth, and intrinsically by plasticity-mediated genetic alteration, e.g., a portion of TECs acquire mesenchymal features through partial endothelial-to-mesenchymal transition (EndoMT) to promote their ability to proliferate and migrate, both serving as crucial targets for normalizing vascular function and relieving tumor hypoxia. [17] Hypoxia caused by the aberrant vasculature is a primary stressor in the TME, significantly contributing to tumor immunosuppression by hypoxia-inducible expression of inflammatory factors and suppression of lymphocyte activity. [49,50]

Abnormal tumor vascularity is characterized by altered EC adhesiveness and extracellular matrix composition. Tumor immune evasion can occur when ECs fail to express proper levels of adhesion molecules that leukocytes bind to, such as intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 (ICAM-1 and VCAM-1), which are necessary for immune cell attachment and extravasation. [1,51,52] It is theorized that inactivation of vascular immune responses plays an important role during development, to allow an embryo to develop the necessary vasculature without excessive immune invasion or targeting by the mother’s immune system; however, tumors take advantage of this process to evade immune detection by reprogramming ECs to adopt this embryonic program. [52] Another hallmark of dysfunctional ECs is the altered basement membrane, through which invading T cells must pass to get into the tumors, with the EC basement membrane component laminins playing a significant role for leukocyte exit from vasculature. [53] It is tempting to speculate that mesenchymal-like activation in TECs not only represses transcription of adhesion proteins but also alters expression of matrix-associated genes, as robust matrix turnover is a common feature in active mesenchymal cells.

Aberrant cell functions of TECs lead to dysfunctional vessels with altered adhesiveness, impeding lymphocyte delivery and activity. This, paired with immunosuppressive signal production and fueled by altered metabolic processes, highlight the role of TECs in promoting tumor immunotherapy resistance and the potential of targeting TEC-intrinsic processes as an immunotherapy-sensitizing strategy.

Rewiring endothelial cells of the tumor vasculature

In contrast to conventional anti-vascular therapy that blocks extrinsic pro-angiogenic factors, including VEGF, we propose that targeting intrinsic gene expression and metabolism in TECs may offer promising opportunities to structurally and functionally rewire tumor vasculature, reactivating anti-tumor immunity and sensitizing tumors to T cell-based immunotherapies (Figure 2).

Figure 2. Rewiring tumor endothelial cells as a promising therapeutic strategy.

Metabolic reprogramming of tumor endothelial cells (ECs) may decrease the production of immunosuppressive metabolites and reduce metabolic competition in the tumor microenvironment (TME) while inhibiting vascular abnormalities, leading to increased T cell activity, persistence, and infiltration. Genetic reprogramming of tumor-associated ECs may downregulate the secretion of immunosuppressive cytokines in the vascular niche, increase EC expression of adhesion proteins, decrease EC expression of immune-inhibitory ligands, induce EC extracellular matrix (ECM) remodeling, and normalize aberrant tumor vasculature, resulting in enhanced T cell adhesion, survival, migration, and infiltration.

Genetic reprogramming of endothelial cells

TECs are genetically unstable, with embryonic characteristics promoting pro-tumor and anti-inflammatory behavior. ECs are one cell type that exhibits robust cell plasticity, which has been well characterized in embryogenesis and pathological settings, including cardiac fibrosis, pulmonary hypertension, vascular inflammation, cerebral cavernous malformation, and cancer. [54-59] Mounting evidence suggests that cell plasticity-mediated genetic alteration, such as EndoMT, is a driving force that induces aberrant vascularity and EC immunosuppressive phenotypes. [17] Recent studies show that c-Met-mediated EndoMT induces aberrant vascularization and tumor chemoresistance [60], and c-Met expression plays a role in communication between TECs during tumorigenesis. [61] Platelet-derived growth factor (PDGF) downregulates VEGF/VEGFR2 signaling through EndoMT, leading to tumor resistance to anti-VEGF therapy. [62] Targeting EC stemness-like characteristics and EndoMT via inhibiting WNT sensitizes tumors to cytotoxic chemotherapy, inhibiting tumor growth and extending survival. [63] Inhibiting radiation-induced EndoMT in colon carcinoma TECs, via Trp53 deletion, decreased abnormal vascular structure, tumor growth, and pro-tumor macrophage polarization. [64] These results collectively indicate that EC plasticity drives vascular abnormality, supportive of a role of EndoMT for vessel function-mediated pro-tumor immunity. Consistent with this role, studies show that TECs produce IL-6 to induce macrophage-mediated tumor immunosuppression [23], and that combination of IL-6 blockade and CD40 activation overcomes tumor resistance to ICB. [65] On the other hand, TME-derived extracellular superoxide dismutase (SOD3)-induced stabilization of WNT target forkhead box M1 (FoxM1), a transcription factor, in TECs improves T cell infiltration in colorectal cancer, suggesting a tumor-specific role of endothelial WNT while highlighting its role in TEC-derived immunosuppression. [66]

Furthermore, kinome-wide genetic screening reveals p21-activated kinase 4 (PAK4) as an important regulator of EC plasticity and aberrant tumor vascularization. [51] Disruption of PAK4 restores expression of adhesion proteins claudin-14 and VCAM-1, normalizes tumor vasculature, decreases intratumoral hypoxia, and improves CAR T cell efficacy to slow tumor growth and prolong survival in glioblastoma. [51] Consistent with these findings, PAK4 knockdown upregulates EC expression of adhesion proteins and increases T cell infiltration, sensitizing pancreatic cancer to ICB. [67] Other studies have supported EC genetic reprogramming as a promising therapeutic strategy. For example, upregulation of stimulator of IFN genes (STING) expression in TECs normalizes tumor vasculature and synergizes with ICB in Lewis lung carcinomas. [68]

These results highlight the potential of targeting cell plasticity to genetically reprogram TECs as a promising strategy to boost lymphocyte infiltration and activation, through multiple mechanisms including vessel normalization and restoration of EC adhesion protein expression. Genetic reprogramming approaches may include pharmacological targeting of kinases and transcriptional factors, virus-mediated gene editing, and nanoparticle delivery of nucleic acids to ECs, as recently reported. [69-71]

Rewiring endothelial metabolism

Targeting the metabolic pathways within ECs fueling angiogenesis, rather than inhibiting the secreted proangiogenic factors themselves, has been a novel approach as an alternative to anti-angiogenic therapy that has been explored in recent years. [36,39,40] It can theoretically affect EC sprouting and reshape vascular structure, while reconditioning the metabolic TME. Glycolysis can produce energy and metabolites anaerobically, providing ECs surviving and dividing advantages in a low-oxygen TME. Disruption of glycolysis via PFKFB3 inhibition improves stabilization of the vascular barrier through increased pericyte adhesion and quiescence, decreasing metastasis and improving chemotherapy efficacy in murine models of liver metastases. [26,36] A recent study also shows that reduction of aerobic glycolysis in TECs normalizes tumor vasculature in colorectal cancer, improving immune infiltration and sensitizing tumors to ICB. [40] Our study identifies PHGDH-mediated serine biosynthesis as preferentially activated in TECs, inducing aberrant vascular growth in glioblastomas via regulating nucleotide synthesis and maintaining redox balance for endothelial glycolysis. Targeting PHGDH normalizes the tumor vascular structure, decreases hypoxia, and promotes T cell recruitment, overcoming tumor resistance to CAR T cell therapy [39], collectively suggesting a central role of EC glucose metabolism for aberrant vascularity and immunity. Rewiring endothelial metabolism may also recondition the immunosuppressive TME, by modulating the level of immunomodulatory metabolites produced by the vascular niche. For example, serine metabolism in TECs contributes to production of lactate and 2-hydroxyglutarate, two immunosuppressants in the TME. [39] Disrupting endothelial fatty acid metabolism has also had promising effects on vascular phenotypes, as ECs use FAO to generate dNTPs required for replication. [28] Inhibition of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme in FAO, disrupts angiogenesis in a mouse retinopathy model [28], implicating disrupting FAO as a potential strategy in normalizing tumor vasculature and improving the tumor immune landscape. Together, these studies show that targeting EC metabolism holds promise to improve immunotherapy efficacy via enhanced vessel delivery-mediated T cell extravasation and reduced metabolite-mediated immunosuppression, however, additional studies are required to elucidate tumor-specific mechanisms and treatment optimization.

Concluding remarks

TECs play a significant role in creating a hypoxic, immunosuppressive TME that drives immunotherapy resistance through secretion of immunosuppressive factors, altered metabolic processes, and aberrant behavior producing structurally abnormal vessels. Therapies designed to obliterate or normalize the vasculature by targeting vascular growth factors have shown promise but fail to produce long-term effects in many cancers and tumors often develop resistance. [1,2,4,8] Here, we highlight the potential for genetic and metabolic EC reprogramming as a novel vascular-targeting strategy to relieve immunosuppression while illuminating further areas of study necessary to explore to optimize this treatment approach (see Outstanding Questions). The precise mechanisms underlying spatiotemporal regulation of vascular abnormality and dysfunctional immunity remain obscure. Recent advances in single-cell RNA analysis of ECs in healthy and diseased tissues paired with functional genome-wide screens may contribute to better understanding of EC plasticity trajectory and molecular resolution of tumor vascularity, while identifying new therapeutic targets (see Clinician’s Corner). [72] Integration of single-cell RNAseq with spatial transcriptomics will provide insight into dynamic interaction between vascularity and immunity in the vascular niche.

Outstanding questions.

• What is the most effective therapeutic window of vascular reprogramming for cancer immunotherapy?

• What are the new and key drivers that spatiotemporally induce genetic and metabolic alteration in tumor ECs?

• What are the mechanisms of resistance that will drive insensitivity to genetic or metabolic reprogramming of tumor ECs?

• How will new therapies targeting vascular reprogramming combine with current standard of care?

• What will be the long-term effects of vascular reprogramming?

• Will new methods of vascular reprogramming therapy universally benefit all patients with all cancer types, or only be beneficial to treat certain types of cancers or a subgroup of cancer patients? How we will identify the subtypes or subgroups?

Clinician’s corner.

• Vessel normalizing low-doses of anti-angiogenic treatments have been shown to moderately improve tumor sensitivity to cytotoxic therapies and immune checkpoint blockade. Genetic or metabolic reprogramming of tumor ECs is a newer strategy for vascular-targeting treatment that may further improve the anti-tumor immune response. Therefore, combining additional vascular-reprogramming approaches with immunotherapy is a promising strategy against solid tumors.

• Genetic reprogramming of tumor ECs may normalize tumor vasculature to improve T cell infiltration and enhance cancer immunotherapy. For example, pharmacological PAK4 inhibitors such as the KPT-9274, an allosteric PAK4 inhibitor that has been tested in phase I clinic trials for lymphoma and acute myeloid leukemia, hold promise.

• Targeting endothelial metabolism, e.g., using PHGDH inhibitors, may recondition the vascular microenvironment to boost T cell infiltration and activation, overcoming tumor resistance to immunotherapy.

• While these novel endothelial cell-reprogramming vessel normalization techniques show promise in pre-clinical studies of aggressive tumor types, e.g. glioblastomas, the tumor- and patient-specific parameters that would determine success of these studies in sensitizing tumors to immunotherapy in humans have yet to be defined.

Altered epigenetic landscapes also support aberrant EC behavior in cancer, affecting expression of cytokine-driven cell adhesion molecules and chemokines important for CD8⁺ T-cell extravasation. [73]-[74] Several epigenetic-targeting drugs have been approved for cancer therapy and tested in clinical trials with other treatments, however, lack of target specificity alongside variability between cancer types and other factors complicate treatment efficacy. [75] Future development of approaches specifically targeting EC epigenetics is vital to improve treatment efficacy and anti-tumor immunity.

Tumor immune infiltration can also be regulated by high endothelial venules (HEV), a vascular structure that allows for higher movement of lymphocytes in and out of circulation and establishes a niche for cytotoxic T cell expansion. [76-78] HEV levels are generally a positive prognostic factor in cancer [77]. A recent work shows that HEVs are major sites of lymphocyte entry into tumors at baseline and upon treatment with ICB. [79] However, what triggers HEV formation or lack thereof in tumors is not fully understood, which could be regulated by interaction with interferon-γ-expressing lymphocytes and with regulatory T cells. [80-82] Better understanding of the underlying mechanisms might change vascular-targeted therapies and immunotherapy in the future.

For decades, conventional therapeutic approaches targeting angiogenic factor-driven vascular growth have been explored; however, they fall short in eliminating the immunosuppressive forces of the vascular niche, allowing tumors to develop therapy resistance. In light of this limitation, we propose reprogramming mechanisms within TECs as a promising vascular-targeting approach that may disrupt tumor evasion of host immunity and empower T cell-based immunotherapy. Moving forward, critical considerations include: i) the development of tailored EC-reprogramming therapeutics, encompassing small-molecule and nucleic acid–nanoparticle drugs; ii) the precise spatial resolution of heterogeneous immunosuppression within tumor vascular niches; and iii) an exploration of the intricate interactions between TECs, immune cells, and other stromal components in the vascular niche, such as fibroblasts and pericytes. These aspects demand further investigation to advance our understanding and application of reprogramming strategies in the complex context of tumor immunotherapy.

Highlights.

• The tumor vascular niche drives intratumoral immunosuppression, leading to immune evasion and immunotherapy resistance.

• Tumor endothelial cells educate myeloid suppressor cells and lymphocytes by angiocrines.

• Aberrant endothelial metabolism induces T cell exclusion and inactivation.

• Abnormal vasculature in tumors forms a pathophysiological barrier that hampers T cell infiltration.

• Genetic and metabolic reprogramming of endothelial cells may reverse tumor immunosuppression and overcome tumor resistance to immunotherapy.

Glossary

Angiocrines

chemokines, cytokines, growth factors, soluble extracellular matrix components, and other signaling molecules produced by vascular endothelial cells to regulate tissue functions through paracrine and juxtracrine signaling to other cells.

CAR T cells

T cells genetically engineered to express a chimeric antigen receptor (CAR), which is designed to recognize specific antigens, i.e. a cancer-specific antigen, to target tumors.

Endothelial cells (ECs)

cells that line blood and lymphatic vessels often in a single layer, making up a barrier between the vessel lumen and surrounding tissues.

Endothelial-to-mesenchymal transition (EndoMT)

a form of transdifferentiation by which endothelial cells lose endothelial cell characteristics and gain a mesenchymal phenotype.

Extravasation

the process by which immune cells exit the vasculature, by crossing through the endothelial cell layer, and entering the surrounding tissue.

Fatty acid oxidation (FAO)

the process by which fatty acids are broken down into energy.

High endothelial venules (HEV)

typically found in lymphoid organs, this specialized type of blood vessel allows for easy movement of lymphocytes into and out of the vasculature.

Hypoxia

a state of insufficient tissue oxygen levels.

Hypoxia-inducible factor 2-alpha (HIF-2α)

stabilized in response to low oxygen levels (hypoxia), this subunit of the transcriptional activating HIF complex mediates angiogenesis through regulating EC proliferation, differentiation, and expression proangiogenic factors such as VEGF.

Immune checkpoint blockade (ICB)

a type of immunotherapy that involves activating T cells by blocking proteins that trigger an “off switch” in T cells. Targets include cytotoxic T lymphocyte associated protein 4 (CTLA-4) as well as programmed cell death protein 1 (PD-1) and its ligand, programmed cell death ligand 1 (PD-L1).

Intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 (ICAM-1 and VCAM-1)

adhesion molecules expressed on endothelial cells and leukocytes that allow for cell-cell interactions, facilitating leukocyte extravasation and signal transduction.

Interleukin-6 (IL-6):

a pro-inflammatory cytokine, often expressed by macrophages, fibroblasts, and endothelial cells.

p21-activated kinase 4 (PAK4)

regulates many cellular functions, including proliferation, migration, and survival. This kinase is expressed by many cell types and has also been shown to play a role in regulating tumor immunity.

Perfusion

regional flow of blood and lymphatic fluid through a tissue. Poor perfusion in tumors means there is inadequate vascularization or circulation of blood flow, resulting in decreased delivery of oxygen, nutrients, immune cells, or drugs through the tumor.

TECs

endothelial cells of the tumor-associated vasculature.

Tumor microenvironment (TME)

the region surrounding a tumor. This environment includes many structures and cell types, including extracellular matrix, blood vessels (including endothelial cells, pericytes), fibroblasts, immune cells, signaling molecules, and others.

Vascular endothelial growth factor (VEGF)

a signaling protein that stimulates the growth of blood vessels, i.e. angiogenesis.