Anti‐angiogenic therapy enhances cancer immunotherapy: Mechanism and clinical application

Immunotherapy, specifically immune checkpoint inhibitors, is revolutionizing cancer treatment, achieving durable control of previously incurable or advanced tumors. However, only a certain group of patients exhibit effective responses to immunotherapy. Anti‐angiogenic therapy aims to block blood vessel growth in tumors by depriving them of essential nutrients and effectively impeding their growth. Emerging evidence shows that tumor vessels exhibit structural and functional abnormalities, resulting in an immunosuppressive microenvironment and poor response to immunotherapy. Both preclinical and clinical studies have used anti‐angiogenic agents to enhance the effectiveness of immunotherapy against cancer. In this review, we concentrate on the synergistic effect of anti‐angiogenic and immune therapies in cancer management, dissect the direct effects and underlying mechanisms of tumor vessels on recruiting and activating immune cells, and discuss the potential of anti‐angiogenic agents to improve the effectiveness of immunotherapy. Lastly, we outline challenges and opportunities for the anti‐angiogenic strategy to enhance immunotherapy. Considering the increasing approval of the combination of anti‐angiogenic and immune therapies in treating cancers, this comprehensive review would be timely and important.

[10][11] Therefore, anti-angiogenic therapy aims to hinder the development of tumor vessels, depriving tumors of essential nutrients and effectively impeding their growth and metastasis. 12][15] Immunotherapies, specifically immune checkpoint inhibitors (ICIs), are currently considered as a revolution for cancer management, achieving durable control in cancer patients with advanced stages.7][18][19][20] Therefore, it is essential to enhance the efficacy of immunotherapy.2][23][24] For example, the combination of the angiogenesis inhibitor bevacizumab plus ICI atezolizumab is better in prolonging the lifespan of patients with unresectable hepatocellular carcinoma (HCC), than either sorafenib or atezolizumab alone. 25ased on these clinical findings, more attention is being paid to uncovering how anti-angiogenesis can enhance the efficacy of immunotherapy in treating tumors.
Here, we concentrate on the synergistic effect between anti-angiogenic and immune therapies in tumor management, dissecting the direct effects and underlying mechanisms of tumor vessels on the recruitment and activation of immune cells.Furthermore, we explore how anti-angiogenic treatments might potentially amplify the benefit of immunotherapy, particularly ICIs.Lastly, we outline challenges and opportunities for anti-angiogenic agents to synergize with immunotherapy.We believe this review is important and timely, considering the increasing approval of a combination of anti-angiogenic and immune therapies for treating cancers.

| Anti-angiogenic therapy
Endothelial cells (ECs) can rapidly form new vessels from existing vasculature, which is referred to as angiogenesis. 2,26As early as 1971, Dr. Folkman suggested that angiogenesis was essential for tumor development and proposed the concept of anti-angiogenesis as an anticancer therapy. 27Since then, the regulatory mechanisms and therapeutic targets underlying tumor angiogenesis have been extensively investigated.So far, numerous molecules have been recognized as regulators of angiogenesis.[8][9][10][11] Therapies targeting VEGF or the VEGF receptor (VEGFR) can be categorized into two types: (i) Drugs such as neutralizing monoclonal antibodies or soluble receptor decoys, that bind to VEGF, disrupting its interaction with VEGFR.(ii) Inhibiting VEGFR downstream signal transduction by either monoclonal antibodies against VEGFR or receptor tyrosine kinase inhibitors (TKI). 28Bevacizumab, which works by neutralizing VEGFA, was approved for treating patients with late-stage colorectal cancer in 2004. 29This is the first clinically approved agent for anti-angiogenic therapy.Until now, there are a dozen anti-angiogenic drugs applied for treating various cancers in clinical practice.
Anti-angiogenic drugs aim to reduce the overall vascular network supplying the tumor. 2 While this is beneficial in starving the cancer of essential nutrients and oxygen, it also results in persistent and severe hypoxia.Hypoxia can activate various cellular responses.Firstly, hypoxia up-regulates the levels of bFGF and PDGF, which also display pro-angiogenic effects, and thus overcomes the effects of anti-VEGF(R) therapy. 30,31This is called compensatory resistance.Secondly, it triggers the process called epithelial-mesenchymal transition (EMT). 32EMT activation in tumor cells is linked to enhanced invasiveness and contributes to therapeutic resistance. 32,33Lastly, hypoxia affects the immune response within the tumor, a point that will be elaborated later.5][36] Moreover, hypoxia plays a role in fostering an immunosuppressive tumor microenvironment, 37 also favoring cancer cell growth. 38,394][15] The tumor vessels are distinct from those in normal tissues and are characterized by excessive, dilated, tortuous, and hyperpermeable.These abnormalities are caused by the rapid sprouting of immature vessels with poor cell-cell contacts and diminished pericyte coverage, and lead to impaired perfusion, increased interstitial fluid pressure, and inadequate immune-cell infiltration and drug delivery. 2,7,10,11tudies have demonstrated that when VEGF signaling is inhibited, tumor vasculature might display a transient normalization, such as reduced vessel branching and permeability, improved vessel perfusion and oxygen supply. 40][8][9][10][11] For instance, low doses of bevacizumab, a VEGF inhibitor, have been shown to improve blood perfusion and enhance the delivery and efficacy of chemotherapy agents in mice models. 41,42In human ovarian carcinoma, a certain dose of axitinib, a VEGFR-targeting TKI, induced a temporary vascular normalization and increased tumor oxygenation, whereas EGFR-targeting TKI erlotinib had no such effect. 43This brief period of normalized vasculature facilitated doxorubicin to efficiently enter the tumor nest. 43,44Similarly, it has been demonstrated that a lower dose of neutralizing antibody of VEGFR2 showed a more potent effect on normalizing tumor vessels and increasing immune stimulatory M1-like macrophages. 45Moreover, this lower-dose anti-VEGFR2 treatment enhanced the impact of vaccine-based anti-cancer immunotherapy. 44,45owever, only transient vascular normalization can be achieved with anti-VEGF(R) agents.The normalization window is typically short, lasting only days, and varies regarding the dosage of anti-angiogenic drugs and tumor types. 4,44Therefore, it is crucial to extensively investigate and develop alternative targets and drugs that can induce stable vascular normalization.

| Immunotherapy
Immunotherapies have revolutionized the clinic management of advanced cancers.][48] Cytotoxic T lymphocyte antigen 4 (CTLA-4), a transmembrane protein in T cells, initially showed the potential as an anti-cancer target. 49,50CTLA-4 acts as an inhibitory receptor and downregulates immune responses, considered as an immune checkpoint molecular. 51CTLA-4 binds to CD86/CD80 presented on antigen-presenting cells (APCs), and blocks the interaction between the costimulatory receptor CD28 and CD86/CD80. 50,52Therefore, by blocking CTLA-4, the inhibitory signals on T-cells are reduced, allowing enhanced anti-tumor immune responses mediated by CD28. 53Ipilimumab, an antibody targeting CTLA-4, prevents its binding to CD80/CD86, thereby improving the activation and proliferation of T cells. 54It has received approval for treating advanced colorectal cancer, 55 HCC, 56 melanoma, 48 etc.
8][59][60] When binding with PD-L1/L2, PD-1 could cause T-cell exhaustion and even apoptosis, thereby preventing excessive immune activation. 58While the PD-L1 level is typically low within nonmalignant cells, certain tumor cells can exhibit high levels of PD-L1 and escape immune surveillance.7][68][69][70][71] While most attention has focused on PD-L1 as a target, PD-L2 can also interact with PD-1 and contribute to immunosuppression in certain circumstances, 72 suggesting that targeting PD-L2 may also have potential therapeutic implications.It is worth highlighting that only a subset of cancer patients respond to PD-1/PD-L1 therapy, 73,74 but the mechanism remains obscure. 73esides PD-1, PD-L1 and CTLA-4, several immune checkpoint proteins are also currently being investigated.These include Lymphocyte Activation Gene 3 (LAG-3) and galectin 1 (Gal-1).LAG-3, also referred to as CD223, is a suppressive receptor in lymphocytes.It is especially expressed in exhausted T cells and suppresses their proliferation. 75Its classical ligand is the class II molecule of major histocompatibility complex (MHC). 76Currently, there are over 20 LAG-3-targeted therapies being evaluated in clinical trials. 75Studies have shown that combining antibodies against PD-1 and LAG-3 synergistically enhances the anti-tumor immune response. 77In 2022, a combined therapy of relatlimab (an antibody against LAG-3) and nivolumab (an antibody against PD-1) was approved to treat patients with advanced melanoma. 78Gal-1, a type of galectin lectin, is up-regulated in most tumor tissues.Gal-1 primarily binds to receptors such as CD7, CD43, CD45, TCR, and Fas, thereby restraining the functions of immune cells, and promoting immune evasion of the tumor. 79Gal-1 inhibitors have shown significant anti-tumor effects, including GM-CT-01 and GR-MD-02. 80In preclinical trials (Phase I/II, NCT00054977 and NCT00110721), GM-CT-01 plus 5fluorouracil significantly improved patient survival.Similarly, in a recent Phase I study (NCT02575404), GR-MD-02 plus pembrolizumab (antibody against PD-1) resulted in positive clinical outcomes in patients with melanoma, NSCLC, and squamous cell carcinoma of the head and neck (HNSCC).To date, there are currently no approved Gal-1 inhibitors for cancer treatment.
Adoptive cell therapy (ACT), based on tumorinfiltrated lymphocytes or T cells expressing genetically modified T-cell receptors or chimeric antigen receptors, is an alternative strategy to recognize and eliminate LI and FANG maligned cells. 81These ACTs are primarily used for the treatment of hematological malignancies. 46,47,82,83Limited lymphocyte infiltration and immunosuppressive microenvironment, which are associated with abnormal tumor vasculature, are major challenges to the effectiveness of ACT in solid tumors.

| The combination of anti- angiogenesis and ICIs
Anti-angiogenic therapies plus ICIs, is one of the many strategies currently being applied in clinics and also >90 clinical trials and shows promising outcomes. 44A clinical trial called IMpower150 investigated atezolizumab (anti-PD-L1) plus bevacizumab (anti-VEGFA) in advanced NSCLC patients.The research showed increased survival benefits in comparison to chemotherapy alone. 84Actually, this drug combination is approved for the treatment of various cancers, such as NSCLC, HCC, RCC, and so on. 25,85,86Another trial, JVDF, examined the effect of ramucirumab (anti-VEGFR2) plus pembrolizumab (anti-PD-1) in patients with advanced NSCLC, gastric cancer, and urothelial carcinomas.The results also demonstrated notable enhancement in survival and response rates in comparison with chemotherapy alone. 87A recent study reaffirmed that combining ramucirumab and pembrolizumab significantly improves OS and response rates in patients with NSCLC. 88o far, a few combinations of anti-angiogenic agents and immune therapies have been approved for clinical practice (Table 1).These data have shown promising results with improved response rates and prolonged survival compared with monotherapy approaches.Nonetheless, it is important to acknowledge that the optimal timing, sequencing, and dosing of these therapies are still under investigation.Additionally, the efficacy of this combination varies depending on the individual patient characteristics.Thus, understanding the mechanism underlying this combination therapy is particularly important.

| MECHANISM OF SYNERGISTIC EFFECT BETWEEN ANTI-ANGIOGENIC AGENTS AND IMMUNE THERAPIES
Limited or absence of T-cell infiltration within tumors is a significant factor that compromises the effectiveness of various immunotherapies, such as ICIs and ACT therapy. 82,89The tumor vasculature plays a crucial role as the entry point for circulating leukocytes, 44 and therefore, abnormal vessels within tumor tissues may contribute to the immune escape of tumor cells. 84,90Mechanistically, the abnormal vascular structure contributes to compromised lymphocyte infiltration.Tumor ECs (TECs) go through a phenotypic change that is distinguished by a loss of adhesion proteins and the expression of immune suppressive molecules, creating a barrier for anti-tumor immunity (Figure 1).In this section, we comprehensively discuss these issues.The aberrant structure of tumor vessels and its induced hypoxic and acidic tumor microenvironment has been introduced in the review before.Here, we further discuss the effect of hypoxia and acidosis on tumor immunity.
Tumor hypoxia gives rise to a wide range of impacts on tumor immunity. 1One such effect is the impairment of innate immune cells. 91,92Macrophages play opposite roles in cancer, promoting tumor growth or exerting anti-tumor activities. 935][96] In a hypoxic tumor environment, NK cells also display diminished cytotoxicity and impaired cytokine production, and thus weakened ability in recognizing and eliminating cancer cells. 97,98Furthermore, hypoxia impairs the activity of both CD4 þ and CD8 þ T cells, thus suppressing the adaptive immune response. 99,100Moreover, hypoxic conditions promoted the naïve CD4 þ T cells to differentiate into regulatory T cells (Tregs), contributing to tumor immune evasion. 101It is worth noting that there is a hypothesis suggesting that anti-angiogenic drugs can induce hypoxia, and augment the effectiveness of ICIs by increasing tumor immunogenicity. 102Tumor Mutational Burden (TMB, mutations per megabase) indicates the level of tumor mutations.It is generally believed that tumors with high TMB possess more surface neoantigens, thereby triggering a heightened immune response. 103,104Only a few studies suggest that hypoxia-induced stress might lead to mutagenesis and increased mutation load within the tumor.An experimental study on cell lines demonstrated that DNA repair genes were transcriptionally downregulated upon hypoxia exposure.Hypoxia also led to an increase in insertions and deletions and the generation of potential neoantigens. 105,106However, further research is still necessary to fully evaluate the role and molecular mechanisms of hypoxia concerning the efficacy of immunotherapy.
T A B L E 1 Approved combinations of anti-angiogenic drugs and ICIs.Acidic conditions within the tumor tissues also significantly impact tumor immunity. 107Tumors often exhibit an acidic extracellular pH as a result of enhanced glycolysis 108 and lactic acid secretion, coupled with insufficient vessel perfusion to remove lactic acid. 1091][112] Additionally, acidic conditions within the tumor microenvironment impair antigen presentation by dendritic cells (DCs), leading to reduced activation and priming of T cells. 100,101Moreover, acidic conditions contribute to the accumulation of immunosuppressive cells.For instance, acidosis enhances the recruitment and activity of Tregs. 113,114Lactic acid promotes the expression of monocarboxylate transporter 1 (MCT1) on the surface of Tregs, facilitating the uptake and utilization of lactate as an energy source.This metabolic adaptation enables Tregs to sustain their suppressive function and survival within the tumor microenvironment. 113,115,116Acidic conditions also polarize TAMs toward an immunosuppressive M2 phenotype. 117,118

| Adhesion proteins expressed in ECs
Immune cells need to extravasate from the circulation into the tissues, 119 thus vessels are considered as the gatekeeper of immune cell infiltration. 120,121The extravasation of immune cells relies on the expression of EC adhesion proteins, including vascular cell adhesion molecule 1 (VCAM1), intercellular adhesion molecule-1 (ICAM-1), and selectins (E-and P-selectin). 122In non-inflamed tissues, ECs generally remain in a quiescent state with low adhesiveness, thus limiting leukocyte adhesion.][127] However, vessels are less adhesive in various malignancies.TECs express reduced adhesion proteins implicated in leukocyte-EC interactions. 2,44,128For example, VCAM-1 is constitutively expressed in ECs of lung blood vessels distant from the tumors, while it is absent in TECs in the tumors. 129In RCC patients, the levels of ICAM-1 and -2 are decreased in TECs compared to that in adjacent non-tumor tissues. 130Similarly, VCAM-1 gene transcription and expression on ECs were suppressed when co-cultured with melanoma cells. 129Of note, IL-1β and TNFα, secreted by tumor cells or mesenchymal cells, facilitate tumor progression by triggering NF-κB and AP1 signaling. 131Thus, given the critical effect of NF-κB and AP1 on inducing EC adhesion molecules, there is a

Line of therapy
Treatment a

Clinical trials ID c
Non-squamous non-small- seemingly contradictory relationship between prevalent activated NF-κB and AP-1 signaling and the downregulation of EC adhesiveness within tumors.Pro-angiogenesis factors like VEGF and bFGF may mediate the unresponsiveness of EC to inflammatory cytokines.The TNFα-induced up-regulation of ICAM1 and VCAM1 was abolished when ECs were pre-incubated with VEGFA or bFGF. 1283][134][135] Besides, endothelial TLR2 was also found to be required for the cytokine-induced expression of adhesion molecules. 136Nonetheless, the detailed mechanism underlying the loss expression of adhesion molecules in TECs remains obscure.

| Antigen presentation by ECs
Various models provide evidence supporting the emerging role of ECs as non-hematopoietic "semiprofessional" APCs, as the expression of genes involved in antigen capture, processing, and presentation. 11The APC function of EC is contextually dependent. 11,120For instance, ECs in the kidney express HLA-DR, which is an MHC II molecule associated with antigen presentation. 11,137,138Of note, unlike professional APCs such as DCs, ECs in the kidney typically do not express CD80/CD86, which bind with CD28 and are essential for T-cell actiavtion. 139Therefore, these ECs primarily activate antigen-experienced T cells.
In addition, liver sinusoidal ECs (LSECs) exert a different PD-L1 is induced by IFN-γ in both human and mouse ECs, while PD-L2 was detected only by human ECs but not mouse ECs. 146TNFα also up-regulated the levels of PD-L1 and PD-L2 in ECs. 44Antibodies that block PD-L1 and PD-L2 on ECs showed a synergistic effect with PHA in the activation of CD8 þ T cells. 146Similarly, blocking PD-L1 on mouse ECs enhanced the activity of CD8 þ cytotoxicity T cells. 147Physiologically, the presence of PD-L1 or PD-L2 on ECs allows the extravasation of T cells without damaging the blood vessels excessively. 44In various malignancies, PD-L1 is expressed on ECs and correlated with the decreased number and energetic phenotype of tumor-infiltrated T cells. 148However, the regulatory mechanism governing the levels of PD-L1 and PD-L2 in tumor ECs remains unclear.Further investigation is also necessary to understand the mechanism underlying the increased PD-L1/L2 levels in TECs.
The Fas ligand (FasL), also known as CD95L, 149 serves as a mediator of cell death and exerts an immunosuppressive function in tumor vasculature.In human tumors, the level of FasL is correlated with the limited infiltration of CD8 þ T cells. 150In mice, suppressing FasL either through genetic manipulation or pharmacologically significantly enhances the infiltration of CD8 þ T cells. 150VEGFA, IL-10, and PGE2 were shown to induce the expression of FasL in ECs. 150 Additionally, ECs in tumors have been found to express Gal-1, 151,152 TIM3, 44,56 and stabilin 1, 153,154 which are also identified as immunosuppressive molecules.

| High endothelial venules
High endothelial venules (HEVs) play an essential role in facilitating the entry of lymphocytes into lymphoid tissues and are primarily located in secondary lymphoid organs. 155,156Unlike regular blood vessels, HEVs have tall, plump ECs with prominent nuclei.These ECs express specific adhesion molecules and chemokines that promote immune cell trafficking. 155Furthermore, HEVs possess immunomodulatory functions beyond immune cell recruitment. 155,157For instance, during inflammation, ECs from HEVs display increased expression of MIF, an important factor in M1/M2 macrophage polarization, 158,159 and thrombospondin 1, which has been shown to inactivate T cell. 160,161umor-associated HEVs (TA-HEVs) are also observed in tumor tissues and play a pivotal role in recruiting T cells and B cells into tumors.For example, one study, based on 146 patients with breast cancer, revealed that the presence of TA-HEVs was correlated with lower recurrent risk and longer survival time of patients. 162TA-HEVs found in melanoma were also associated with increased lymphocyte. 163Furthermore, a study based on metastatic melanoma patients treated with combination therapy of anti-PD-1 and anti-CTLA-4, revealed an intriguing correlation: the presence of TA-HEVs was found to be a predictor of improved response to the combination therapy. 163These findings offer an important understanding of the role of TA-HEVs in the regulation of lymphocyte infiltration and activation within the context of cancer immunity.

| Anti-angiogenic agents regulate the vessel-induced immunosuppression
The tumor vessel-induced immunosuppressive microenvironment arises from structural abnormalities and vascular ECs' multiple immune inhibitory functions.Therefore, we summarize the effects of anti-angiogenic drugs by normalizing blood vessel structure and functions (Figure 2). 3.2.1 | Inducing normalization of vascular structure As we described before, anti-angiogenic therapies targeting VEGF signaling are primarily used to exhaust or destroy the tumor vasculature.These agents could also induce transient "vascular normalization" and relief of tumor hypoxia and acidosis.In mouse breast cancer, a VEGFR2 antibody at a low dose is more effective than higher doses in inducing a normal structure of tumor vessels, and relief of hypoxic stress, thus polarizing M1-TAM and facilitating T-cell infiltration. 45,164Similarly, in mouse breast cancer, pancreatic tumor and melanoma models, a bispecific antibody (A2V) and simultaneous blockade of Angpt2 and VEGFA, induced a vascular regression but normalized the residual tumor vessels, thus facilitating lymphocyte recruitment into tumor tissues. 165Moreover, vascular targeted tumor necrosis factor superfamily member 14 (LIGHT) induced more pericyte cover and vessel maturation, thus increasing the integrity and function of tumor vessels, 166,167 and synergizing with ICIs.In addition to these effects, LIGHTinduced HEV formation in tumors is associated with increased intratumoral lymphocyte infiltration. 167,168oreover, anti-angiogenic agents promote pericyte migration, recruitment, and increased coverage of the tumor vascular barrier by targeting the PDGF/PDGFR signaling, thereby facilitating vascular normalization. 169dditionally, pericytes secrete a variety of immunosuppressive proteins, including NO, PGE2, and TGF-β, which assist tumors in evading immune responses, highlighting the intricacy of pericytes on tumor immunity. 170,171 3.2.2| Up-regulating endothelial cell adhesion One study based on a phase I clinical trial showed that ipilimumab (anti-CTLA-4) plus bevacizumab (anti-VEGFA) displayed favorable clinical outcomes than monotherapy, which were associated with increased adhesion between TECs and infiltration of lymphocytes. 172 Mchanistic investigations revealed that combination therapy altered the levels of various cytokines and chemokines. 173Further in vitro studies demonstrated that TNFα and IL1α up-regulated ICAM1, E-selectin and VCAM1 levels in TECs, thus facilitating lymphocyte adhesion with TECs.Furthermore, VEGF could decrease the levels of ICAM1 and VCAM1 induced by TNFα, which could be reversed by bevacizumab. 173Another study showed that the angiogenesis inhibitor platelet factor-4 could abolish bFGF-mediated ICAM-1 downregualtion. 174Additionally, in a model of RCC, ICAM-1 level was dramatically increased upon sunitinib and bevacizumab treatments.Further in vitro investigations showed that TKI targeting VEGFRs increases the transendothelial migration of leukocytes. 175.2.3| Regulating endothelial expression of immunosuppressive molecules PD-L1 is a prominent immunosuppressive molecule in TECs.Anlotinib, a TKI targeting VEGFRs and FGFRs, effectively decreased the level of PD-L1 on ECs.This inhibition resulted in increased infiltration of CD8 þ T cells and suppressed tumor growth. 148Interestingly, other research showed increased endothelial PD-L1 levels following treatment with anti-VEGF agents. 176Furthermore, Gal-1 has been associated with the increased expression of PD-L1 in TECs. 177Through genetic and pharmacological interventions, Nambiar et al. showed that inhibition of Gal-1 decreased TEC PD-L1 expression, which was implicated in increased tumor infiltration of T cells, leading to improved responses to anti-PD1 therapy. 177s for FasL, in a mouse model, the suppression of FasL through genetic or pharmacological approaches resulted in an up-regulated proportion of CD8 þ T cells over Treg cells. 150Similarly, pharmacological blockade of PGE2 and VEGF also led to a significantly increased ratio of CD8 þ T cells over Treg cells.This effect relied on the reduction of EC FasL expression. 150 3.2.4 | Inducing the formation of TA-HEVs Several studies have revealed that inhibitors of tumor angiogenesis can stimulate immune responses by inducing TA-HEV formation. Fr instance, in a glioblastoma (GBM) study, an EC-targeted peptide, CGKRK, introduced LIGHT into tumor vessels.LIGHT facilitated the normalization of the brain cancer vasculature and triggered the formation of TA-HEVs.When CGKRK-LIGHT was combined with a VEGF antibody and an ICI, it further enhanced the formation of HEVs and the presence of T cells in GBM, leading to the reduction of tumor burden.167 Similarly, in the tumor tissues of mouse insulinoma, vessel-targeted LIGHT could induce TA-HEV formation and the emergence of tertiary lymphoid structures, increasing T-cell infiltration and sensitizing tumors to ICI treatment.168 In overt melanoma metastases, LIGHT also induced the generation of TA-HEVs, which sensitized refractory lung metastases to anti-PD-1 inhibitors.163,178 Furthermore, the combination of anti-PD-L1 with anti-VEGFR2 induced HEVs in RT2-PNET pancreatic neuroendocrine tumor and PyMT breast cancer models.176

| OTHER COMBINATIONS OF ANTI- ANGIOGENIC AND IMMUNE THERAPIES
Besides synergizing with ICIs, the combination of antiangiogenic agents with other immune strategies has shown promising progress in both preclinical and clinical settings.This further supports the idea that remodeling the tumor immune microenvironment through antiangiogenic approaches enhances the efficacy of immunotherapy.
][181] However, the poor perfusion of the tumor vessels often hinders the presence of T cells in tumor tissues.Studies have provided evidence supporting the idea that blocking VEGF-VEGFR signal transduction can enhance the efficacy of ACT.For instance, VEGF antibodies combined with ACT in B16 melanoma xenografts have been shown to halt tumor development and improve the overall survival of tumor-bearing mice. 182erapeutic cancer vaccines are a type of immunotherapy designed to stimulate the immune system to identify and attack tumor cells.Unlike preventive vaccines that prevent infections, therapeutic cancer vaccines are designed to provoke an immune response against existing cancer cells.For example, co-administration of neoantigen-based DC vaccine and lenalidomide, an angiogenesis inhibitor, exhibited obvious anti-tumor effects in the mouse xenografts derived for colon cancer. 183his combination therapy also attenuated tumor growth and progress in lymphoma 89 and myeloma xenografts. 82,89urthermore, lenalidomide combined with DNA vaccine decreased the presence of myeloid-derived suppressor cells and Treg cells. 184These preclinic investigations provide further rationale for clinical application.

| EFFECTS OF IMMUNOTHERAPY ON TUMOR VASCULATURE
We have primarily explored how anti-angiogenic agents enhance immunotherapy efficacy.Concerning the synergistic mechanisms involved, recent literature has also revealed that immune checkpoint blockade can reshape the vascular system by impacting T cells.Tian et al. discovered that immune checkpoint blockade can induce Th1 cells to secrete IFN-γ, promoting vascular normalization. 35Similarly, Zheng and colleagues demonstrated that anti-CTLA-4 therapy induced the production of IFNγ by CD8 þ T cells, subsequently enhancing vascular perfusion, 185 of which the mechanism was unexplored.Consequently, when combined with ICIs, anti-angiogenic agents trigger a positive feedback loop where ICIs activate T cells, leading to tumor vascular normalization.In turn, normalized blood vessels promote T cell activation, leading to more vascular remodeling, ultimately resulting in long-term tumor control effects.

| CONCLUSION AND PROSPECTIVE
Anti-angiogenic therapy plus immunotherapy has shown significant benefits in comparison with monotherapy in clinical treatment.It has evolved into the standard for treating various cancers.The success of anti-angiogenic therapy has highlighted its effectiveness in enhancing immune therapy outcomes by targeting VEGF signaling.However, there are still questions that need to be addressed.Notably, these would also be opportunities to advance anti-angiogenic therapy.
Firstly, the brief existence of treatment windows and resistance to VEGF-targeted therapy restricts its clinical application.The normalization window of tumor vasculature through low-level VEGF blockade is transient and unpredictable.Therefore, finding ways to stabilize vascular normalization is crucial.Exploring alternative targets beyond VEGF for anti-angiogenic therapy could be a solution.
Next, our knowledge about the regulatory mechanism between blood vessels and the immune response is still limited.Inflammation activates ECs through cytokines like TNFα, NF-κB and AP-1 signaling pathways.This activation leads to the upregulation of adhesion proteins such as selectins, ICAM1, and VCAM1, along with the secretion of chemokines, facilitating leukocyte extravasation and tissue infiltration.In TECs, the expression of these adhesion proteins is downregulated.Nonetheless, the activation of the NF-κB or AP-1 pathway is evident in tumor cells, which presents a seemingly contradictory result.Although VEGF and bFGF have been implicated as important regulators in this process, further research is needed to clarify the specific mechanisms involved.
Furthermore, for PDL1, LAG-3, gal-1 and FasL, it is also crucial to explore novel immune regulatory molecules to leverage the potential of EC functionality fully, for instance, the antigen presentation ability of ECs.By gaining deeper insights into the immune regulatory function of ECs, innovative therapeutic strategies can be developed to target the interaction between angiogenesis and immune responses.
These challenges have prompted researchers to investigate the mechanisms underlying anti-angiogenic therapy.As our understanding of tumor vasculature and the immune microenvironment deepens, a wide range of new drug targets are anticipated to emerge, enhancing the functionality of tumor vasculature.This advancement has significant potential for improving the overall effectiveness of cancer treatment, particularly when combined with immunotherapies.

3. 1 |
Blood vessel is the key regulator of the immune microenvironment 3.1.1| Physical features of tumor vessels on cancer immunity