Tumor angiogenesis and anti-angiogenic therapy

Abstract

Anti-angiogenic drugs (AADs), which mainly target the vascular endothelial growth factor-A signaling pathway, have become a therapeutic option for cancer patients for two decades. During this period, tremendous clinical experience of anti-angiogenic therapy has been acquired, new AADs have been developed, and the clinical indications for AAD treatment of various cancers have been expanded using monotherapy and combination therapy. However, improvements in the therapeutic outcomes of clinically available AADs and the development of more effective next-generation AADs are still urgently required. This review aims to provide historical and perspective views on tumor angiogenesis to allow readers to gain mechanistic insights and learn new therapeutic development. We revisit the history of concept initiation and AAD discovery, and summarize the up-to-date clinical translation of anti-angiogenic cancer therapy in this field.

Introduction

As one of the largest tissue mass, blood vessels play critical roles in homeostasis maintenance, nutrient exchange, metabolism, and tissue growth through various mechanisms. The ubiquitous role of blood vessels suggests that targeting vessel growth may be an effective approach in treating a wide range of diseases. Among numerous blood vessel-targeting therapies for various diseases, targeting tumor angiogenesis is the most validated therapy, with 20 years of clinical experience since the first US Food and Drug Administration-approved anti-angiogenic drug (AAD), which has been routinely used to treat a broad spectrum of solid tumors. The mechanism of tumor angiogenesis has been summarized by several comprehensive reviews, including reviews from the same series in this journal. This review provides historical views and references on tumor angiogenesis, and offers perspectives on new therapeutic development in the field of anti-angiogenic cancer therapy.

Historical Overview of Tumor Angiogenesis

The foundations for the field of tumor angiogenesis were laid by studies published a century ago. However, these studies had little impact at the time, partly owing to technological limitations. In 1971, the young surgeon Judah Folkman^[1] proposed inhibiting angiogenesis to treat cancer. This innovative idea provided a major impetus for research in this field and inspired many investigators. After decades of preclinical studies, various tumor-derived angiogenic factors have been identified, isolated, and studied extensively. In 2004, the US Food and Drug Administration approved bevacizumab, an anti-vascular endothelial growth factor (VEGF) neutralizing antibody, as the first AAD for treating metastatic colorectal cancer in humans.

Folkman’s concept of targeting tumor angiogenesis to treat cancer

In the late 1960s and early 1970s, Folkman observed that solid tumors contained an exceptionally high number of fragile and hemorrhagic blood vessels. As well as conducting his surgical work in the clinic, Folkman et al^[2,3,4,5] performed experiments on rabbits and mice and observed that in the absence of neovascularization tumors were viable but remained small, typically 2–3 mm in diameter. Based on these findings, Folkman^[1] hypothesized that tumor growth is dependent on angiogenesis and that inhibition of tumor angiogenesis would provide a new therapeutic approach for the treatment of cancer. Earlier observations also supported Folkman’s hypothesis. For example, in 1945, Algire et al^[6] observed the vascularization process in a mouse mammary gland tumor model, recording initial tumor vascularization and subsequent hypervascularization during tumor growth. Based on these in vivo results, the authors concluded that the vascular supply is essential to support rapid tumor growth, and tumor cells produce signaling molecules to elicit the growth of new capillary endothelium. They postulated that unlike normal cells, malignant cells have the ability to stimulate continued vascular proliferation.

In 1971, Folkman et al^[3] isolated a tumor angiogenic factor (TAF) from tumor homogenates. TAF was shown to consist of nucleic acids and possibly other molecular entities. Therefore, the same group attempted to develop an antibody against ribonucleic acid to inhibit angiogenesis.^[1] While these were undoubtedly pioneering efforts, it became apparent that further biochemical purification and characterization of TAF was required to identify the key mediators of tumor angiogenesis and effectively target this process. Subsequently, it became clear that the key angiogenic molecules were proteins,^[1] not nucleic acids.

Development of angiogenesis assays

Folkman et al^{[3,4,7,8,9,10,11,12]} developed elegant animal models to facilitate the study of tumor angiogenesis. In both rabbit and mouse models, implantation of a piece of solid tumor tissue in the cornea induced tumor angiogenesis [Figure 1]. Since corneal tissue lacks pre-existing blood vessels, tumor angiogenesis in the cornea must represent new vasculature. Tumor cells produce angiogenic stimuli to induce endothelial cell proliferation and migration, which are essential for angiogenic vessel formation.^[3] Tumor implants induced DNA synthesis in resting endothelial cells, which was validated using electron microscopy and autoradiography.^[1,3] TAFs appeared to not only stimulate tumor neovascularization but also maintain tumor vasculature. Withdrawal of TAFs resulted in regression of the established tumor vasculature and kept tumors in a dormant state, typically smaller than 2–3 mm and comprising less than one million cells.^[1,3] Based on these findings, Folkman et al^[1,4] proposed the concept of anti-angiogenesis-induced avascular tumor dormancy, a harmless and relatively stable state. Moreover, without neovascularization, tumor cannot spread and metastasize to other tissues and organs, emphasizing the crucial role of tumor angiogenesis in cancer progression.

Figure 1.

Tumor angiogenesis in the cornea. Implantation of a tiny piece of solid tumor into the corneal micropocket of the experimental animals instigates neovascularization from the pre-existing limbal vessel. Because the cornea is an avascular tissue, blood vessels in the cornea and tumor are newly formed angiogenic vessels. This model provides an in vivo tool to discover novel angiogenesis inhibitors.

Isolation of key angiogenic stimulators

According to Folkman’s hypothesis, solid tumors release pro-angiogenic factors to stimulate pre-existing vessels in the surrounding tissues. Testing this hypothesis required the establishment of reliable angiogenesis assays. In 1979, Folkman et al^[13] developed an in vitro endothelial assay involving the long-term culture of capillary endothelial cells from rats, calves, and humans. Tumor-derived conditioned media and gelatin-coated plates enabled the successful and prolonged culture of these cells. Long-term culture of capillary endothelial cells with tumor-conditioned media provides an in vitro system to discover proangiogenic factors. The major technological challenge in protein discovery during this period was the isolation of a protein to homogeneity in order to obtain a partial amino acid sequence. The sequence not only enabled comparisons with available sequence databases to determine whether the protein was novel but also allowed the design of probes for molecular cloning. A disadvantage of sequencing technology at this time was the length of time taken to obtain the sequence data. As proteins can have multiple biological effects, it was not uncommon for different research groups to converge on the same protein.

In 1984, using the capillary endothelial cell proliferation assay, Folkman et al^[14] isolated the first TAF–basic fibroblast growth factor (bFGF or FGF-2), from chondrosarcoma. It proved to be identical to FGF, which had been previously isolated from the pituitary of mammals.^[15,16] Isolation of bFGF from tumors validated early observations that tumors produce vasoproliferative factors, as well as Folkman’s concept of tumor-derived angiogenic factors promoting neovascularization. Notably, genetic deletion of either acidic fibroblast growth factor (aFgf, or FGF-1) or bFgf did not lead to obvious vascular defects,^[17,18] suggesting redundant functions within the FGF family.

In addition to these pro-angiogenic factors, a potent tumor-derived vascular permeability factor (VPF) was discovered by Dvorak et al^[19] in 1983, although sequencing was not complete until 1990.^[20,21] In 1989, Ferrara et al^[22] isolated and cloned an endothelial cell mitogen from the conditioned media of pituitary cells, which they named VEGF. Cloning of VPF revealed it to share identical sequences with VEGF.^[21] Subsequent studies have shown that VPF/VEGF is a potent tumor angiogenic factor.^[23]

The discoveries of bFGF and VEGF were important milestones in angiogenesis research and galvanized the search for additional tumor-derived angiogenic factors. This leads to the discovery of additional members of the FGF, VEGF, and platelet-derived growth factor (PDGF) families, including aFGF and another 20 members of the FGF family, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PlGF), PDGF-A, PDGF-B, PDGF-C, and PDGF-D.^{[24,25,26,27,28,29,30,31,32,33,34,35,36,37]} In addition to these angiogenic factors, the angiopoietin family members angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2), which bind to the endothelial receptor Tie2, were also discovered.^[38,39,40]

Discovery and isolation of angiogenesis inhibitors

Angiogenesis inhibitors can be categorized into two groups: endogenous and exogenous. Endogenous angiogenesis inhibitors are often protein molecules, such as angiostatin, endostatin, and soluble receptors, that exist in the body to prevent excessive vessel growth.^{[41,42,43,44,45,46,47,48,49]} Exogenous inhibitors are pharmacological molecules that block angiogenic responses of proangiogenic factors.

The existence of endogenous angiogenesis inhibitors was first proposed based on several observations: (1) Most adult tissues, except reproductive organs, lack angiogenesis; (2) Several avascular tissues such as cartilage and cornea avoid neovascularization;^{[7,8,9,10,50]} and (3) Transplantation of avascular cartilage into the cornea blocks tumor angiogenesis. Folkman et al^[7,8] developed an elegant model involving the simultaneous implantation of tumor tissue and a tiny drug delivery system containing angiogenesis inhibitors into the same cornea [Figure 1]. Using this model, the first endogenous angiogenesis inhibitor was isolated in 1976^[8] and subsequently identified as a tissue inhibitor of metalloproteinases.^[51,52,53]

In addition to the corneal angiogenesis model, Folkman and colleagues developed the chick chorioallantoic membrane (CAM) assay to identify angiogenesis inhibitors.^{[50,54,55,56]} Robust angiogenesis occurs during embryonic development, and chick embryonic development can be studied in a petri dish.^[50,57] When a cartilage fragment was implanted in the CAM, diffusible cartilage-derived angiogenesis inhibitors created an avascular zone.^[50] Similar to the corneal angiogenesis model, the implantation of inhibitor-embedded polymers into the developing CAM regressed angiogenesis. Another important angiogenesis assay, the in vitro capillary endothelial cell proliferation and migration system, was also used to identify pro-angiogenic factors and inhibitors; angiostatin and endostatin were discovered by inhibiting FGF-2-driven capillary endothelial proliferation.^[41,42]

Surgical removal of a large primary tumor mass in patients often boosts metastatic growth.^[58] Folkman et al^[41] hypothesized that this is due to the production by these tumors of inhibitors that restrict metastatic tumor growth in remote organs. Consequently, metastatic nodules remain in a microscopic avascular dormant state, usually consisting of a few hundred cells.^[41,59,60] Surgical removal of primary tumors eliminates these inhibitors, triggering the growth of these metastatic nodules. This interesting phenomenon can be reproduced in mouse tumor models, in which angiostatin and endostatin were found to act as circulating angiogenesis inhibitors.^[41,42]

Endogenous angiogenesis inhibitors have several common features: (1) Protein fragments. They are often proteolytic fragments of large parental protein molecules that are abundant in the body.^[46,48,49] For example, angiostatin is a group of plasminogen fragments containing kringle domains,^{[41,45,61,62,63]} endostatin is a C-terminal fragment of collagen XVIII, and tumstatin is a fragment of collagen IV.^[42] Collectively, experimental evidence indicates that proteolytic processing suppresses neovascularization by generating angiostatic molecules; (2) Enigmatic mechanisms. Unlike angiogenic factors and their specific receptors, endogenous angiogenesis inhibitors often lack defined signaling pathways. Although various mechanisms have been proposed, none seem to be solely responsible for the broad-spectrum angiostatic activity. Soluble receptors, which are generated by either alternative splicing or proteolytic processing and neutralize specific ligands, are an exception to this. For example, soluble VEGF receptor (VEGFR)-1 and VEGFR-2 neutralize their respective ligands and therefore block angiogenesis;^[64,65,66] (3) Broad-spectrum inhibition. Endogenous angiogenesis inhibitors usually block a common pathway of angiogenesis and therefore neutralize the pro-angiogenic activities of various angiogenic factors;^{[46,67,68,69]} and (4) Nontoxicity. As endogenous angiogenesis inhibitors exist naturally in the body, they generally do not produce adverse effects. To our knowledge, the only endogenous angiogenesis inhibitor approved for clinical use as an anti-cancer drug is endostatin (Endostar). In 2006, endostatin in combination with chemotherapy was approved by the National Medical Products Administration in China to treat non-small cell lung cancer (NSCLC)^[70] and it is now one of the main components of first-line NSCLC therapy in China.^[71]

Numerous exogenous angiogenesis inhibitors have been obtained from plants, food, and fungi. TNP-470, an analog of the fungus-derived antibiotic fumagillin, potently inhibits angiogenesis and tumor growth.^[72] TNP-470 also displays anti-obesity and anti-diabetic effects by modulating adipose tissue vascular function.^[73,74] Epigallocatechin-3-gallate in green tea is an orally active inhibitor of angiogenesis and tumor growth,^[75] and may therefore have been reducing cancer incidence for thousands of years.

Despite these discoveries, neutralizing antibodies against VEGF have the advantages of monospecific targets and long half-life in the body.

Successful Translation of Antiangiogenic Therapy in the Clinic

The concept of angiogenesis-dependent tumor growth is generally accepted, although alternative theories of pre-existing vessel-dependent tumor growth via a vascular co-option mechanism have been proposed.^[76,77] The fact that an avascular tumor lacking intra-tumoral neovascularization remains dormant provides definitive evidence of angiogenesis-dependent tumor growth.^[4] This theory suggests inhibition of tumor angiogenesis as a universal approach for treating all solid tumors. Indeed, decades of preclinical research support this concept, and robust suppression of tumor growth by angiogenesis inhibitors has been observed in almost every experiment.^[68] In some tumor models, over 80%–90% tumor suppression can be achieved with a single anti-angiogenic agent.^[78] The impressive effects of anti-angiogenic monotherapy make this therapy a potent anticancer modality and an attractive candidate for clinical development. Today, almost all clinically available AADs contain anti-VEGF components that block VEGF-VEGFR interactions.

The key issue is the successful translation of these preclinical findings into clinical practice. Clinically, limited therapeutic benefits have been observed following anti-VEGF-based monotherapy in most cancer types,^[79] with the exception of clear cell renal cell carcinoma (ccRCC), in which bevacizumab monotherapy significantly improves progression-free survival (PFS) and overall survival (OS).^{[80,81,82,83]} Likewise, anti-angiogenic single-agent tyrosine kinase inhibitors (TKIs), including sunitinib, sorafenib, and pazopanib, achieve objective response rates (ORR) of 20%–47%, PFS of 8.4–11.0 months, and OS of 26.4–28.4 months.^{[84,85,86,87]} In patients with glioblastoma multiforme (GBM), bevacizumab monotherapy has only modest clinical effects on PFS.^{[88,89,90,91]}

Understanding the differences between human tumors and animal tumor models may provide important clues for improving the clinical outcomes of anti-angiogenic therapy. These differences include: (1) Assessment of drug benefits. In preclinical models, the anti-cancer effects of AADs are often determined by measuring tumor volumes. In contrast, in clinical settings, improvements in PFS and OS are the gold standard criteria for demonstrating therapeutic benefits. The size of primary tumors does not always correlate with survival rate;^[92,93] (2) Intrinsic differences between animal tumor models and human tumors. Experimental animal models typically involve cancer cell lines that have undergone long-term culture and have therefore lost the original features of the primary tumors from which they were isolated. These tumor cell lines exhibit accelerated growth rates and following implantation give rise to relatively large tumors in a relatively short period. In contrast, human tumors often grow relatively slowly, with months or even years passing before a clinically detectable mass is formed. The variation in tumor growth rates may also dictate the difference in angiogenic profiles within tumors and the subsequent discrepancy in drug responses; (3) Cancer hosts. In experimental animal tumor models, genetically identical animals (often mice) are utilized. In contrast, each human patient has a unique genetic background; (4) Prevention versus treatment. In experimental animal models, drug treatment is often initiated at the time of tumor cell implantation. This is considered a preventive intervention rather than a therapeutic treatment. In contrast, the treatment of humans involves established tumors with a relatively large tissue mass; (5) Tumor progression. Clinical trials of AADs are often conducted in patients with advanced cancer in whom other therapeutic options have failed. At an advanced stage of cancer progression, patients suffer from metastasis and systemic diseases such as cancer cachexia. These hard-to-treat patients therefore respond poorly to anti-cancer drugs. Conversely, animal models are often treated at an early stage of cancer development and lack metastasis and systemic disease; and (6) Age. Human cancers often occur in aged individuals, whereas experimental tumors are frequently grown in relatively young animals [Table 1]. The clinical challenges associated with anti-angiogenic therapy for cancer treatment have been discussed elsewhere.

Table 1.

Differences between human tumors and mouse tumor models in response to antiangiogenic therapy

Characteristics	Mouse tumor models	Human tumors
Tumor
Growth rate	Fast	Relatively slow
Genetic alterations	Diverse mutations	Relatively fewer mutations
Heterogeneity	Relative homogenous	Highly heterogenous
Cancer host
Genetic background	Homozygosity	Highly heterozygous
Age	Relative young animals	Often aged population
Tumor site	Often subcutaneous	Primary site
Metastasis	Lack of metastasis	Often with metastasis
Cachexia	Often lack of cachexia	Often with cachexia
Cancer stage	Relative early stage	Often advanced stage
Antiangiogenic therapy
Treatment schedule	Early intervention	Established large tumors
Effectiveness assessment	Often tumor size suppression	Survival improvement
Therapeutic regimen	Monotherapy	Often combination therapy

Combination with chemotherapy

In the clinic, most AADs are administered alongside chemotherapeutics, and this combination shows improved effects beyond those of chemotherapy alone.^[93] However, the mechanism underlying these effects remains unclear. Several hypotheses have been proposed to explain the beneficial mechanisms underlying the combination therapy. The most prominent of which states that AAD-induced vascular normalization enhances the therapeutic effects of chemotherapeutics.^[94] Unlike healthy vasculature, tumor vasculature possesses distinctive features, including vascular network disorganization, high permeability, lack of perivascular cells, lack of basement membrane, immature and unstable plexuses, inappropriate remodeling, and poor vascular perfusion.^[95,96] These vascular features are likely caused by tumor hypoxia, acidosis, metabolites, inflammation, and high growth rates of cancer cells. Despite the presence of exceptionally high numbers of microvessels and ongoing angiogenesis, tumor tissues are severely hypoxic. Indeed, tumor tissues, especially those located in the central region, often undergo necrosis due to hypoxia and poor blood perfusion.^[97] In response to anti-angiogenic agents, the disorganized and premature microvasculature undergo marked remodeling by regressing excessive vascular networks and sustaining functional vessels.^[94] AAD-treated tumors therefore exhibit decreased vascular density, but the remaining vessels have been reported to increase, instead of decrease, blood perfusion via a mechanism of vascular normalization. These remaining vessels lack excessive sprouts and have relatively large-diameter lumens, which are structurally similar to those observed in healthy tissues. If the vessels remaining after AAD treatment increase blood perfusion, the distribution of simultaneously delivered chemotherapeutics will also increase, thus enhancing their cytostatic effects. This mechanism is supported by experimental evidence from preclinical cancer models.

Another hypothesis proposes that AADs diminish the toxicity of chemotherapeutics.^[98] Chemotherapeutics exhibit broad toxicity profiles that have effects on multiple organs and tissues, including the suppression of bone marrow (BM) hematopoiesis.^[99] It has been estimated that a substantial number of patients with cancer die of chemotherapy rather than malignant disease.^[100] Tumor-derived circulating VEGF (cVEGF) also suppresses BM hematopoiesis by inducing dilation of the sinusoidal vasculature.^{[98,101,102,103]} Thus, cVEGF aggravates the toxicity of chemotherapeutics on BM hematopoiesis.^[98] Neutralizing tumor VEGF in patients with cancer increases BM hematopoiesis, thereby increasing their tolerance of chemotherapeutics. Other potential mechanisms include: (1) Anti-angiogenic activity of chemotherapeutics may result in additive effects when administered alongside AADs;^[104] (2) Dual targeting of cancer cells and vascular endothelial cells by combination therapy may produce enhanced anti-tumor activity; and (3) Anti-angiogenic therapy may markedly alter the tumor microenvironment (TME), enhancing the therapeutic effect of chemotherapy and reducing drug resistance.

Combination with immunotherapy

Immunotherapy is an emerging and effective therapeutic modality for treating various cancers. In particular, immune checkpoint inhibitors (ICIs), including antibodies targeting programmed cell death-1 (PD-1), PD-1-ligand 1 (PD-L1), and cytotoxic T-lymphocyte antigen 4 (CTLA-4), have demonstrated notable effects on long-term survival in a subpopulation of patients with cancer, including melanoma, lung cancer, and renal cell cancer.^[105,106] However, several challenging issues need to be resolved to enhance the therapeutic effects: (1) Similar to most other therapeutics, immunotherapy lacks beneficial effects in the majority of cancer patients;^[107] (2) Development of drug resistance;^[108] and (3) Not all cancer types respond to immunotherapy. Because of these unresolved issues, combination therapy with other therapeutic modalities, such as chemotherapy, radiation therapy, and anti-angiogenic therapy, has been proposed.^{[105,109,110]}

Several phase III clinical trials have demonstrated that the combination of anti-angiogenic components with ICIs produces superior survival benefits relative to their monotherapy. For example, in chemotherapy-treated NSCLC patients, a combination of atezolizumab (an anti-PD-L1 antibody) with bevacizumab improved OS to a greater extent than either monotherapy.^[111,112]. Similarly, the atezolizumab–axitinib combination markedly improved PFS compared with sunitinib for treating metastatic RCC.^[113] The combination of sunitinib with nivolumab (an anti-PD-1 antibody) or ipilimumab (an anti-CTLA-4 antibody) also had greater therapeutic efficacy in RCC relative to sunitinib alone.^[114] Patients with unresectable hepatocellular carcinoma (HCC) treated with atezolizumab combined with bevacizumab demonstrated improved OS and PFS compared with those of patients treated with sorafenib alone.^[115] These trial results demonstrate that the combination of ICIs and AADs produces additive and synergistic effects resulting in improved survival.

Currently, the mechanisms underlying the beneficial effects of combining anti-angiogenic therapy with ICIs remain elusive, although several hypotheses have been proposed. Notably, tumor-derived VEGF is considered as an immunosuppressive factor that alters the tumor microenvironment, creating an unfavorable environment for an anti-tumor immune response.^[116,117] VEGF-induced immunosuppressive effects include: (1) Promoting M1 to M2 polarization of tumor-associated macrophages (TAMs); (2) Recruiting regulatory T cells (Tregs); (3) Recruiting myeloid-derived suppressor cells (MDSC); (4) Inhibiting dendritic cell maturation and thus inactivation of cytotoxic T lymphocytes (CTLs); and (5) Upregulating PD-1 in CTLs. Thus, anti-VEGF therapy may increase the immunosensitivity of tumors by altering the tumor microenvironment.

Combination with other anti-cancer therapeutics

As described above, tumors employ multiple factors to induce neovascularization, and current clinically available AADs are mainly designed to target the VEGF signaling pathway. It is believed that tumors could potentially develop resistance to anti-VEGF drugs by switching to non-VEGF-dependent angiogenesis.^{[118,119,120]} Dual blockade of VEGF and Ang2 has shown survival advantages over monotherapies in preclinical models of several cancer types, including breast cancer, melanoma, and glioblastoma, but was ineffective in RCC.^{[121,122,123,124,125]} The clinical benefit of combined VEGF and Ang2 inhibitor treatment therefore warrants further validation in rigorously designed clinical trials. It remains unclear why the additive benefit of this combination is dependent on the tumor type. Simultaneous blocking of VEGF and FGF receptor (FGFR), VEGF and Notch, and VEGF and c-Met also produces enhanced anti-tumor activity relative to monotherapy. The use of these non-VEGF inhibitors shows promise for treating anti-VEGF-resistant tumors in certain experimental models.

Preclinical evidence has also shown that blocking epidermal growth factor receptor (EGFR) overcomes anti-VEGF resistance, indicating that combination therapy involving the simultaneous inhibition of VEGF and EGFR may be effective.^[126,127] However, randomized phase III studies combining cetuximab with bevacizumab or panitumumab with bevacizumab for colorectal cancer treatment have not shown therapeutic benefits.^{[128,129,130]} In a phase II trial, the combination of bevacizumab and erlotinib showed efficacy in the treatment of NSCLC.^[131] In two phase III trials, the combination of bevacizumab with trastuzumab (a human epidermal growth factor receptor 2 blocking antibody) and chemotherapy resulted in only a modest improvement in PFS in patients with breast cancer.^[132,133]

Concluding Remarks and Perspectives

AADs have become important therapeutic agents for the treatment of various cancers. The clinical success of AADs in the treatment of cancer and eye diseases provides one of the best examples of hypothesis-driven research changing clinical practice to the benefit of millions of patients. However, the clinical benefits of anti-VEGF-based AADs in the treatment of most cancer types are relatively small. Improvements in survival are urgently required in clinical practice. How can more effective next-generation AADs be developed? Would multi-target drugs that block the VEGF-VEGFR pathway be more effective? Clinical experience with anti-angiogenic TKIs that simultaneously block several signaling pathways, including the VEGF pathway, shows that these multi-target AADs may not necessarily be more effective in cancer therapy than anti-VEGF single-target drugs. As VEGF expression levels are always higher in tumors than in adjacent healthy tissues,^[134] targeting the VEGF-VEGFR pathway is an obvious approach for the suppression of tumor angiogenesis. Non-VEGF angiogenic factors and cytokines contribute to anti-VEGF drug resistance during treatment. Therefore, drugs that block non-VEGF angiogenic pathways should be used in combination with anti-VEGF drugs. Perhaps, genetic mutations in cancer cells and the constitution of the TME collectively determine the optimized AAD combination. For example, a recent study demonstrated that functional KRAS mutations in epithelial cancers augment Ang2 expression to escape the anti-VEGF response.^[135] Combining an anti-VEGF AAD with an anti-Ang2 drug effectively inhibited KRAS-mutated cancers; both monotherapies were ineffective. This study provides an example of personalized anti-angiogenic therapy by selecting a subpopulation of patients who are likely to benefit from AAD therapy. Similarly, an in-depth analysis of the cellular and molecular components of the tumor microenvironment will likely provide important clues for the selection of potentially effective AADs and potentially responsive cancer patients. Another important issue in terms of the survival benefits of AADs relates to their non-tumor targets. Some preclinical studies have shown that the survival benefits of AADs are not related to their anti-tumor effects, but instead occur by improving systemic cancer diseases through effects on blood vessels located outside tumors.^[92,98,100] Given the complexity of genetic, epigenetic, and TME heterogeneity, the simultaneous targeting of multiple angiogenic pathways and the selection of responsive patient subpopulations remain challenging issues. Addressing these challenges helps to unlock the full potential of anti-angiogenic therapy to improve patient benefit.

Funding

This study was supported by grants from the European Research Council (ERC) advanced grant ANGIOFAT (No. 250021), the Swedish Research Council (Nos. 2021-06122, 2020-06121, 2020-03427, and 2019-01502), the Swedish Cancer Foundation (Nos. 200734, 232684), the Karolinska Institute Foundation (Nos. 2020-02080, 2018-00904), the Karolinska Institute distinguished professor award, the NOVO Nordisk Foundation-Advance grant, the NOVO Nordisk Foundation (Nos. 0078219, 0057158), the National Key Research & Development Program of China (No. 2020YFC0846600), the Hong Kong Centre for Cerebro-cardiovascular Health Engineering, the National Natural Science Foundation of China and the Swedish Research Council Cooperative Research Project (No. 8211101233), and the Horizon Europe grant-PERSEUS (No. 101099423).

Conflicts of interest

None.