Although specific targeted drugs for tumor cells have been introduced into clinical trials in the past decade or so, their application is greatly limited by the high heterogeneity of tumor cells and the complexity of molecular alterations. Since the growth and metastasis of solid tumors depend on the formation of neovascularization, anti-angiogenic tumor treatment strategies are theoretically characterized by broad anti-tumor spectrum, less resistance to drugs and easy access to target sites. In this paper, we will briefly discuss the issues of tumor angiogenesis and structural characteristics, the regulation of angiogenesis by the body, and the current status of research and development of anti-angiogenic tumor therapy. I. Angiogenesis and structural characteristics of tumors Tumor cells are highly metabolically active cells, which require sufficient nutrient supply for sustained growth. In the early stage of tumor development, tumor cells can maintain their growth through tissue infiltration, but when the diameter of tumor exceeds 1-2mm, the tumor must form new blood vessels to provide its own nutrition, otherwise it will be in a tiny and dormant state below 1-2mm in diameter for a long time. Angiogenesis of tumor refers to the formation of new capillaries through “budding” on the basis of the original microvessels. The vascular system of normal mature tissues is relatively static (except for the cyclical changes of female endometrium), and the renewal of endothelial cells is also extremely slow (about 250-300 days). In contrast, in tumor angiogenesis, the proliferative renewal cycle of endothelial cells can be as short as a few days. Tumor angiogenesis is a complex process involving multiple factors and cells, and its basic steps can include: (1) the balance between pro-angiogenic and anti-angiogenic factors that locally maintain the vascular state is disrupted, the activity of pro-angiogenic factors is up-regulated, and endothelial cells proliferate; (2) the activity of various hydrolases such as metalloproteinases and tissue fibrinogen activator in the vascular basement membrane is up-regulated, causing the basement membrane and (3) up-regulation of adhesion molecules on the surface of endothelial cells and activation of related pathways leading to invasion of endothelial cells into the stroma of surrounding tissues and their proliferation and migration; (4) elevated expression of vascular endothelial growth factor receptors, which promote the remodeling of endothelial cell shape and the formation of lumen-like structures; (5) in the presence of related genes, by promoting and relaxing the relationship between endothelial cells and surrounding (5) Under the action of related genes, the formation of blood vessels is completed by promoting and relaxing the interaction between endothelial cells and surrounding cells (e.g. smooth muscle cells, fibroblasts). In addition to angiogenesis, which is the main mode of tumor blood vessel formation, there is another mode called vasculogenesis. In this mode of generation, tumor microvascular endothelial cells originate from endothelial precursor cells (precusor) in the bone marrow or circulatory system, which can localize at the tumor site and differentiate into endothelial cells stimulated by certain factors and form vascular-like structures by proliferation. Some factors secreted by tumor tissues, such as vascular endothelial growth factor (VEGF), can promote the release of precursor cells from the bone marrow and promote the formation of tumor microvasculature. Although the main angiogenesis process of tumor vessels is generally similar to that of normal physiology, there are great differences between the two in terms of structure, cell composition and spatial and temporal regulation of the angiogenesis process. As the process of tumor angiogenesis is a disorderly state without normal control, compared with normal blood vessels, the structure of tumor neovascularization lacks integrity, with weak walls, lack of smooth muscle and complete basement membrane structure. There are large gaps between endothelial cells and strong permeability; the structure of vascular network is disordered, with a large number of blind ends, short-circuiting between arteries and veins and local expansion of vessels, leading to increased exudation and high pressure between tissues, and also easy penetration of cancer cells and formation of distant metastasis. In recent studies on the vascular composition of tumors, it has been found that in melanoma and prostate cancer, the phenomenon of “vasculogenic mimicry” (VM) exists, which is an interconnected ring-like pathway composed of an outer stroma surrounding the mass of tumor cells. VM is usually found in highly aggressive melanomas, where the tumor cells are easily shed and hematogenous metastases occur. In addition to the “angiogenic mimicry”, which does not involve endothelial cells at all, there are also “mosaic blood vessels” in the tumor tissue, in which tumor cells and endothelial cells are arranged interdependently on the inner surface of the tumor vascular lumen. The formation of mosaic blood vessels may be related to the existence of large gaps between the endothelial cells of tumor vessels and the exposure of cancer cells around the vessels to the lumen after the endothelial cells are shed and participate in the formation of the inner cell layer of the vessels. The abnormal structure of tumor blood vessels and the abnormal composition of tumor blood vessel cells increase the heterogeneity and complexity of tumor blood vessels, which is a new challenge for anti-angiogenic tumor treatment strategy. Regulation of tumor angiogenesis Tumor angiogenesis is a waterfall response mediated by tumor cells and vascular endothelial cells through different forms of paracrine and autocrine induction of various cytokines in vivo, and whether this response can occur depends on the balance between angiogenesis-promoting factors and angiogenesis-inhibiting factors (i.e., the switch system that regulates vascular homeostasis proposed by Hanahen et al.) When the equilibrium between the two is disrupted and the expression or production of angiogenesis-promoting factors is higher than that of angiogenesis-inhibiting factors, tumor blood vessels start to form. There are many endogenous pro-angiogenic substances in the body, which can be broadly classified into heparin-binding growth factors, such as VEGF and FGF, non-heparin-binding growth factors, such as transforming growth factor (TGF-, ), epidermal growth factor (EGF), inflammatory mediator, etc. according to their biochemical and physiological characteristics (see Table I). etc. (see Table I for details). In particular, VEGF, FGF, PDGF and other heparin-binding growth factors are most closely related to tumor angiogenesis. Table I Endogenous pro-angiogenic substances heparin-binding growth factors: VEGF, PIGF, FGF-1, FGF-2, pleiotrophin, HIV-tat, PDGF, HGF/SF Non-heparin-binding growth factors: TGF-, TGF-, EGF, IGF-I Inflammation-mediating factors: TNF-, IL-8, IL-3, prostaglandin E1, E2 Enzyme molecules: PD-ECGF/TP, COX-2, Angiogenin Hormone molecules: Oestrogens, proliferin Oligosaccharide molecules: Hyaluronan, Gangliosides Hematopoietic factors: EPO, G-CSF, GM-CSF Cell adhesion molecules: VCAM-1, E-selectin Others: Nitric, Oxide, Ang-1 (1) VEGF and VEGF receptor VEGF is the most potent growth factor with pro-angiogenic activity, and during mouse embryonic development, if the VEGF plays an important role in tumor angiogenesis, and many tumor cells have high expression of VEGF. Numerous studies have reported that the level of VEGF in peripheral blood is directly related to tumor prognosis. VEGF acts mainly through the corresponding receptors KDR and Flt-1, which are both tyrosine kinase receptors (RTK), and VEGF binds to KDR to promote endothelial cell proliferation and angiogenesis mainly through MAPK signaling pathway. In addition to promoting endothelial cell migration, VEGF binding can also activate matrix metalloproteases (MMPs) and regulate KDR activity through binding to VEGF. The elevated VEGF expression in tumor cells is associated with the production of hypoxia-inducible factors by tumor tissue hypoxia, which can upregulate VEGF expression. VEGF receptor expression is increased during endothelial cell proliferation. Traditionally, the expression of VEGF receptors was thought to be endothelial cell-specific, but more and more studies have recently shown that tumor cells can express VEGF receptors along with secreted VEGF, with the consequence that VEGF secreted by tumor cells can promote vascularization through paracrine (acting on endothelial cells) and autocrine (acting on the tumor cells’ own VEGF receptors) pathways. VEGF secreted by tumor cells can promote vasculogenesis and tumor cell proliferation through paracrine (acting on endothelial cells) and autocrine (acting on tumor cells’ own VEGF receptors) pathways. (2) Basic fibroblast growth factor (bFGF) bFGF induces endothelial cell proliferation and migration by upregulating endothelial cell expression and secretion of collagenase, uPA and its receptor. bFGF and VEGF have synergistic effects on pro-angiogenesis, with the latter increasing bFGF production in endothelial cells, while the pro-angiogenic effect of VEGF in vitro and its ability to induce fibrinogen activator (PA) are also dependent on the ability of VEGF to induce the pro-angiogenic effect of endothelial cells. The ability of VEGF to promote angiogenesis and induce fibrinogen activator (PA) in vitro is also dependent on bFGF production by endothelial cells. Clinical studies have shown that bFGF expression is elevated in many tumor tissues. It has been suggested that bFGF levels in body fluids are a potential indicator of tumor prognosis and recurrence. (3) Other pro-tumor angiogenic factors are listed in Table I. In addition to VEGF and bFGF mentioned above, there are dozens of endogenous angiogenic factors or substances in the body that are involved in the pro-angiogenic process to varying degrees, such as platelet-derived growth factor (PDGF) and tumor necrosis factor (TNF-), both of which originate from mononuclear macrophages, and the former can promote endothelial cell proliferation chemotaxis, participate in the aggregation of vascular peripheral cells and the development of the microvascular system, while the latter can act as a growth factor to induce angiogenesis at low doses. Recently, the role of Axon guidance molecules in the development of vascular regeneration has received increasing attention. Axon guidance receptors and ligands can be divided into four main families: neuropilins (NRP)/semaphorins, ephrins, eobo/Slit, and netrin/Unc5. For example, NRP can act as a receptor for VEGF heterodimers, can enhance the activation of VEGF receptor KDR via VEGF165, can as a positive regulator of the VEGF signaling pathway involved in angiogenesis, and also as ephrin B2 and its receptor Eph B4 play an important role in arteriovenous vascular differentiation. Recently, it has been suggested that Eph/ephrin interaction plays an important role in the progression of malignant tumors and vascular regeneration, and soluble Eph B4 can inhibit tumor growth in experimental animals, which may be used as a new anti-angiogenic target. In order to regulate the balance of angiogenesis, in addition to many pro-angiogenic substances, there are also a variety of substances that inhibit angiogenesis in human body, a considerable part of which comes from the hydrolysis fragments of some proteins, such as endostatin, the carboxy-terminal fragment of XVIII collagen, and Angiostatin, the fibrinogen degradation fragment ( See Table II), most of them showed strong angiogenesis inhibitory activity, and some of them have entered clinical trials. Table II Endogenous angiogenesis-inhibiting substance protein fragments: Angiostatin: fibrinogen degradation fragment 38KD Kringle-containing region Endostatin: fragment of zinc-binding region of XVIII collagen 20KD AaAT: fragment of antithrombin 3 (antithrombin 3) Vasostatin: calreticulin ( N-terminal fragment of calreticulin 10KD Protactin: 16KD fragment of prolactin PF4: N-terminal fragment of platelet factor 4 Alphastatin: 24 amino acid fragment of fibrinogen-derived small peptide Tumstatin: a peptide fragment of the type IV collagen 3 chain, molecular weight Canstatin: a peptide fragment of the 2 chain of type IV collagen, molecular weight 24 KD Arresten: a peptide fragment of the 1 chain of type IV collagen, molecular weight 26 KD PEX: hydroxyl-terminated hydrolysis fragment of Mmp2, molecular weight 20 KD Soluble mediator Molecules: TSP-1 (platelet response protein-1), Troponin I (myogenin I), IFN , PEDF (pigment epithelial factor), IP-10 (thymidine phosphatase 10), IL-12, IL-4, VEGI (VEGF (VEGF inhibitor), TIMP-1, TIMP-2; PAI-1 (fibrinogen activation inhibitor-1), Retinoic acid, Ang, 2-Methoxyoestradiol Due to the limitation of space, only representative endogenous anti-angiogenic inhibitors are briefly described below, more and more detailed information can be found in the relevant literature. (1) Angiostatin (Angiostatin) Angiostatin was isolated by O’Reilly in 1994 from serum and urine samples of mice transplanted with Lewis lung cancer. Angiostatin inhibits the growth of different tumors in a tumor-bearing mouse model, and its mechanism of angiogenesis inhibition may be mainly related to its downregulation of VEGF expression, its ability to inhibit endothelial cell proliferation by binding to endothelial cell surface ATP synthase; subunits, and its ability to reduce the activity of intracellular protein kinases erk-1 and erk-2 induced by bFGF and VEGF. Angiostatin is commonly injected subcutaneously, with a wide range of effective and safe doses and no toxic side effects have been found. (2) Endostatin (endothelial inhibitor) Endostatin was first isolated from the serum of mice with endothelial cell tumors by O’Reilly in 1997, and is a fragment of the carboxy-terminal non-collagenous region of macromolecular collagen XVIII, consisting of 183 amino acid residues with a relative molecular mass of 20 kD. Endostatin specifically inhibited endothelial cell proliferation and significantly inhibited tumor growth and metastasis. Treatment of bovine pulmonary artery endothelial cells with endostatin caused apoptosis and significantly reduced the production of the anti-apoptotic proteins bcl-2 and bcl-xl, but this effect was not observed in other non-endothelial cells, suggesting that endostatin may selectively cause apoptosis in endothelial cells. Recent studies have revealed that endostatin may also inhibit endothelial cell proliferation by competing with fibroblast growth factor and by blocking the transition from G0/G1 to S phase in different ways. In addition to its antitumor activity alone, endostatin, as a new class of antitumor agents, can also be used in combination with conventional chemotherapy and radiotherapy, with significant synergistic effects. Currently, endostatin has entered clinical trials in different tumors and has shown good efficacy in lung cancer and breast cancer. China SFDA has approved recombinant endostatin to be marketed as an experimental drug. (3) Alphastatin Alphastatin is an endogenous substance with angiogenesis inhibiting function discovered by Carolyn et al. in 2004 after analyzing the effect of different fibrinogen fragments on angiogenic activity, consisting of 24 amino acids, which is the smallest endogenous peptide angiogenesis inhibitor found at this stage. Alphastatin inhibits angiogenesis mainly by inhibiting the migration and tubularization of endothelial cells, but the specific mechanism of action is still unclear. alphastatin has strong angiogenic inhibitory activity and has good anti-tumor activity (0.025 mg/kg/d) at low doses in animal experiments, which has good potential for clinical application. (4) Interferons (TNF-//) IFN is a cytokine with significant inhibition of angiogenesis, which can down-regulate the expression of bFGF and VEGF in many tumor cells, thus inhibiting angiogenesis. can directly inhibit the proliferation and migration of human epidermal microvascular endothelial cells and human capillary endothelial cells. In addition, IFN and IFN also inhibited the formation of neovascularization induced by tumor cells and lymphocytes. It was found that the inhibitory effects of IFN and IFN on bFGF expression were cell density-dependent, while the anti-proliferative activity of IFN was non-cell density-dependent. IFN has entered phase III clinical trials as an anti-angiogenic antitumor agent. III. Anti-angiogenic tumor therapy Current status of research and development As early as a century ago, it was found that tumor growth was often accompanied by an increase in vascular distribution, and it was proposed that angiogenesis might be a key factor in the process of tumorigenesis. In the 1940s, the presence of tumor-derived pro-angiogenic factors was hypothesized, followed by the suggestion that tumor growth is primarily dependent on neovascularization. In the 1970s, Folkman et al. proposed the idea of treating tumors by inhibiting angiogenesis, and subsequent research has become increasingly active as related work progresses, especially in the last decade. 2004, the first anti-angiogenic drug avastin (a monoclonal antibody whose target molecule is VEGF) was officially approved by the U.S. FDA for marketing. It has been used in combination with chemotherapy drugs for the treatment of metastatic colorectal cancer and has achieved good efficacy, confirming the feasibility of anti-angiogenesis in tumor treatment strategy. As the generation of neovascularization is a complex process involving cell proliferation, apoptosis and extracellular matrix degradation and remodeling, theoretically, any part of the angiogenesis process can be blocked. According to the mechanism of action of anti-angiogenic products, they can be broadly divided into: 1) Vascular endothelial cell inhibitors: such as endostatin and angiostatin, which can inhibit endothelial cell proliferation and induce apoptosis; Linomide can inhibit endothelial cell migration. 2) Inhibitors of angiogenic factors or related receptors: such as antibodies against VEGF and VEGF receptors, soluble VEGF receptors, etc., which can inhibit endothelial cell proliferation and migration by inhibiting VEGF activity. 3) Inhibitors of extracellular matrix degradation: Inhibitors of MMPs, for example, block the activity of MMPs and inhibit matrix degradation, thereby blocking the migration and invasion of endothelial cells and tumor cells. 4) Inhibitors of adhesion molecules: Antibodies against adhesion molecules, for example, can block endothelial cell adhesion and inhibit blood vessel formation. 5) Intracellular signaling blockers: Since VEGF receptors and bFGF receptors are tyrosine kinase receptors, factors that bind to the receptors can activate kinase activity and cause cell proliferation, etc. Therefore, tyrosine kinase inhibitors can block the proliferation and migration of endothelial cells by blocking tyrosine kinase activity. However, the specificity of tyrosine kinase inhibitors is the key to determine whether this class of drugs can be used clinically. Among the many anti-angiogenic therapeutic targets mentioned above, targeting VEGF and VEGF receptors has attracted the most attention. This is because VEGF and VEGF receptor play the most important role in angiogenesis, and both of them are highly expressed in many tumor cells and tumor vascular endothelial cells, so they are relatively the most ideal targets for anti-angiogenesis. 2, anti-angiogenesis clinical trials There are many anti-angiogenesis inhibitors in clinical trials, for more information, please visit the website: http://www. cancer.gov/clinicaltrials/developments/anti-angio-table. The fastest, the VEGF antibody avastin is the only anti-tumor vascular regenerative agent approved by the FDA for clinical use. FDA approval of avastin was preceded by a large, randomized, double-blind clinical phase III trial in which avastin was used as a first-line agent in combination with blous-IFL chemotherapy in patients with colorectal tumors. In the trial, the median overall survival time for patients in the blous-IFL and avastin combination group was extended from 15.6 to 20.3 months compared to the IFL only + placebo group. This was accompanied by an increase in progression-free survival, drug effect rate and duration of efficacy. Although patients tolerated avastin better, gastrointestinal perforation or impaired healing of the incision occurred in 2% of patients. Also for patients over 65 years of age, the likelihood of concomitant arterial embolism was increased twofold relative to chemotherapy alone. The mechanism by which these reactions occur is unclear, and it is speculated that it may be that cytotoxic drugs destroy blood vessels and that blocking VEGF exacerbates this destruction. Phase III clinical trials of avastin for the treatment of non-small cell lung cancer, renal cell carcinoma, and metastatic breast cancer are also underway. In addition to avastin, other VEGF inhibitors are in clinical trials. Many targeted small molecule drugs targeting the VEGF receptor, such as SU11348 and Baf43-9006, have made important advances. SU11348 inhibits the activity of VEGFRs, PDGFR, C-kit, and Flt-3, and significant efficacy has been reported in the treatment of imatinib-resistant gastrointestinal tumors. baf43-9006 was initially thought to be a raf kinase inhibitor, and later studies suggested that it could also inhibit VEGFR activity. Results from clinical trial III in patients with advanced renal cell carcinoma showed that Baf43-9006 alone resulted in a significant increase in progression-free survival. AG-01376 has similar inhibitory effects to SU11348 on kinase activity. In a phase II clinical trial in which it was used alone to treat metastatic renal cell carcinoma, it was 46% effective. PTK787 is also a VEGF receptor tyrosine kinase inhibitor. It has entered clinical phase III in combination with FOLFOX4 chemotherapy regimens for the treatment of patients with colon cancer. It has shown a statistically significant improvement in progression-free survival time as assessed by a survey of patients treated with PTK787. A recombinant drug of endogenous peptide endostatin with strong anti-angiogenic activity has been approved for marketing by SFDA in China in 2005 for experimental treatment of tumors. The various major anti-angiogenic products in development and their mechanisms of action are shown in Table III.