Extracellular vesicles in tumor angiogenesis and resistance to anti‐angiogenic therapy

Abstract

Tumor angiogenesis plays an important role in the development of cancer as it allows the delivery of oxygen, nutrients, and growth factors as well as tumor dissemination to distant organs. Although anti‐angiogenic therapy (AAT) has been approved for treating various advanced cancers, this potential strategy has limited efficacy due to resistance over time. Therefore, there is a critical need to understand how resistance develops. Extracellular vesicles (EVs) are nano‐sized membrane‐bound phospholipid vesicles produced by cells. A growing body of evidence suggests that tumor cell‐derived EVs (T‐EVs) directly transfer their cargoes to endothelial cells (ECs) to promote tumor angiogenesis. Importantly, recent studies have reported that T‐EVs may play a major role in the development of resistance to AAT. Moreover, studies have demonstrated the role of EVs from non‐tumor cells in angiogenesis, although the mechanisms involved are still not completely understood. In this review, we provide a comprehensive description of the role of EVs derived from various cells, including tumor cells and non‐tumor cells, in tumor angiogenesis. Moreover, from the perspective of EVs, this review summarized the role of EVs in the resistance to AAT and the mechanisms involved. Due to their role in the resistance of AAT, we here proposed potential strategies to further improve the efficacy of AAT by inhibiting T‐EVs.

We provide a comprehensive description of the role of extracellular vesicles (EVs) derived from various cells, including tumor cells and non‐tumor cells, in tumor angiogenesis. Moreover, from the perspective of EVs, this review summarized the role of EVs in the resistance to AAT and the mechanisms involved. Due to their role in the failure of AAT, inhibiting T‐EVs may serve as an effective method to increase the efficiency of AAT in the future.

Abbreviations

AAT

anti‐angiogenic therapy

BC

breast cancer

CAFs

cancer‐associated fibroblasts

ECs

endothelial cells

EVs

extracellular vesicles

FGF2

fibroblast growth factor 2

MSC

mesenchymal stem cell

NPC

nasopharyngeal carcinoma

PC

pancreatic cancer

P‐EVs

platelet‐derived EVs

TAMs

tumor‐associated macrophages

T‐EVs

tumor cell‐derived EVs

TME

tumor microenvironment

1. INTRODUCTION

Tumor angiogenesis, the formation of new blood vessels from pre‐existing vessels, provides oxygen and nutrients to actively proliferate tumor cells. Due to the critical role of angiogenesis in tumor development, angiogenesis inhibitors, also known as AAT, have been developed to treat cancer. Over the past three decades, several anti‐angiogenic drugs have been developed and approved by the United States Food and Drug Administration (US FDA) for several types of advanced cancers. ¹ However, this potential strategy has shown limited efficacy, with survival benefits ranging from only a few weeks to several months. Many patients who initially respond to AAT eventually develop resistance over time. Therefore, it is vital to understand how primary or acquired resistance develops. ²

EVs are nano‐sized membrane‐bound phospholipid vesicles that are likely to be produced by all types of cells. ^{3 , 4} As a critical intercellular communicator, EVs carry different bioactive molecules (lipids, nucleic acids, and proteins) and can deliver those cargoes to receipt cells to participate in many biological processes, particularly in the development of cancers. ⁴ A growing body of evidence now suggest that tumor cell‐derived T‐EVs directly transfer bioactive cargoes to ECs to promote tumor angiogenesis. ^{5 , 6} Moreover, T‐EVs exert pro‐angiogenic effects via other cells, such as fibroblasts and immune cells. However, the emerging role of EVs from non‐tumor cells (e.g., endothelial cells, fibroblasts, macrophages, and cardiomyocytes) in tumor angiogenesis has received only scant attention previously. Recent studies reported that T‐EVs play a critical role in the development of resistance to AAT, but the underlying mechanisms remain obscure. In this review, we summarized recent studies on tumor angiogenesis mediated by EVs derived from both tumor cells and non‐tumor cells. Moreover, we discussed the resistance of AAT from the perspective of EVs and proposed potential strategies to further improve the efficacy of AAT. We hypothesized that, due to the important role of T‐EVs in the resistance of AAT, EVs might be served as an effective and potential target to enhance the efficiency of AAT in the future.

2. BIOGENESIS AND CLASSIFICATION OF EVS

EVs are a diverse population of membrane vesicles generated via diverse mechanisms. On the basis of their formation mechanism and size, EVs are generically categorized as exosomes and microvesicles (MVs), and apoptotic body. ⁷ Exosomes have a complex biogenesis process. As shown in Figure 1, the plasma membrane buds inwards and then endocytosed, thus forming a lipid bilayer structure known as the early endosome. Subsequently, these endosomes continue to invaginate, forming vesicles containing many intraluminal vesicles (ILVs), known as multivesicular bodies (MVBs). Then, MVBs fuse with the plasma membrane of mother cells and then release exosomes into the extracellular environment. ⁸ Unlike exosomes, microvesicles arise through direct outward budding and fission of the plasma membrane without going through pathways such as ILV and MVB. Apoptotic bodies are vesicles formed by the decomposition of cell contents during the process of programmed cell death. After arriving at the receptor cell, EVs can release their cargo into the cytoplasm of recipient cells to regulate their biological activities. ⁹ In 2018, the International Society for Extracellular Vesicles (ISEV) put forward the point that, unless the source of EVs in this experiment can be clearly identified, the EV should be classified according to size, which are “small EVs” (sEVs, <200 nm) and “large EVs” (lEVs, >200 nm). ¹⁰

FIGURE 1.

The biogenesis of extracellular vesicles (EVs). Exosomes originate from the endocytic pathway, and the plasma membrane invaginates to form endosomes, and endosomes continue to invaginate to form multivesicular bodies (MVBs), then MVBs fuse with the plasma membrane and release exosomes into the extracellular environment. Microvesicles are released directly from the plasma membrane to the outside of the cell. Apoptotic bodies are produced by apoptosis.

3. TUMOR CELL‐DERIVED EVs IN ANGIOGENESIS

3.1. Effects of T‐EVs on endothelial cells

A previous study has found that ECs take up EVs through endocytosis and promote angiogenesis through the phosphoinositide 3‐kinase/Akt signaling pathway. ¹¹ We recently found that following clathrin‐mediated endocytosis by ECs, T‐sEVs are transported to the perinuclear region in a typical three‐stage pattern. Importantly, T‐sEVs frequently interact with and finally enter lysosomes, followed by a quick release of their carried miRNAs. ¹² The proliferation, migration, and tube formation of ECs are finely controlled by T‐EVs via multiple pathways. T‐EV‐mediated angiogenesis depends on their bioactive cargoes, such as proteins, miRNAs, and lncRNAs (Table 1). Among these cargoes, miRNA is the most reported molecule in T‐EV‐mediated angiogenesis. The tumor promoter miRNA‐23a is found in T‐EVs secreted by some types of cancer cells. T‐EV‐derived miRNA‐23a downregulates prolyl hydroxylase domain 2 (PHD2), a key oxygen sensor that negatively regulates hypoxia‐inducible factor (HIF) protein, thus leading to enhance angiogenesis. ²⁵ In addition, miRNA‐23a targets and damages ZO‐1, the EC junction protein, to increase vascular permeability. ³⁷ Another study confirmed that miRNA‐23a in NPC‐derived EVs can promote angiogenesis by inhibiting TSGA10 (an anti‐angiogenic factor). ³⁸ The high level of miRNA‐23a in EVs endows EVs with enhanced pro‐angiogenic effects in vivo and in vitro. ²⁶ MiR‐143‐3p and miR‐145‐5p are two tumor suppressors that inhibit the proliferation of colorectal cancer cells with mutated insulin‐like growth factor 1 receptor (IGF1R). ³⁹ Another study revealed that miR‐143‐3p and miR‐145‐5p within EVs derived from lung adenocarcinoma cells could increase tube formation in ECs by reducing the level of CAMK1D in ECs. ²⁴

TABLE 1.

Cargoes in tumor cell‐derived EVs activate angiogenesis through different signaling pathways.

Cargo	Signaling pathways	Key role	References
Protein
YAP	–	Promotes angiogenesis	[13]
Shh; Gli	Shh/RhoA		[14]
VEGF‐A	–		[15]
EPHB2	Ephrin‐B reverse signaling/STAT3		[16]
CD39/CD79	A2BR/ADORA2B		[17]
Glypican‐1, Syndecan‐4	–		[18]
Gelatinases, Plasminogen; VEGF/TGF‐β; CXCR4 chemokine receptor	–	Promotes angiogenesis and formation of ECM	[19]
PFKFB3	–	Promotes angiogenesis and proliferation and migration of ECs	[20]
VEGF‐A	Akt and ERK1/2	Tube formation	[21]
mRNA
VEGF‐A mRNAs		Activates VEGF‐A–VEGFR	[22]
miRNA
miRNA‐155	Downregulates FOXO3a and c‐MYB; Upregulates VEGF	Promotes angiogenesis	[22, 23]
miRNA‐143‐3P; miRNA‐145‐5p	CAMK1D		[24]
miRNA‐23a	ZO‐1 Protein, PHD2 SIRT1 TSGA10	Damage ECs' junctions; Upregulate VEGF, VEGFR2 and MMP9	[25, 26, 27]
miRNA‐210‐3P	EphinA3 Protein/p‐Akt/Akt Inhibits SMAD4 and STAT6	Promotes proliferation of ECs	[28, 29]
miRNA‐26a	Inhibits PTEN; PI3K/Akt		[30]
miRNA‐130a	Downregulates c‐MYB	Promotes angiogenesis	[31]
miRNA‐549a	VEGFR2–ERK–XPO5 axis	Increases the permeability of ECs	[32]
miRNA‐141‐3p	SOCS5 JAK–STAT3	Promotes angiogenesis	[33]
LncRNA
IncRNA‐p21	Silent miRNA‐23a, miRNA‐146b, miRNA‐330, miRNA‐494	EC adhesion; Tube formation	[34]
MALAT1	Targets VEGF‐A, VEGF‐D, ENA‐78, IL‐8	Promotes angiogenesis	[35]
RAMP2‐AS1	miR‐2355‐5p/VEGFR2 Axis		[36]

Abbreviations: A_2BR, ADO receptor subtypes 2B; Akt (PKB), protein kinase B; CXCR4, chemokine (C‐X‐C motif) receptor 4; ENA‐78 (CXCL5), recombinant human C‐X‐C motif chemokine 5; EPHB2, ephrin type B receptor 2; ERK, extracellular signal‐regulated kinase; ERK1/2, extracellular regulated protein kinases 1/2; FOXO3a, Forkhead box O3; IL‐8, interleukin 8; JAK, Janus Kinase; MALAT1, metastasis‐associated lung adenocarcinoma transcript 1; PFKFB3, 3 6‐phosphofructo‐2‐kinase/fructose‐2,6‐biphosphatase; PHD2, prolyl hydroxylase 2; PTEN, phosphatase and tensin homolog deleted on chromosome 10; Shh, sonic Hh; SOCS5, suppressor of cytokine signaling 5; STAT3, signal transducer and activator of transcription 3; TGF‐β, transforming growth factor‐β; TSGA10, testis‐specific gene antigen 10; VEGF‐A, vascular endothelial growth factor‐A; VEGFR‐A, vascular endothelial growth factor receptor‐A; XPO5, exportin‐5; YAP, yes kinase‐associated protein; ZO‐1, zonula occludens‐1.

The production and content of EVs are susceptible to the growth environment of parent cells, including hypoxia. ⁴⁰ Hypoxic or normoxic microenvironment conditions will lead to different cargo profiles in T‐EVs. Compared with T‐EVs under the normoxic microenvironment, T‐EVs under the hypoxic microenvironment are more likely to induce angiogenesis and vascular leakage. ⁴¹ Changes in the cargoes of T‐EVs from parent cells under normal and low oxygen environments are shown in Table 2. Myeloid/lymphoid or mixed‐lineage leukemia3 (MLL3) and disheveled binding antagonists of beta‐catenin 2 (DACT2) are tumor suppressors ^{51 , 52} that are downregulated in various types of cancer. Studies have revealed that inhibition of MLL3 and DACT2 will promote the migration and epithelial‐to‐mesenchymal transition (EMT) of tumor cells. T‐EV‐derived miR‐181a activates the YAP–VEGF pathway by inhibiting MLL3 and DACT2 to promote angiogenesis. ⁵³ The level of miR‐181a in T‐EVs under hypoxic has been shown to be higher than it is under normoxic in papillary thyroid cancer. ⁵⁴ YAP, the Hippo pathway effector, works as a crucial signal transducer to mediate VEGF–VEGFR2 signaling during angiogenesis. ⁵³ The binding of VEGF and VEGFR activates YAP/TAZ in ECs, leading to cytoskeletal remodeling, cell migration, and protein transmission, which is necessary for angiogenesis. YAP knockout will impair the transfer of VEGFR2 and change the distribution of VEGFR2, which influences angiogenesis. ⁵³ In the hypoxic microenvironment, miR‐23a carried by liver cancer cell‐derived EVs can inhibit the SIRT1 in the ECs from inducing angiogenesis. ²⁷ In addition, EVs released by NPC cells under hypoxia can carry HIF‐1α, which are taken up by other cells, and then propagate hypoxic signals, ⁵⁵ forming a cycle of hypoxic signal transduction.

TABLE 2.

Changes of the cargoes in EVs under hypoxia compared with normoxic conditions.

Extracellular vesicle origin	Protein change	Nucleic acid change	Signaling pathways	Implication	References
Glioblastoma	Upregulates TSP‐1 and LOX	Upregulates miR‐182‐5p and KCNJ3	Target Kruppel‐like factor 2 and 4; accumulate VEGFR	Tumor angiogenesis; tumor neovascularization	[42, 43]
Esophageal squamous carcinoma		Upregulates miR‐340‐5p			[44]
Colorectal cancer	Upregulates Wnt4		β‐catenin signaling	Facilitates EC proliferation and migration	[45]
Lung adenocarcinoma cancer		Upregulates miR‐103a	PI3K/AKT and STAT3 Axis	Increases M2‐like macrophage polarization and pro‐angiogenic factor expression	[46]
Renal cell carcinoma	Upregulates CA9		Increases MMP2 expression	HUVEC migration and tube formation	[47]
Leukemia		Expresses miR‐92a	Reduces integrin α5 expression	Enhances EC migration; Stimulate tube formation	[48]
Chondrosarcoma	Increases VEGF	Upregulates miR‐181a			[49]
Retinoblastoma		Upregulates miR‐181b	PDCD10 and GATA6	Stimulate tube formation	[50]

Abbreviations: CA9, carbonic anhydrase 9; GATA6, GATA binding protein 6; KCNJ3, potassium voltage gated channel subfamily J member 3; LOX, protein‐lysine 6‐oxidase; PDCD10, programmed cell death‐10; TSP‐1, thrombospondin‐1; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

3.2. Effects of T‐EVs on fibroblasts

Fibroblasts represent the majority of tumor stromal cells, referred to as CAFs. T‐EVs can induce the transdifferentiation of fibroblasts into CAFs, which play a role in angiogenesis. ^{56 , 57} Melanoma cell‐derived EVs, which are enriched with miR‐155‐5P, are capable of inducing the transdifferentiation of fibroblasts into CAFs. miR‐155‐5P downregulates the expression of SOCS1, a kind of tumor suppressor, ⁵⁸ and then inhibits the JAK2/STAT3 signaling pathway, ⁵⁶ leading to trigger the angiogenic switch of CAFs. A similar phenomenon also exists in lung cancer. ⁵⁷ MiR‐210 in lung cancer cell‐derived EVs ⁵⁷ induce the transdifferentiation of fibroblasts into CAFs and upregulate the level of angiogenic factors (e.g., VEGF‐A, MMP‐9, and FGF2) through the JAK2/STAT3 signaling pathway, thus promoting angiogenesis. ⁵⁹

3.3. Effects of T‐EVs on macrophages

Macrophages in the TME can differentiate into TAMs, and most of them exhibit the M2‐like phenotype. The differentiation of macrophages is influenced by induction conditions and the type of cargoes in the T‐EVs. ⁶⁰ For example, EVs derived from head and neck cancer cells, which are enriched with adenosine A_2B receptor, mediate the differentiation of macrophage into an M2‐like phenotype. ¹⁷ The tumor suppressor phosphatase and tensin homolog (PTEN) ⁶¹ and hypoxia are both considered to be critical factors within the TME. Under hypoxic conditions, EVs derived from ovarian cancer cells can induce macrophages to differentiate into an M2‐like phenotype ⁶² and inhibit PTEN to upregulate some angiogenic factors (e.g., VEGF‐A and Ang‐1) in lung cancer. ⁴⁶ Anx‐II in EVs derived from BC cells was previously shown to activate macrophages to promote angiogenesis in a tPA‐dependent manner. In addition, macrophages create a pre‐metastatic niche by releasing inflammatory factors such as IL‐6 and TNF‐α. ⁶³ The pre‐metastatic niche destroys the integrity of the matrix and is conducive to the formation of tumor angiogenesis.

4. NON‐TUMOR CELL‐DERIVED EVS IN ANGIOGENESIS

4.1. Effects of TAM‐derived EVs on angiogenesis

Clinical research has shown that the proportion of TAM is highly correlated with tumor angiogenesis, invasion, and prognosis in the center of the tumor and the tumor‐infiltrating front. ⁶⁴ Signaling molecules (primarily nucleic acids, such as miRNA‐155‐5p, miRNA‐221‐5p, miRNA‐942, and miRNA‐130b‐3p) that are involved in the process of TAM‐derived EVs mediated angiogenesis are listed in Table 3. The communication network effectively remodels immune cells to the immune‐suppressing phenotype, and promotes the angiogenesis driven by cancer cells. Angiogenesis in pancreatic ductal cancer is highly susceptible to the influence of EVs derived from the M2‐like phenotype of macrophages. The increased level of miR‐155‐5p and miR‐221‐5p in TAM‐derived EVs promote tumor angiogenesis by inhibiting E2F2 in ECs both in vitro and in vivo. ⁶⁵

TABLE 3.

TAM‐derived EVs in tumor angiogenesis and mechanisms that underlie these events.

Tumor type	Cargo of TAM‐EVs	Signaling pathway	Results	References
Pancreatic ductal adenocarcinoma	microRNA‐155‐5p and microRNA‐221‐5p	E2F2	Promotes the growth of tumors; Increases vascular density	[65]
Lung adenocarcinoma	miRNA‐942	FOXO1/β‐catenin	Facilitates angiogenesis; Promotes LUAD cell invasion and migration	[66]
Lung cancer			Facilitates angiogenesis; Promotes tumor growth	[46]
Gastric cancer	miRNA‐130b‐3p	MLL3/GRHL2	Vessel‐like tube formation of HUEVCs; GC cells proliferation, migration, invasion	[67]

4.2. Effects of MSC‐derived EVs on angiogenesis

The precise effect of MSC‐derived EVs on angiogenesis remains controversial currently. A previous study has shown that MSC‐EVs suppress angiogenesis in BC. ⁶⁸ Other studies have revealed that MSC‐derived EVs, carrying miR‐100, are internalized by BC cells, and that these EVs reduce the expression of VEGF by regulating the mTOR/HIF‐α axis, thereby inhibiting angiogenesis. ⁶⁹ In contrast, some studies have shown that, in the TME of BC, MSC‐EVs activate YAP and TAZ proteins to promote tumor angiogenesis and tumor progression through the Hippo signaling pathway. ⁷⁰ Furthermore, external stress, such as hypoxia, ⁷¹ may promote MSC‐EVs to induce angiogenesis. The disparity may result from differences between the cellular context and variation of the external microenvironment.

4.3. Effects of CAF‐derived EVs on angiogenesis

Fibroblast‐derived EVs begin to play the pro‐angiogenic role after fibroblasts are transdifferentiated into CAFs in the TME. The levels of T‐box transcription factor 5 (TBX5) and Thioredoxin‐interacting protein (TXNIP) were negatively correlated with angiogenesis, indicating their negative role in angiogenesis. In cervical cancer, miR‐10a‐5p from CAF‐derived EVs promotes angiogenesis through the hedgehog signaling pathway by targeting TBX5 and inhibiting the expression of TBX5. ⁷² Another study showed that CAF‐derived EVs deliver miR‐135b‐5p to colorectal cancer cells, thereby inhibiting TXNIP and promoting angiogenesis. ⁷³ However, the mechanisms of CAFs‐derived EVs promote angiogenesis remain to be explored, perhaps mainly determined by internal cargoes, including membrane proteins, cytosolic, nuclear proteins, and nucleic acids, especially non‐coding RNAs. ⁷⁴

5. THE RELATIONSHIP BETWEEN EVS AND AAT FAILURE

Several bioactive molecules (e.g., VEGF, PlGF, FGF, PDGF) are known to be involved in tumor angiogenesis. Among them, since VEGF is the primary regulator of angiogenesis and promotes proliferation, migration, and tube formation in ECs, VEGF/VEGFR has become the principal target for AAT. ⁷⁵ Representative drugs include bevacizumab and sorafenib. However, the impact of these agents on improving overall survival has been disappointing due to the developed resistance over time. Moreover, the mechanisms mediating AAT resistance are far from understood. Recent studies have shown that EVs might play a critical role in the developed resistance of AAT. We here summarize the relationship between EVs and the resistance of AAT to provide new strategies for improving drug‐resistant AAT.

After bevacizumab treatment, BC cells can actively secrete EVs that are carried with VEGF_90k. VEGF_90K is a unique 90 kDa form of VEGF, which might be a larger crosslinked form of VEGF₁₆₅ because the antibody that specifically recognizes VEGF₁₆₅ can detect VEGF_90k. ⁷⁶ Bevacizumab binds more weakly to EV‐associated VEGF_90K than soluble VEGF_90K (which does not bind to EVs). Furthermore, VEGF_90k on the surface of EVs interacts well with VEGFR on the surface of ECs and mediates direct and sustained VEGFR activation, thus promoting tumor revascularization after drug resistance. The phenomenon that EVs derived from BC cells are not sensitive to anti‐VEGF therapy may explain AAT resistance. The ability of BC cell‐derived EVs to stimulate ECs migration was largely unaffected by bevacizumab. However, BC cell‐derived EVs were highly sensitive to bevacizumab when treated with Hsp90 (the connection between VEGF_90K and EVs) inhibitor 17AAG (17‐N‐allylamino‐17‐demothoxygeldanamycin). Hsp90 is also located on the surface of T‐EVs. In addition, a movie showing the 3D rotation of the T‐EV demonstrates that Hsp90 and VEGF are located along the vesicle surface. VEGF_90k can be co‐immunoprecipitated with recombinant Hsp90, indicating that Hsp90 directly binds to VEGF_90k as the EV surface protein. Compared with the monotherapy group, using bevacizumab and 17AAG, tumor volume was significantly smaller in tumor‐bearing mice. VEGF_90K restores sensitivity to bevacizumab once Hsp90 is inhibited, thus suggesting the critical role of Hsp90 in EV‐mediated AAT resistance. ⁷⁷

Another study found that CAF‐derived EVs in the tumor stroma promoted angiogenesis through the VEGFR2–AKT–ERK signaling pathway in ECs both in vivo and in vitro. However, the VEGF bound to CAFs‐derived EVs is not neutralized by bevacizumab, which may also be one of the reasons for the resistance of tumor AAT therapy. VEGF is anchored on the surface of EVs via the link of heparan sulfate proteoglycans (HSPGs). When heparinase was used to release VEGF bound to CAFs‐derived EVs, the VEGF bound to bevacizumab increased, indicating that the use of heparanase can restore the sensitivity of tumor cells to bevacizumab to some degree. ⁷⁸ Interestingly, bevacizumab had little effect on the number of glioblastoma‐derived EVs. Annexin A2 is a protein that can promote angiogenesis and tumor progression. The mechanism is that annexin A2 carried by T‐EVs promotes EC migration and invasion in a tPA‐dependent manner, which are important steps in angiogenesis. ⁶³ The expression of annexin A2 was increased in glioblastoma‐derived EVs after treatment with bevacizumab. Similarly, glioblastoma‐derived EVs did not bind to bevacizumab, which appears to be one of the ways in which a glioblastoma protects itself from AAT. In addition to T‐EVs, annexin A2 was also found in perivascular cell‐derived EVs (TPC‐EVs), which also play a role in tumor angiogenesis. ⁶³ More interestingly, the experiment used the combination of GW4869, an inhibitor that reduces EV biogenesis, ⁷⁹ and bevacizumab. The combination therapy substantially increased anti‐tumor efficacy compared with bevacizumab use alone. ⁸⁰ We do not deny the role of bevacizumab targeting VEGF in AAT, however EVs could weaken the effect of AAT to some extent. Following AAT in ovarian cancer cells, tumor cells secreted EVs carrying VEGF₁₈₉ which had a strong targeting effect on tumor‐associated ECs (TAECs). Data have shown that VEGF₁₈₉ on the surface of EVs was not neutralized by bevacizumab both in vivo and in vitro. Blocking the uptake of EVs by ECs did not eliminate the tube formation of ECs, whereas blocking the binding between VEGF₁₈₉ and VEGFR2 eliminated EV‐mediated angiogenesis. ⁸¹ This finding indicated that the role of EVs in the resistance to AAT is based not only on the classical intercellular receptor‐ligand binding, but also on the endocytosis of parente cells‐derived EVs by recipient cells. In addition to VEGF₁₈₉, VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉ amino acid variants are the most common VEGF isoforms. Among them, VEGF₁₂₁ is freely secreted, VEGF₁₈₉ is membrane‐bound, and VEGF₁₆₅ exists in both soluble and membranous forms. VEGF₁₈₉ is the most abundant isoform in EVs isolated from the body fluids of colorectal or renal cancer patients. ⁸² The general principle and situation are depicted in Figure 2.

FIGURE 2.

Bevacizumab restores sensitivity to vascular endothelial growth factor (VEGF) on the surface of tumor cell‐derived extracellular vesicles (T‐EVs) and cancer‐associated fibroblast (CAF)‐derived EVs after using heparinase and Hsp90 inhibitor 17AAG, which can disrupt the connection between VEGF and EV.

Vandetanib, an inhibitor of VEGFR‐2, EGFR, and rearranged during transfection (RET) tyrosine kinases, inhibits the angiogenesis of hepatocellular carcinoma (HCC)‐associated ECs. After treatment with vandetanib, TAECs release EVs that are enriched with VEGF, thus increasing the formation of vascular networks around HCC cells and the level of VEGF in tumor tissues. Moreover, EV‐VEGF is not accessible to anti‐VEGF antibodies and can trigger VEGF signaling in ECs. T‐EVs have been shown to package increasing quantities of VEGF and other factors in response to anti‐VEGF therapy. ⁸³

We should note that the inhibition of VEGF bound to EVs cannot completely block out T‐EV‐induced angiogenesis, ⁸³ because T‐EVs may carry other pro‐angiogenic factors (e.g., VEGF/VEGFR, FGF/FGFR, PDGF/PDGFR). ⁸⁴ In a previous study, patients who received anti‐angiogenic tyrosine kinase inhibitors still had good vessel networks in tumors, probably because TAEC‐derived EVs recruited endothelial progenitor cells (EPC) through the Gas6/Axl signaling pathway and mediated revascularization in colorectal cancer after drug discontinuance or drug resistance. A previous study has shown that activation of the Gas6/Axl signaling pathway promotes tumor angiogenesis by recruiting EPCs. Specifically, Gas6 carried by tumor perivascular cell‐derived EVs (TPC‐EV) induces the phosphorylation level of the Axl receptor of EPCs and subsequently activates its downstream effectors, such as Akt and Erk1/2. ⁸⁵

As the presence of VEGF/VEGFR on T‐EVs is known to induce a lower affinity for anti‐angiogenic drugs, a promising strategy that would enhance the therapeutic effect of AAT is to develop new drugs that specifically bind to VEGF/VEGFR on T‐EVs. Alternatively, targeted inhibition of the connection between VEGF and EVs, such as Hsp90 and HPSGs, or the combination of bevacizumab and heparinase, such as HSPGs, may release the VEGF originally connected to EVs, which is not sensitive to anti‐angiogenic drugs, and restore the sensitivity of anti‐angiogenic drugs. As mentioned above, VEGF is not the only signal for T‐EVs to mediate tumor angiogenesis, simply blocking the connection between T‐EVs and VEGF may insufficiently improve the effect of AAT. Due to the important role of T‐EVs in the resistance to AAT, the use of GW4869, an inhibition for the biogenesis and secretion of EVs, may serve as an effective strategy to enhance the efficiency of AAT in the future. The combination of T‐EV inhibition and AAT may be a promising strategy to effectively suppress tumor growth in the clinic, yielding new methods to overcome the resistance of AAT. In addition, cargoes of EVs (e.g., Hsp90) may be used as biomarkers to predict susceptibility to AAT and assist in evaluating response.

6. CONCLUSION AND PROSPECTS

This review discussed the pro‐angiogenic effects of EVs secreted from both tumor and non‐tumor cells. The interrelation between EVs and tumor angiogenesis is complex and forms a specific network (Figure 3). We also discussed the potential role of EVs in the resistance of AAT as well as the underlying mechanisms. Due to the important role of T‐EVs in AAT resistance, strategies that target EVs, such as restoring the sensitivity of VEGF on EV surfaces to anti‐angiogenic drugs or inhibiting the release of EVs, might be served as effective methods to enhance the efficiency of AAT in the future. In addition to T‐EVs, EVs released by non‐tumor cells, such as TAMs, can also be considered targets for AAT (e.g., induce M2‐type macrophages into M1‐type macrophages). Although EVs released by various cells induce tumor angiogenesis in vitro, strong empirical evidence in vivo is lacking. This suggests that there is still a long way from bench to bed, and the anti‐tumor angiogenesis effect of targeting EVs is worthy of further study in vitro and in vivo.

FIGURE 3.

Tumor cells‐derived extracellular vesicles (EVs) and non‐tumor cells‐derived EVs mediate tumor angiogenesis through different signaling pathways.

AUTHOR CONTRIBUTIONS

Zi‐Wu Ye and Zi‐Li Yu contributed equally to this study. Zi‐Wu Ye drafted the manuscript and drew the figures. Zi‐Wu Ye and Zi‐Li Yu discussed and revised the manuscript. Gang Chen and Jun Jia designed the study. All authors read and approved the final manuscript.

FUNDING INFORMATION

National Key R&D Program of China (2019YFA0210500), National Natural Science Foundation of China (81922038, 81801842).

CONFLICT OF INTEREST STATEMENT

All other authors declare that they have no competing interests.

ETHICS STATEMENT

Approval of the research protocol by an Institutional Reviewer Board: N/A. Informed Consent: N/A. Registry and the Registration No. of the study/trial: N/A. Animal Studies: N/A.

Ye Z‐W, Yu Z‐L, Chen G, Jia J. Extracellular vesicles in tumor angiogenesis and resistance to anti‐angiogenic therapy. Cancer Sci. 2023;114:2739‐2749. doi: 10.1111/cas.15801