Blood vessels of the microvasculature are composed of two major cell types: endothelial cells and perivascular cells known as pericytes (Karamysheva, 2008). Pericytes are known for regulating endothelial cell proliferation, differentiation, and migration through paracrine regulators and vasoactive agents (Kelly-Goss et al., 2014). Under physiologic conditions, endothelial cells exist in a quiescent non-proliferative state (Kruger-Genge et al., 2019). However, in response to vascular injury, inflammation, or hypoxia, angiogenesis is induced through a cascade of highly regulated sequential events (Kruger-Genge et al., 2019). Quiescent endothelial cells are initially activated through increased levels of pro-angiogenic factors (Kruger-Genge et al., 2019). During the activation phase, pericytes are detached from the vessel wall and blood vessels dilate and tight junctions of endothelial cells are disrupted allowing endothelial cells to proliferate and elongate to form the new blood vessel (Mazurek et al., 2017). Simultaneously, proteases remodel the interstitial matrix enabling endothelial cell migration and fusion of newly developed blood vessels (Carmeliet and Jain, 2011). Subsequently, the proliferative activity of endothelial cells is reduced to restore the quiescent state of endothelial cells, and pericytes are recruited to the newly formed blood vessel (Rust et al., 2019). The interaction between endothelial cells and pericytes during angiogenesis is regulated, in part, by Ang-1/Tie-2, TGF-β, and PDGF signaling (Kelly-Goss et al., 2014). The presence of pericyte coverage of endothelial cells supports the maturation and stabilization of blood vessels (Gerhardt and Betsholtz, 2003). Table 1 summarizes the activity of major pro-angiogenic factors.

Two basic types of angiogenesis exist, sprouting and intussusception (Adair TH, 2010). Sprouting angiogenesis represents the major mechanism of angiogenic growth and is characterized by sprouts of endothelial cells growing through the branching morphogenesis process (Teleanu et al., 2019; Lugano et al., 2020). Alternatively, intussusception angiogenesis involves the splitting of existing blood vessels to form new ones (Adair TH, 2010).

The role of angiogenesis in cancer growth and metastasis was first introduced by Judah Folkman who described the growth of blood vessels as an essential process for the growth of solid tumors (Folkman, 1971). Angiogenesis establishes vascular networks to supply oxygen and nutrients essential for tumor growth and metastasis (Lugano et al., 2020). Tumors secrete various pro-angiogenic factors to promote the sprouting of new blood vessels from existing vasculature thus enabling tumor growth and metastatic spreading to distant organs (Yonenaga et al., 2005). Though tumor blood vessels carry distinct molecular markers in the endothelium, several other markers are shared by vessels in non-malignant tissues (Ruoslahti, 2002).

Although angiogenesis plays a key role in tumor vascular growth, non-angiogenic mechanisms of vascularization exist to meet the demands for oxygen and nutrients by tumors (Stessels et al., 2004). Vasculogenesis is the process of the formation of blood vessels from circulating cells (Brown, 2014). The main driver of vasculogenesis is the stromal cell-derived factor-1 (SDF1/CXCL12) upregulated in response to tumor hypoxia and increased levels of hypoxia-inducible factor-1 (HIF-1) (Brown, 2014). Vasculogenesis is mediated by the recruitment of endothelial progenitor cells (EPCs) or bone marrow-derived hematopoietic cells leading to the formation of new blood vessels in the tumor microenvironment (Lugano et al., 2020). EPCs may originate from hematopoietic stem cells, myeloid cells, circulating mature endothelial cells, or other circulating progenitor cells. Regularly, VEGF in the tumor microenvironment mobilizes VEGFR-2-positive EPCs from the bone marrow to initiate vasculogenesis (Lugano et al., 2020). In addition, cancer cells themselves have unique characteristics to form vessel-like channels within the tumor in a process known as vascular mimicry (Ruoslahti, 2002). These vascular structures lack endothelial cells and serve as alternate channels to supply blood and nutrients to tumor cells (Lugano et al., 2020). Like in the case of vasculogenesis, hypoxia promotes vascular mimicry (Andonegui-Elguera et al., 2020). Vascular co-option has also been found to be an important approach to establish tumor vasculature, especially in the more aggressive types of tumors (Qian et al., 2016). In the latter procedure, tumor cells obtain blood supply by hijacking blood vessels in the surrounding normal tissue along with the migration and invasion of cancer cells (Donnem et al., 2013; Qian et al., 2016). Further, cancer stem cells trans-differentiation to endothelial cells and vascular smooth muscle-like cells has been observed in different types of tumors to promote tumor vascularization (Lugano et al., 2020).

Unlike normal blood vessels, tumor vasculature displays multiple functional and structural abnormalities characterized by unusual leakiness, high tortuosity, and poor coverage by pericytes (Dudley, 2012; Goel et al., 2013). These abnormalities mediate chaotic blood flow and support the hematogenous dissemination of tumor cells while impairing the delivery of chemotherapeutic drugs (Goel et al., 2013). Though the measurement of microvascular density is the gold standard approach for quantification of angiogenesis (Tahergorabi and Khazaei, 2012), the maturity and stability of blood vessels are being increasingly recognized in the assessment of tumor vasculature (Fakhrejahani and Toi, 2012). Intratumoral hypoxia triggers the formation of dysfunctional blood vessels thus facilitating cancer cell metastasis and reducing the efficacy of treatments (Kugeratski et al., 2019). Hypoxia increases cell adhesion, coagulant properties, endothelial intracellular gaps, and endothelial permeability, all of which are crucial for the processes of intravasation, and extravasation needed for cancer cell metastasis (Evans et al., 2012). Endothelial cells exposed to hypoxia demonstrated an amplified pro-inflammatory phenotype, characterized by an increased expression of inflammatory cytokines (Tellier et al., 2015). In the microenvironment of solid tumors, hypoxia has been shown to stimulate autophagy in tumor-associated blood vessels which in turn can alter metabolic pathways and surface markers of endothelial cells (Verhoeven et al., 2021). Additionally, hypoxic cancer-associated fibroblasts induced blood vessel abnormalities by altering the secretion of various pro- and anti-angiogenic factors leading to changes in endothelial cell function and promoting angiogenesis (Kugeratski et al., 2019). Figure 1 illustrates angiogenic activity in the tumor microenvironment.

Targeting angiogenesis in cancer therapy is an appealing approach to stop the growth and metastasis of solid cancers. Nevertheless, clinical evidence showed variable sensitivity to angiogenesis inhibitors over different tumor types. The main objectives to conceive and prepare this review paper were to 1) provide a summary of the state of angiogenesis inhibitors in the treatment of breast cancer, 2) analyze the factors attributing to the lack of efficacy of anti-angiogenic drugs, 3) explore new potential drug targets for angiogenesis inhibitors through non-VEGF/VEGFR signaling, and 4) describe novel approaches for targeting tumor vascularization and their potential implementation in breast cancer. The next part of this review describes available evidence for the effect of angiogenesis inhibitors in breast cancer treatment.

Bevacizumab (Avastin^®) is a humanized anti-VEGF monoclonal antibody approved in combination with chemotherapy for the treatment of several advanced solid cancers (Kazazi-Hyseni et al., 2010). It binds selectively to circulating VEGF, thereby inhibiting VEGF binding to its receptor (Kazazi-Hyseni et al., 2010). The AVF2119G clinical trial was the first to provide published data regarding the clinical usefulness of bevacizumab in the treatment of metastatic breast cancer (Miller et al., 2005). The results of this phase III randomized trial showed that the addition of bevacizumab to capecitabine in second-line therapy improved response rate compared to capecitabine treatment alone (19.8 vs. 9.1%, p = 0.001) (Miller et al., 2005). However, this combination neither improved progression-free survival (PFS) (median, 4.86 vs. 4.17 months) nor OS (median, 15.1 vs. 14.5 months) (Miller et al., 2005). The E2100 was an open-label, randomized, phase III clinical trial that investigated the efficacy and safety of the combination of paclitaxel and bevacizumab compared to paclitaxel as a first-line treatment for metastatic breast cancer (Miller et al., 2007). Findings from the E2100 trial indicated a significantly improved PFS for the combination arm compared to paclitaxel alone, while OS was similar in both treatment arms (median, 25.2 vs. 26.7 months; p = 0.16) (Miller et al., 2007). The results from the AVF2119G and E2100 trials granted accelerated approval of bevacizumab use in the treatment of metastatic breast cancer by the US Food and Drug Administration (FDA). Subsequently, several phase III clinical trials have evaluated bevacizumab with chemotherapy revealing no significant improvement in OS (Miles et al., 2010; Brufsky et al., 2011; Robert et al., 2011). The failure of achieving a survival advantage along with serious tolerability issues created controversy over the real value of bevacizumab treatment in metastatic breast cancer and further brought its approval into question. Ultimately, the US FDA had revoked the indication of bevacizumab to treat patients with metastatic breast cancer in 2011 (Sasich and Sukkari, 2012).

After the withdrawal statement, results from other clinical trials on the use of bevacizumab in breast cancer were published. The BEATRICE study was an open-label, randomized, phase III clinical trial that assessed the addition of bevacizumab to chemotherapy in adjuvant settings in patients with operable TNBC (Cameron et al., 2013). Results from the BEATRICE study revealed no improvement of OS compared to patients receiving chemotherapy alone (Cameron et al., 2013; Bell et al., 2017). Rather, grade III adverse events were increased in the bevacizumab arm (Cameron et al., 2013). The E5103 study was a double-blind, phase III trial of adjuvant chemotherapy with and without bevacizumab in breast cancer patients with lymph node-positive and high-risk lymph node-negative disease (Miller et al., 2018). The findings of the study failed to show improvements in invasive disease-free survival or OS upon the addition of bevacizumab to chemotherapy in adjuvant settings in breast cancer patients with high-risk HER2-negative disease (Miller et al., 2018).

Other studies investigated the value of adding bevacizumab to chemotherapy in the neoadjuvant treatment of breast cancer (Bear et al., 2012; von Minckwitz et al., 2012; Clavarezza et al., 2013). The primary endpoint in these studies was the pathologic complete response (pCR) rate. Overall, findings from these clinical trials showed a favorable response for using bevacizumab with chemotherapy in neoadjuvant treatment in terms of increased pCR rates (Bear et al., 2012; von Minckwitz et al., 2012; Clavarezza et al., 2013; Tampaki et al., 2018).

Recently, Martin et al. examined the addition of bevacizumab to endocrine drugs as first-line treatment in metastatic hormone receptor-positive breast cancer through pooled data analysis from the LEA and CALGB 40503 trials (Martin et al., 2019). The addition of bevacizumab significantly improved PFS compared to endocrine treatment alone, however, there was no difference in OS between both groups. Besides, a significantly higher rate of grade III/IV adverse events was observed for the combination treatment (Martin et al., 2019). Besides, a phase II trial of nab-paclitaxel and bevacizumab, followed by maintenance therapy with bevacizumab and erlotinib, for patients with metastatic TNBC was conducted by Symonds et. al. (Symonds et al., 2019). No significant difference was seen for either PFS or OS for patients enrolled and none of them achieved complete response (Symonds et al., 2019).

Ramucirumab (Cyramza^®) is a monoclonal antibody targeting VEGFR-2 (Singh and Parmar, 2015). It was first approved by the US FDA in 2014 as monotherapy for the treatment of metastatic gastric cancer (Casak et al., 2015). Ramucirumab approval was thereafter expanded to combination treatment with chemotherapy for gastric cancer, non-small cell lung cancer (NSCLC), and colon cancer. The drug is also approved as monotherapy for hepatocellular carcinoma (HCC), and most recently in combination with erlotinib for patients with epidermal growth factor receptor (EGFR)-positive NSCLC (Effing and Gyawali, 2020).

Few clinical trials investigated ramucirumab treatment in breast cancer. In phase II, randomized, open-label study, the addition of ramucirumab to capecitabine in previously treated patients with locally advanced and metastatic breast cancer failed to improve PFS and OS compared to capecitabine therapy alone (Vahdat et al., 2017). The frequency of adverse effects was increased in the combination group and included headache, anorexia, constipation, epistaxis, and hypertension (Vahdat et al., 2017). Another phase II, randomized, open-label clinical trial revealed no difference in survival for the combination of ramucirumab and eribulin versus eribulin monotherapy in patients with advanced breast cancer (Yardley et al., 2016b). The ROSE/TRIO-12 was a randomized, placebo-controlled, phase III trial evaluating the addition of ramucirumab to first-line docetaxel treatment in HER2-negative metastatic breast cancer (Mackey et al., 2015). In agreement with findings from previous phase II trials, ramucirumab neither improved PFS nor OS compared to docetaxel treatment (median, 9.5 vs. 8.2 months; p = 0.077 and 27.3 vs. 27.2 months; p = 0.915, respectively). Higher rates of toxicity were reported in patients receiving ramucirumab treatment (Mackey et al., 2015).

Tyrosine kinase inhibitors (TKIs) are small molecules that inhibit the kinase domain of RTKs thus inhibiting receptor activation and downstream signaling (Fakhrejahani and Toi, 2014). Several angiogenesis inhibitors are small-molecule TKIs.

The activation of compensatory pro-angiogenic pathways in response to anti-VEGF therapy is a well-established mechanism of acquired resistance in tumors (Bergers and Hanahan, 2008; Ramadan et al., 2020). Blockade of the VEGF/VEGFR signaling pathway can aggravate hypoxia resulting in the upregulation of alternative angiogenic factors such as PDGFs, FGFs, chemokines, interleukin-8 (IL-8), Delta-like ligand-4, and ephrins (Bergers and Hanahan, 2008; Lord and Harris, 2010; Carmeliet and Jain, 2011). Collectively, these angiogenic factors may rescue tumor vascularization despite the presence of the VEGF/VEGFR inhibitor (Bergers and Hanahan, 2008; Carmeliet and Jain, 2011; Ramadan et al., 2020).

Typically, vasculogenesis is a minor pathway in the development of tumor vasculature at which angiogenesis is the primary pathway. However, upon the inhibition of angiogenic growth, vasculogenesis may become crucial to maintaining tumor vasculature (Brown, 2014). Hypoxia-induced by anti-VEGF therapy leads to the recruitment of pro-angiogenic bone marrow-derived cells (BMDCs) to the tumor microenvironment (Lord and Harris, 2010; Ramadan et al., 2020). BMDCs can restore vascularization of tumors thus enabling them to overcome hypoxia and become resistant to anti-VEGF drugs (Bergers and Hanahan, 2008; Lord and Harris, 2010). Several BMDCs have been identified in the tumor microenvironment such as tumor-associated macrophages (TAMs), pro-angiogenic monocytic cells, myeloid cells, and Tie-2-expressing macrophages (Lord and Harris, 2010). TAMs were associated with high VEGF expression and high microvessel density in ductal breast carcinoma (Longatto Filho et al., 2010). Tripathi et al. revealed that TAMs were recruited to tumor microenvironment in an animal model of breast cancer by eotaxin and oncostatin M cytokines (Tripathi et al., 2014). Blocking these cytokines with neutralizing antibodies reduced tumor vascularization and improved sensitivity to bevacizumab (Tripathi et al., 2014). Liu et al. demonstrated that inhibiting SDF1/CXCL12 with a neutralizing antibody decreased infiltration of myeloid cells and correlated with reduced endothelial cell percentage and tumor angiogenesis in a transgenic mouse model of breast cancer (Liu et al., 2010). Obesity was associated with increased IL-6 production from adipocytes and myeloid cells within tumors in murine breast cancer model (Incio et al., 2018). Inhibition of IL-6 normalized tumor vasculature, reduced hypoxia, and restored sensitivity to anti-VEGF therapy.

Heterogeneous pericyte coverage has been described in several types of tumors, at different stages of tumor progression, and even within a single tumor stage (Hida et al., 2013). The reduction in tumor vascularity induced by anti-VEGF therapy enhances the recruitment of pericytes to maintain blood vessel function and integrity (Bergers and Hanahan, 2008). Increased pericyte coverage of these blood vessels supports tumor endothelium to survive and function despite the anti-angiogenic drug (Bergers and Hanahan, 2008; Lord and Harris, 2010). In addition, pericytes can release pro-angiogenic factors in response to PDGF (Lord and Harris, 2010). In the breast cancer vasculature, heterogenous pericyte coverage was identified (Kim et al., 2016). However, the impact of pericyte on resistance to anti-VEGF therapy in breast tumors is largely unknown.

Vasculogenic mimicry and vessel co-option may decrease the dependence on classical angiogenesis by tumors (Schneider and Miller, 2005; Bergers and Hanahan, 2008; Carmeliet and Jain, 2011). These alternative mechanisms render tumors insensitive to anti-angiogenic agents by allowing tumors to obtain the necessary blood supply when classical angiogenesis is limited (Schneider and Miller, 2005; Haibe et al., 2020). Vasculogenic mimicry is associated with aggressive breast cancer phenotypes and poor prognosis (Shen et al., 2017; Haibe et al., 2020). Bevacizumab failed to inhibit vasculogenic mimicry in the HCC1937 breast cancer cell line (Dey et al., 2015). Besides, Sun et al. showed that the administration of sunitinib induced vasculogenic mimicry in animal models of TNBC which ultimately promoted resistance to sunitinib therapy (Sun et al., 2017). Vascular co-option is another mechanism to escape angiogenesis inhibitors and has been shown to drive brain metastasis of breast cancer cells (Ramadan et al., 2020).

Growing evidence supports the concept of the heterogeneity of the endothelium of vessels involved in angiogenesis (Hida et al., 2013). Hida et al. showed that tumor blood vessels are heterogeneous and that tumor-associated endothelial cells had relatively large, heterogeneous nuclei, cell aneuploidy, and chromosomal alterations indicative of cytogenetic abnormalities (Hida et al., 2004; Akino et al., 2009). Altered gene and protein expression profiles in tumor endothelium have also been reported (Aird, 2012). The heterogeneity of tumor endothelial cells may differ by tumor type, tumor microenvironment, and the stage of tumor growth (Hida et al., 2013). Grange et al. showed that breast cancer-derived endothelial cells did not undergo normal cell senescence in culture, had increased motility, and constantly expressed markers of endothelial activation and angiogenesis (Grange et al., 2006). These endothelial cells were resistant to the cytotoxic activity of chemotherapeutic drugs as compared to normal micro-endothelial cells (Grange et al., 2006). The functional abnormalities of tumor-associated endothelial cells and the microvascular heterogeneity could explain, at least in part, the reduced efficacy of anti-angiogenic therapy in breast cancer by enabling endothelial cells an increased pro-angiogenic activity to acquire drug resistance (Grange et al., 2006; Madu et al., 2020).

Lack of response to angiogenesis inhibitors may be explained in terms of the stage of progression, treatment history, and genomic constitution that exist in the tumor microenvironment (Bergers and Hanahan, 2008). An analysis of human breast cancer biopsies demonstrated a plethora of pro-angiogenic factors in late-stage breast cancers including FGF-2, in contrast to earlier-stage tumors which preferentially expressed VEGF (Relf et al., 1997). Thus, resistance to anti-VEGF drugs in advanced-stage breast cancer may be explained by the dominance of FGF-2 and other pro-angiogenic factors in such stage of the disease (Bergers and Hanahan, 2008). Invasive cancers commonly express multiple angiogenic factors and this heterogeneity occurs at an early point in time. Genetic instabilities in the tumor cells may cause alterations of both the amount and type of pro-angiogenic factors expressed in a tumor which could further promote resistance to anti-angiogenic treatments (Schneider and Miller, 2005).

Cancer stem cells are a subpopulation of cancer cells capable of self-renewal, differentiation, and induction of tumorigenesis, metastasis, and drug resistance (Li et al., 2021). The potential of cancer stem cell trans-differentiating into endothelial cells has been reported in a variety of solid tumors (Li et al., 2021). Bussolati et al. showed that breast cancer stem cells were able to differentiate into the endothelial lineage in the presence of VEGF (Bussolati et al., 2009). The stem cells acquired several endothelial markers and organized into capillary-like structures forming vessels in a xenograft animal model (Bussolati et al., 2009). Similarly, Wang et al. showed that breast cancer stem cells may trans-differentiate into endothelial cells that can form capillary-like vascular structures in the cell culture system and participate in tumor angiogenesis (Wang et al., 2017). An earlier study demonstrated that microRNA-27a (miRNA-27a) expression promoted tumor angiogenesis and metastasis in vivo by mediating endothelial trans-differentiation of breast cancer stem-like cells (Tang et al., 2014). Brossa et al. reported the ability of breast cancer stem cells to trans-differentiate to endothelial cells expressing endothelial markers under hypoxic conditions in vitro (Brossa et al., 2015). Notably, treatment with the VEGFR inhibitor sunitinib but not the VEGF inhibitor bevacizumab impaired the endothelial differentiation ability of breast cancer stem cells both in vitro and in vivo. Mechanistically, sunitinib, but not bevacizumab, suppressed HIF-1α required for endothelial differentiation under hypoxic conditions (Brossa et al., 2015). Together, increasing evidence suggests that cancer stem cell endothelial trans-differentiation supports tumor vascularization and partly contributes to the failure of anti-angiogenic drugs.

Interleukins (ILs) are a family of cytokines known to play essential roles in the regulation of several immune cell functions such as differentiation, activation, proliferation, migration, and adhesion (Turner et al., 2014). Interactions of ILs and their receptors in endothelial cells have been shown to regulate angiogenesis through pro-angiogenic and anti-angiogenic activity (Ribatti, 2019).

IL-6 is a pleiotropic cytokine that binds to its membrane-bound receptor (IL-6R) to activate a distinct JAK/STAT signaling pathway (Taher et al., 2018). Serum IL-6 levels were elevated in breast cancer patients compared to controls (Barron et al., 2017; Raghunathachar Sahana et al., 2017), and correlated with advanced stage of the disease (Raghunathachar Sahana et al., 2017). Additionally, serum IL-6 and VEGF correlated positively in breast cancer patients (Raghunathachar Sahana et al., 2017). Higher expression of IL-6R was demonstrated in clinical specimens for patients with high-grade invasive ductal carcinoma (Bharti et al., 2018). Recent evidence showed that the IL-6/IL-6R pathway is activated in hypoxic breast cancer cells and that inhibition of IL-6R using siRNA significantly blocked angiogenesis and invasion in different models (Bharti et al., 2018). IL-6R siRNA also reduced expression of MMP-2/9 in breast cancer cells (Bharti et al., 2018). A recent study by Hegde et al. showed that a crosstalk between IL-6 and VEGFR-2 signaling pathways exists in myoepithelial and endothelial cells isolated from clinical human breast tumors (Hegde et al., 2020). IL-6 epigenetically regulated VEGFR-2 expression through induction of proteasomal degradation of DNA methyltransferase 1 leading to promoter hypomethylation and angiogenic activity (Hegde et al., 2020).

IL-8 is a pro-inflammatory cytokine that exerts its biologic activity through binding to its CXCR1 and CXCR2 receptors (Waugh and Wilson, 2008). IL-8 enhanced the proliferation of cancer cells and produced a pro-angiogenic activity (Waugh and Wilson, 2008). Serum IL-8 levels were significantly higher in breast cancer patients compared with healthy subjects and were associated with advanced disease (Benoy et al., 2004). High levels of IL-8 are secreted by stromal cells into the microenvironment of breast cancer patients compared to controls (Razmkhah et al., 2010). Evidence from preclinical studies showed that IL-8 mediated invasion and angiogenesis of breast cancer cells (Lin et al., 2004). Cancer-associated adipocytes express high levels of IL-8 in breast cancer stroma thus promoting the pro-angiogenic effects of breast adipocytes (Al-Khalaf et al., 2019). In this context, IL-8-expressing adipocytes increased vascularity of tumor xenografts as indicated by increased expression of CD34, an endothelial cell marker (Al-Khalaf et al., 2019). Neutralization of IL-8 or inhibiting its target receptors had been shown to reduce breast cancer growth and angiogenesis (Lin et al., 2004). Nannuru et al. showed that silencing of CXCR2 expression reduced tumor vascularity and inhibited spontaneous lung metastasis in an orthotopic animal model of breast cancer (Nannuru et al., 2011). Further, CXCR1 blockade with the small molecule inhibitor, repertaxin reduced metastasis in an animal model of breast cancer (Ginestier et al., 2010).

The platelet-derived growth factor (PDGF) family consists of four gene products (PDGF-A, -B, -C, and -D) that are combined into five different isoforms: PDGF-AA, -BB, -CC, -DD, and -AB (Bartoschek and Pietras, 2018). These factors bind and activate their respective RTKs, PDGFR-α, and PDGFR-β. PDGF family plays a key role in a wide range of oncologic activities essential for cancer growth including angiogenesis, fibrosis, and cellular migration (Bartoschek and Pietras, 2018).

High expression of PDGFs was correlated with an advanced presentation, increased recurrence, and poor survival in patients with invasive breast cancer (Jansson et al., 2018; Bottrell et al., 2019). PDGF is an important regulator for the motility of vascular smooth muscle cells induced by breast cancer cells (Banerjee et al., 2006). Besides, the expression of HIF-1α in invasive breast cancer was significantly associated with angiogenesis and expression of PDGF-BB (Bos et al., 2005). Earlier evidence showed that PDGFRs are expressed by breast cancer cells and endothelial cells in metastatic bone lesions in animal models (Lev et al., 2005). Imatinib remarkably inhibited PDGFR activation in breast cancer cells and tumor-associated endothelial cells and reduced microvessel density in the tumors (Lev et al., 2005). Recently, Wang et al. provided evidence from cell culture and animal studies that the downregulation of PDGF-B greatly contributed to the metformin-induced vessel normalization in breast cancer (Wang et al., 2019).

Fibroblast growth factors (FGFs) belong to a large family of growth factors that includes 23 members (Hui et al., 2018). FGFs are key regulators of numerous physiological processes such as angiogenesis, wound healing, and embryonic development. These functions are mediated by the binding of FGFs with their receptors (FGFRs), which belong to the RTK family (Hui et al., 2018). Growing evidence signifies the oncogenic impact of FGFs and FGFRs to promote cancer development and progression by mediating cancer cell proliferation, survival, epithelial-to-mesenchymal transition, invasion, and angiogenesis (Wesche et al., 2011).

Dysregulations of the FGF/FGFR axis have been reported in breast cancer (Navid et al., 2020). FGF/FGFR signaling induced angiogenic activity in breast cancer cells through promoting the secretion of VEGF, enhancing HIF effects, and downregulation of thrombospondin 1 (Mattila et al., 2006; Shi et al., 2007). Chen et al. showed that dipalmitoylphosphatidic acid, a bioactive phospholipid, induced anti-angiogenic activity, and inhibited tumor growth in an experimental xenograft model of breast cancer (Chen J. et al., 2018). These effects were attributed to transcriptional inhibition of FGF-1 expression leading to the downregulation of HGF (Chen J. et al., 2018). In the same context, Cai et al. showed that neutralizing FGF-2 by a disulfide-stabilized diabody inhibited tumor growth and angiogenesis in a mouse model of breast cancer (Cai et al., 2016). The antitumor activity was associated with a significant decrease in microvessel density and the number of lymphatic vessels (Cai et al., 2016). Formononetin, an FGFR-2 inhibitor, demonstrated anti-angiogenic activity in breast cancer in both ex vivo and in vivo angiogenesis assays (Wu et al., 2015). Besides, formononetin significantly inhibited angiogenesis in vivo by reducing microvessel density and phosphorylated FGFR-2 levels in tumor tissue (Wu et al., 2015). Recent evidence showed that FGF-2-positive tumors are resistant to clinically available drugs targeting VEGF and PDGF (Hosaka et al., 2020). The resistance is mediated by the ability of FGF-2 to recruit pericytes onto tumor microvessels through a PDGFR-β-dependent mechanism in breast cancer and fibrosarcoma models. Dual targeting of the VEGF and PDGF produced a superior antitumor effect in FGF-2-positive breast cancer (Hosaka et al., 2020).

Angiopoietins (Angs) represent an imperative family of vascular growth factors that produce their biological effects through binding to the RTKs, Tie-1, and Tie-2 (Akwii et al., 2019). Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are best characterized for their role in angiogenesis and vascular stability (Akwii et al., 2019). Ang-1 regulates the organization and maturation of newly formed blood vessels and promotes quiescence and structural integrity of vasculature (Brindle et al., 2006). Alternatively, Ang-2 antagonizes the effects of Ang-1 resulting in vessel destabilization (Brindle et al., 2006).

Ramanthan et al. indicated that high Ang-2 gene expression in breast cancer patients was associated with reduced survival (Ramanathan et al., 2017). In addition, a strong correlation existed between Angs and VEGF genes in breast cancer tissues (Ramanathan et al., 2017). Besides, serum levels of Ang-2 were significantly higher in breast cancer patients compared to healthy control subjects. High Ang-2 serum levels had shorter survival than that of the low Ang-2 expression group (Li et al., 2015). Evidence from preclinical models also demonstrated that Ang-2 mediated initial steps of breast cancer metastasis to the brain (Avraham et al., 2014).

He et al. showed that targeting Ang-2 with miRNA-542-3p reduced tumor growth, angiogenesis, and metastasis in animal models (He et al., 2014). Besides, Wu et al. showed that oral administration of methylseleninic acid reduced microvessel density and increased pericytes coverage by inhibiting Ang-2 in a breast cancer animal model (Wu et al., 2012). Dual inhibition of VEGF-A and Ang-2 using a bispecific antibody promoted vascular regression and normalization in a model of metastatic breast cancer (Schmittnaegel et al., 2017). Dual inhibition of Ang-1 and TGF-βR2 was also shown to suppress tumor angiogenesis in breast cancer in vivo (Flores-Perez et al., 2016).

Notch receptors belong to a highly conserved signaling pathway that relies on cell-cell contacts to mediate a response to environmental signals in multicellular animals (Aster et al., 2017). Four different Notch receptors are expressed in humans, each is encoded by a different gene. In addition, four functional Notch ligands exist and belong to two families: members of the Delta family of ligands; Dll-1 and Dll-4, and members of the Serrate family of ligands; Jag-1 and Jag-2 (Aster et al., 2017). In breast cancer, Notch signaling promotes cell proliferation, self-renewal, anti-apoptotic effects, and angiogenesis (Aster et al., 2017; Mollen et al., 2018). Notch expression has been associated with the progression and recurrence of breast cancer (Mollen et al., 2018). Proia et al. showed that blocking Notch-1 function with a specific antibody inhibited functional angiogenesis and breast cancer growth in animal models (Proia et al., 2015).

HGF is a member of the plasminogen-related growth factor group and is a known angiogenic factor (Nakamura and Mizuno, 2010). It is primarily expressed and produced by stromal cells, such as fibroblasts in mammary tissues (Jiang et al., 2003). The angiogenic actions of HGF are mediated by binding to its RTK, MET on endothelial cells (Organ and Tsao, 2011; Zhang et al., 2018). In the activated endothelial cells, MET is upregulated thus modulating cell dissociation, motility, proliferation, and invasion (Peruzzi and Bottaro, 2006). HGF regulates VEGF expression in tumor cells promoting angiogenic activity (Matsumura et al., 2013). Earlier studies showed that targeting HGF with retroviral ribozyme transgene or HGF antagonist reduced the growth and angiogenesis of breast tumors in vivo (Jiang et al., 2003; Martin et al., 2003).

Syndecans are transmembrane proteoglycans composed of a core protein and a glycosaminoglycan side chain to which growth factors are attached (Szatmari and Dobra, 2013). Syndecan-1 is the major syndecan found in epithelial malignancies (Szatmari and Dobra, 2013). Syndecan-1 ligates with several pro-angiogenic factors such as VEGF, FGFs, Wnt, and HGF, which act as signaling co-receptors (Szatmari and Dobra, 2013). Expression of syndecan-1 in breast tumors was associated with adverse prognosticators, metastasis, and reduced OS in patients (Kind et al., 2019; Qiao et al., 2019; Sayyad et al., 2019). Besides, stromal syndecan-1 expression increased vessel density and area and promoted the growth and angiogenesis of triple-negative tumors in vivo (Maeda et al., 2006). Schönfeld et al. showed that targeting syndecan-1 with an antibody-drug conjugate reduced the growth of TNBC in animal models when combined with chemotherapy (Schonfeld et al., 2018).

A potential strategy to sensitize tumor endothelium to angiogenesis inhibitors is by targeting pericytes to achieve tumor vascular normalization (Lord and Harris, 2010; Meng et al., 2015; Zirlik and Duyster, 2018). Normalization of tumor vasculature prevents cancer cell metastasis, improves the delivery of systemic anticancer therapies, increases the efficacy of local therapies, and enhances recognition by the host immune system. Pericyte coverage of tumor blood vessels is heterogeneous. In certain tumors, high pericyte coverage of the tumor vasculature causes resistance to anti-angiogenic therapies. Alternatively, low pericyte coverage detected in the vasculature of certain tumors reduces vascular stability and increases vascular permeability which impairs the delivery of anticancer therapies to tumor cells and allows them to metastasize (Meng et al., 2015). Earlier studies showed that combining VEGFR and PDGFR inhibitors targeting endothelial cells and pericytes, respectively, improved the efficacy of anti-angiogenic therapy and reduced tumor growth in animal tumor models (Bergers et al., 2003; Erber et al., 2004). In a xenograft model of breast carcinoma, tumor vascularization was enhanced by increasing the pericyte-endothelium association via a mechanism involving the TGF-β-fibronectin axis (Zonneville et al., 2018). In addition, Keskin et al. showed that pericyte targeting in established mouse breast tumors increased Ang-2 expression and that targeting Ang-2 signaling along with pericyte depletion restored vascular stability and decreased tumor growth and metastasis (Keskin et al., 2015). Although data from preclinical studies showed that pericyte targeting could be a novel strategy to normalize tumor vasculature, this strategy should be carefully considered as lack of pericyte coverage may disrupt vascular integrity and promote cancer metastasis (Lord and Harris, 2010; Zirlik and Duyster, 2018). Assessment of pericyte coverage of tumor vasculature and the identification of the appropriate pericyte-targeted therapy are potential challenges to pericyte targeting (Meng et al., 2015).

MicroRNAs (miRNAs) are critical regulators of signaling pathways involved in angiogenesis and cancer metastasis by interacting with the target mRNAs (Gallach et al., 2014). To date, there are groups of well-characterized miRNAs implicated in regulating endothelial cell function and angiogenesis, making them attractive targets in tumor angiogenesis (Gallach et al., 2014). Liang et al. showed that miRNA-153 suppressed breast tumor angiogenesis through targeting HIF-1α and Ang-1 in breast cancer cell lines and animal model. MiRNA-153 inhibited the proliferation, migration, and tube formation of endothelial cells and decreased the microvessel density (Liang et al., 2018a; Liang et al., 2018b). Lu et al. reported that miRNA-140-5p inhibited tumor invasion and angiogenesis by silencing VEGF-A in breast cancer cells both in vitro and in vivo (Lu et al., 2020). MiRNA-29b inhibited proliferation, migration, and tube formation of endothelial cells. Systemic administration of miRNA-29b potently suppressed breast tumor growth and vascularization by targeting Akt and downregulating VEGF and c-Myc in breast cancer cells (Li et al., 2017). Mimics of miRNA-497 suppressed the proliferation and tube formation of endothelial cells in vitro (Wu et al., 2016). Moreover, the overexpression of miRNA-497 reduced VEGF and HIF-1α protein levels and suppressed angiogenesis in vivo (Wu et al., 2016). Zou et al. showed that miRNA-145 inhibited growth and angiogenesis of TNBC in vivo via post-transcriptional regulation of N-Ras and VEGF (Zou et al., 2012).

Importantly, miRNAs can be transported between cancer cells and stromal cells through extracellular vesicles known to mediate cell-to-cell communication in the tumor microenvironment (Kuriyama et al., 2020). Extracellular vesicles are classified into exosomes, microvesicles, and apoptotic bodies based on the size or biogenesis of the vesicles (Kuriyama et al., 2020). Under hypoxic conditions, tumor cells release extracellular vesicles to a larger extent compared to cells in a normoxic environment (Kuriyama et al., 2020). Growing evidence points to the role of tumor-derived extracellular vesicles in tumor angiogenesis of breast cancer. Lu et al. recently reported that extracellular vesicles derived from breast cancer cells are highly enriched with miRNA-182-5p which enhanced proliferation and migration of endothelial cells in vitro and angiogenesis and metastasis of breast cancer in vivo (Lu et al., 2021). Microvesicles rich in a special VEGF isoform activated VEGFR and induced angiogenesis while being resistant to bevacizumab (Feng et al., 2017). Exosome-mediated transfer of breast cancer-secreted miRNA-105 efficiently destroyed tight junctions in endothelial monolayers associated with increased vascular permeability (Zhou et al., 2014). Few studies showed that extracellular vesicles can be targeted to prevent breast cancer metastasis and restore the activity of anti-angiogenic drugs (Zhou et al., 2014; Feng et al., 2017). Aslan et al. showed that docosahexaenoic acid decreased the expression of pro-angiogenic genes including HIF-1α, TGF-β, and VEGFR in breast cancer cells and their secreted exosomes (Aslan et al., 2020). Also, docosahexaenoic acid altered miRNA content in breast cancer cells and their derived exosomes in favor of the inhibition of angiogenesis (Aslan et al., 2020). Taken together, miRNAs and extracellular vesicles can be selectively targeted to reduce vascularization in breast cancer providing a novel approach for angiogenesis inhibition (Gallach et al., 2014).

Normal vasculature is needed for immunosurveillance and efficient detection and killing of cancer cells by immune cells. Disorganized tumor vessels create a selective immune cell barrier limiting the extravasation of immune cells, particularly the cytotoxic T lymphocytes into blood vessels and tumor tissue (Yang et al., 2021). Further, hypoxia in the tumor microenvironment promotes lactate accumulation, extracellular acidosis, VEGF overexpression, and VEGFR activation, all of which are known drivers of immune cell tolerance and immunosuppressive status (Mendler et al., 2012; Vaupel and Multhoff, 2017). Endothelial cells are the first to come into contact with immune cells while infiltrating from the circulation into the tumor tissue (Solimando et al., 2020). Interestingly, tumor endothelial cells expressed PD-L1 and produced immunosuppressive activity contributing to tumor immune evasion in a mouse model of melanoma (Taguchi et al., 2020). Further, leukocyte adhesion was remarkably diminished in tumor vessels (Dirkx et al., 2003). Tumors secrete angiogenic growth factors that can downregulate endothelial adhesion molecules essential for the interactions with granulocytes, macrophages, and natural killer cells on the vascular endothelium (Griffioen, 2008). The suppression of these selective adhesion molecules leads to the loss of the adhesive properties of the tumor endothelium thereby impairing immune cell infiltration to tumor tissues. Solimando et al. showed that junctional adhesion molecule-A (JAM-A) is an important factor influencing angiogenesis and extra-medullary dissemination in patients with multiple myeloma and its targeting suppressed multiple myeloma-associated angiogenesis both in vitro and in vivo (Solimando et al., 2019; Solimando et al., 2021). Bednarek et al. recently demonstrated that targeting JAM-A with an antagonistic peptide inhibited the adhesion and trans-endothelial migration of breast cancer cells (Bednarek et al., 2020). In breast cancer, vascular cell adhesion molecule-1 was aberrantly expressed and mediated angiogenesis and metastasis by binding to its ligand α4β1integrin (Sharma et al., 2017). Earlier findings also showed that angiogenic stimuli in the microenvironment of breast cancer may influence the expression of endothelial adhesion molecules to prevent leukocyte infiltration to tumor tissue (Bouma-Ter Steege et al., 2004). Dual VEGF/Ang-2 inhibition normalized tumor vasculature and reprogrammed the tumor immune microenvironment toward the antitumor phenotype in an animal model (Kloepper et al., 2016). Therefore, selective targeting of adhesion molecules and normalizing tumor vasculature could improve immune cell endothelial adhesion and strengthen the antitumor immune response in epithelial tumors, including breast cancer.

A growing body of evidence describes the interplay between immune cells and vasculature in the tumor microenvironment. The immune response and vascular normalization seem to be mutually regulated (Fukumura et al., 2018; Huang et al., 2018). Normalization of the tumor vasculature improves the infiltration of immune effector cells into tumors enhancing antitumor immune activity (Fukumura et al., 2018; Solimando et al., 2020; Yang et al., 2021). Likewise, immunotherapy can promote vascular normalization which further improves the effectiveness of immunotherapeutic drugs and response to anti-angiogenic therapies (Huang et al., 2018; Ciciola et al., 2020; Yang et al., 2021). In preclinical models of breast cancer, immune checkpoint inhibitors induced normalization of tumor vasculature and increased infiltration of immune cells into breast tumors (Tian et al., 2017; Zheng et al., 2020). Together, the combination of anti-angiogenic and immunotherapeutic drugs might be an attractive approach to increase the effectiveness of each class of drugs and reduce the emergence of drug resistance (Fukumura et al., 2018; Huang et al., 2018; Solimando et al., 2020). The combination treatment has shown encouraging results in various cancer types (Ciciola et al., 2020; Madu et al., 2020). In a preclinical study, Allen et al. revealed that treatment with a combination of anti-VEGFR-2 and anti-PD-L1 antibodies sensitized tumors to anti-angiogenic therapy and prolonged its efficacy in breast cancer (Allen et al., 2017). Li et al. recently demonstrated a dose-dependent synergism for the combined treatment of anti-angiogenic therapy and immune checkpoint blockade (Li et al., 2020). In this regard, the combination of low-dose anti-VEGFR2 antibody with anti-programmed cell death protein-1 (PD-1) therapy normalized tumor vasculature, induced immune cell infiltration, and upregulated PD-1 expression on immune cells in syngeneic breast cancer mouse models. Additionally, the combined treatment was effective and tolerable in patients with advanced TNBC (Li et al., 2020). An open-label, randomized, parallel, phase II trial investigated the combination treatment of apatinib, a VEGFR-2 tyrosine kinase inhibitor with the anti-PD-1 monoclonal antibody camrelizumab in patients with advanced TNBC (Liu et al., 2020). The results showed that the combination treatment produced favorable therapeutic outcomes in terms of improved objective response rate and PFS which was associated with increased tumor-infiltrating lymphocytes. The adverse events were manageable and included elevated aminotransferases and hand-foot syndrome (Liu et al., 2020). Multiple clinical trials of combining anti-angiogenic therapy and immune checkpoint inhibitors are underway (Zirlik and Duyster, 2018).

The nanotechnology-based approach is an emerging strategy for the development of therapies targeting tumor angiogenesis which could improve the current pharmacokinetic profiles of anti-angiogenic drugs and favor their selective accumulation in tumors (Banerjee et al., 2011; Darweesh et al., 2019). Compared to the free drug, in vitro and in vivo assays showed that gold nanoparticle-conjugated quercetin inhibited angiogenesis and invasion of breast cancer by targeting the EGFR/VEGFR-2 signaling pathway (Balakrishnan et al., 2016). Radical-containing nanoparticles produced in vitro and in vivo anti-angiogenic activity in a breast cancer model that was mediated by suppressing VEGF in cancer cells (Shashni et al., 2021). Nanoparticles were also utilized to deliver a combination of therapy for breast cancer to produce anticancer and anti-angiogenic activity (Zhao et al., 2017). In a recent study by Gong et al., nanoparticles delivering an inhibitor of sphingosine-1 phosphate receptor-1 dramatically inhibited TNBC growth and angiogenesis in vivo via downregulating STAT3/VEGF axis (Gong et al., 2021). Table 3 provides a list of novel approaches for targeting vascular growth and angiogenesis in breast cancer.