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The most prevalent types of cancer diagnosed and the main reason for deaths due to cancer among women is breast cancer (BC) (1). The detection and treatment of BC have undergone a number of advancements in recent years. Since the early 1990s, there has been a 39% decrease in breast cancer mortality thanks to a combination of better screening, earlier detection/diagnosis, and anti-cancer medicines that have made substantial advances. The prognosis for patients with advanced-stage breast cancer can be improved, though further investigations are required to overcome the threat of cancer resistance to treatment.

Aggressive breast cancers with hypoxic cores account for 40% of cases; these tumours are highly metastatic, and resistant to the majority of treatments. Breast cancer stem cells (BCSCs) multiply in a hypoxic tumor microenvironment, which results in a variety of epigenetic changes that retain cancer stem cells in an undifferentiated state and aid in its development and recurrence (2).Unchecked tumor cell growth outgrows the surrounding vascular system, which leads to a reduction in oxygen delivery relative to demand. Chronic hypoxia refers to this restriction on oxygen diffusion, whereas acute hypoxia occurs when blood arteries abnormally close, leading to reduced perfusion (3–5). Chronic hypoxia causes DNA breakage, malfunctions in the mending systems and mutagenesis (6). On the other hand, short-term hypoxia boosts the generation of Reactive Oxygen Species (ROS), tumor survival, and spontaneous metastasis (7, 8). Cancer cells have demonstrated radio-resistance in vitro and in vivo under both chronic and acute hypoxia (9). Hypoxia-inducible factors (HIF), which is a transcription factor, serves as the catalyst for tumor growth (10, 11). HIFs are heterodimers made up of the oxygen-sensitive HIF–α and a subsequently expressed HIF–β (12). There are three isoforms; HIF1 and HIF2 are well known, while HIF 3 is also present but has not been well researched in relation to cancer (13). HIF-1 and HIF-2 under normoxic circumstances undergo hydroxylation at particular proline residues. This causes the tumor suppressor protein Von-Hippel Lindau (VHL) protein to bind to HIF 1 and 2, which then makes it easier for them to be degraded by the ubiquitin-proteaosome system (11, 14, 15). A variety of genes encoding proteins are used for anaerobic energy production, vascularization, extracellular matrix (ECM) remodelling, suppression of apoptosis, and metastasis progression. They are transcribed when hypoxia occurs due to which HIF–1 and HIF–2 get dimerized with the subunit. They (HIF–1 and HIF–2) are then translocated to the nucleus and where they activate hypoxia responsive elements (HRE) (11, 16). Patients with breast cancer who had higher levels of HIF expression in their primary tumor biopsies are more likely to develop metastases to the bone, lungs, liver, brain, and local lymph and causes major portion of breast cancer related mortalities (17).

Through a series of sequential multistep processes the original tumour transforms into a secondary tumour at a distant site (18). The EMT transcription factors (EMT-TFs), mainly belonging to the SNAIL, TWIST, and ZEB families, which play significant roles in all processes of tumour metastasis, cause the epithelial to mesenchymal transition (EMT), which is critical for cancer spread (19).

EMT type-1, type-2, and type-3 subtypes have been extensively investigated in a variety of physiological and pathological processes. Type -1 EMT is the exchange of Epithelial to Mesenchymal cells and associated with events in embryonic phase such as implantation, embryogenesis and organ development, while Type -2 is the transformation of epithelial to mesenchymal cells that occurs during wound healing and fibrosis driven by inflammation. Type-3 EMT is the transition of epithelial cells to mesenchymal cells and is active in different types of cancers including breast cancer. Therefore, Type-3 EMT is also known as “oncogenic epithelial-mesenchymal transition” (20). Different subtypes of breast cancer show distinct metastatic organ tropisms governed by different molecular mechanisms. Along with distant lymph nodes, common target organs for breast cancer metastasis include the bone, lung, liver, and brain (21). Though all breast cancer subtypes show bone metastasis, Luminal A and B have bone as their major metastatic site. Luminal B subtype is more probable than luminal A subtype to have bone as the first site of relapse when compared to other subtypes. Incidence of bone metastasis is much higher in luminal subtype tumors than in HER-2 positive and basal like subtypes. HER -2 positive subtype is more often positively tropic to liver and luminal B and basal-like subtypes present higher levels of lung -specific metastasis (22). Triple negative breast cancer (TNBC) is often associated with visceral metastases including lung, liver and brain (23). Additionally, it has been reported that hypoxia triggers EMT in different type of cancers including breast cancer, prostate cancer and oral cancer (24). Studies by Peng, Jianheng, et al. (2018). clearly show that TGF-1 and Suppressor of Mothers Against Decapentaplegic (SMAD3) expression levels were both dramatically raised by HIF-1 in breast cancer cells, however SMAD3 overexpression had no effect on either of these proteins’ expression levels (25). Moreover, HIF-1α upregulated the expression of EMT transcription factors, SWIFT and SNAIL. In case of SWIFT in breast cancer cell lines, HIF-1α could directly bind to proximal promoter of SWIFT and enhance transcription (26). Hypoxia triggers a significant up-regulation of angiogenic growth factors and their receptors, which causes endothelial cell migration with enhanced vascular permeability and promotes tumor angiogenesis. Due to the leaky vessels and haphazard arrangement, tumor and stromal cells have limited access to nutrients and oxygen during transformation and proliferation (27). However, tumors make up for this by producing metabolic intermediates that act as precursors for biosynthetic pathways, which allows cancer cells to adapt to these circumstances in the tumor microenvironment (TME) and continue to grow and multiply. Glucose metabolism is switched from the tricarboxylic acid pathway to the oxygen independent glycolysis through the activation of glycolytic pathway regulators such as glucose transport proteins (GLUTs), hexokinase 1 and 2 (HK1, 2), and pyruvate dehydrogenase kinase 1 (PDK1) (28–30). The role of HIF–1 in reshaping the topography of the ECM under hypoxia by collagen deposition and promoting the increased expression of remodelling enzymes such prolyl-4-hydroxylases and lysyl oxidases, which leads to ECM stiffness and metastasis, has been determined by studies. An increasing body of research indicates that hypoxia promotes the chemo-invasive and metastatic potential of breast cancer by activating metalloprotease 2 and 9 (MMP-2 and MMP-9), which also degrade ECM (31–33). According to multiple studies, hypoxic stress is expected to stimulate VEGF (Vascular Endothelial Growth Factor), a crucial regulator of angiogenesis (34). Immunosurveillance in breast cancer, as in many other tumor forms, is functionally represented by the presence of tumor infiltrating lymphocytes (TILs) into the neoplastic cellular mass (35). According to Ono et al., 2012, when compared to the HER–2-/HR+ subtype, TILs were significantly greater in TNBC (triple negative breast cancer) and HER–2+/HR- breast cancer subtypes. Furthermore, compared to TNBC with low TILs levels, the pathological complete response (pCR) rate was considerably higher in TNBC with high TILs scores (36). It has been found that low oxygen levels in the TME cause TILs to become less activated, which results in immunological suppression and less immune detection. Since hypoxia-associated transcription factors have long been suggested as a viable target for immunostimulatory therapy and immunological detection, numerous researchers have found therapeutic strategies for inhibiting these factors (37). In the TME, it has been demonstrated that overexpression of HIF-1 specifically affects many aspects of the immune cell’s capacity to fight tumors (38). The foregoing discussion has briefly outlined the role of hypoxia in the aggressiveness of breast cancer, specifically in the transformation of normal endothelium into tumorigenic endothelium, as well as the various therapeutic approaches by which immune modulators can specifically target breast cancer and how hypoxic tumor endothelial cells can be targeted within the TME.

Growing body of data indicates that, a hypoxic microenvironment may protect tumors from immunological treatments as well as naturally occurring anti-tumor immune responses by limiting anti-tumor immune effector cells and encouraging immune escape. It is clear that hypoxia affects immune cells either directly or indirectly, supporting the TME in a direction that is immunosuppressive (39–41). Understanding the disease biology of Breast Cancer (BC) requires an understanding of the immune surveillance in the tumor microenvironment since it has the potential to either facilitate the eradication of disease or encourage tumor growth. This dual role is sustained by the dynamic interactions between diverse immune effector cells, tumour cells, stromal cells, and soluble substances in the tumour microenvironment (42). Selection favours tumor variants with the genetic trait of escaping immunological detection, while variants lacking it are wiped off by immune surveillance. In addition to genetic variation, deficiencies in antigen presentation methods, T-cell receptor (TCR) signalling, interferon (IFN) signalling pathways, and expression of the MHC class I protein has been lost or mutated, along with tumour antigens (43, 44), which will impair the immune surveillance mechanism in the tumor microenvironment. Inhibition of tumor antigen-specific T cells by intratumoral myeloid-derived suppressor cells (MDSCs), T regulatory cells (Tregs), and the switch from an anti-tumorigenic T-helper type 1 (TH1) to a pro-tumorigenic T-helper type 2 (TH2) immune response are additional factors that significantly support tumor growth. Furthermore, a variety of soluble factors like TGF-β, IL–10 etc are released by tumor/stromal cells inhibit T-cell activation and dendritic function while promoting stromal remodelling and angiogenesis (45, 46). The hypoxic core of the tumors attracts more pro-tumorigenic leukocytes, including MDSCs, tumor-associated macrophages (TAMs), and regulatory T cells. This double down the body’s natural immune defensive measure to evacuate the aggressive tumor (47) ( Figure 1 ). An in-depth study using Balb/c mice found that intra-tumoral hypoxia increases HIF-1 activation, which then sequentially activates the PDL-1, CD73, and CD47 genes to reduce the recruitment and activity of CD8+ T cells, NK cells, and macrophages, ultimately causing activity to evade both innate and adaptive immunity (48). Chemokines and their related receptors are another important hypoxic hotspot (49), which in turn affect tumor endothelial cells and increase the over expression of VEGF, CXCL12, and its receptor CXCR4, making all of them function on endothelial cells in an autocrine way. The expression of the pro-inflammatory chemokines CCL5 and CCL2 increased in HIF–1-deficient tumors, which in turn increased the infiltration of cytotoxic lymphocytes into the tumor (50). Additionally, the CXCR4-CXCR12 axis promotes metastasis to distant organs (51). Thus, hypoxic stress in TME along with activation of HIF transcription factors, is a principal cause for breast cancer angiogenesis, metastasis, immune suppression and overall poor survival rate. In the subsequent sections of this review, a detailed mechanism of hypoxia induced vascular abnormalities that activate endothelial cells (ECs) associated with tumor cells and immune suppression is furnished.

Tumor vasculature is different from healthy blood vessels in a number of aspects, including irregular structural dynamics, high permeability, and convoluted arteries, whereas healthy blood vessels are well-organized and provide for the best perfusion of nutrients and oxygen (52). Specialized mural cells called pericytes are seen in the normal vasculature, whereendothelium covers them uniformly (53). Patchy hypoperfusion and blood vessel leakage are caused by the loosening, unstable, and tumor-related pericyte phenotype (54).

Endothelial cells interact with tumor cells in numerous ways to promote angiogenesis (55) ( Figure 2 ). They serve as the connecting link between cancer cells and immune cells (56). Proangiogenic substances like Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), placental growth factor (PGF), and angiopoietin are released by tumor cells to initiate angiogenesis. According to studies, the hypoxic tumor microenvironment might boost VEGF synthesis, which in turn promotes the growth of new blood vessels (57). When the microenvironment experiences a lack of oxygen, endothelial cells and pericytes are stimulated to create VEGF, which functions in an autocrine and paracrine manner to increase the recruitment and activation of endothelial cells in the tumor site (58). Angiopoietin-2 (Ang–2) plays a critical function in destabilising vasculature for normal or pathological angiogenesis and is also up-regulated by hypoxia. It is only expressed at sites of vascular remodelling. Numerous studies have documented the essential function of Ang-2, a ligand for the endothelial cell-specific tyrosine kinase Tie2, in the vascular permeability and blood vessel instability that leads to tumor growth (59, 60). Thus, newly formed blood vessels have an uneven thickness of the basement membrane, a loose interaction between pericytes and endothelial cells, an increase in interstitial pressure, and ultimate vascular leakage (47). One of the main processes behind angiogenesis is the angiopoietin/Tie (tyrosine kinase) signalling pathway, which is composed of growth factors called angiopoietins. These include Ang-1, a strong angiogenic growth factor that communicates with Tie2, and Ang-2, a vascular disruptor with a negative effect that also uses Tie2 as a conduit (61). To cause pericyte separation from the basement membrane and migration, Ang-2 and Tie2 bind in the hypoxic tumor microenvironment. Mice lacking in pericytes had higher Ang–2 levels, suggesting that pericytes may control Ang–2 levels and limit vascular permeability. This finding highlights the importance of Ang–2 in reducing vessel leakiness (62, 63). In a different study, it was reported that reduced pericyte coverage increased IL-6 expression in the hypoxic tumor microenvironment and MDSC transmigration and circulating malignant cell phenotype (64). Rgs5 overexpression in pericytes and endothelium seen in the hypoxic tumor microenvironment causes the vasculature to become unstable. Better pericyte coverage, less vascular leakage, and adequate oxygen perfusion was all observed in the Rgs5-deficient mice (65). In-depth research found that the development of a receptor complex made up of PDGF-R and VEGF-R2 during PDGF-induced angiogenesis is what causes VEGF to activate VEGF-R2 and suppress PDGF-R signalling in vascular smooth muscle cells (66), which then decouples neo-vasculature from pericyte covering. Researchers have shown that inhibiting VEGF and Ang–2 jointly results in tumor necrosis, vascular regression, intra-tumoral phagocyte antigen presentation, and a reduction in breast cancer-brain metastases (67, 68).

Endothelial Cells (ECs) ECs have specific functions based on their locations and exhibit distinct heterogeneity across vascular beds. They have a critical role in the regulation of immune responses, inflammation, angiogenesis and actively control the degree of vascular relaxation and constriction (69). ECs that line tumor blood vessels are initially derived from the surrounding tissue, and in due course of the tumor progression, they reprogram to a tumoral phenotype (70). Normal physiologic conditions do not often need the activation of ECs, but in tumors, hypoxia and other inflammatory signals cause ECs in the tumor microenvironment to become active, causing aberrant angiogenesis and impeding normal immune surveillance (71). Numerous inflammatory reactions in the tumor microenvironment, such as inflammatory cytokines, chemokines, reactive oxygen species, etc., directly or indirectly stimulate the tumor endothelium. Tumor endothelium has a very different genetic profile than healthy endothelium ( Table 1 ), with the main variations influencing a number of cell adhesion molecules (such as ICAM1, VCAM1, E-selectin), antigen presentation, and chemokines (such as CCL2, CCL18, CXCL10, and CXCL11) and cytokines involved (such as TNFα, IFNγ, and IL-1) in immune cell recruitment. All of these elements have a deleterious impact on the immune surveillance of tumor cells in the tumor microenvironment. This unique character of TEC that enables them to avoid immune cell extravasation and, unresponsiveness of TECs to pro-inflammatory stimulation is Endothelial cell anergy, also called vascular immune checkpoint (72). ECs lining tumour blood arteries have a very different metabolic profile from ECs in normal tissue. A stronger dependence of tumour ECs on glucose metabolism is supported by the transcriptional elevation of the glycolytic pathway gene, PFKFB3, in comparison to other normal EC. According to a report, tumor ECs and tumor associated macrophages (TAMs) engage in fierce competition for glucose, and the tumor ECs’ intake of the metabolite boosts the angiogenic response in the tumor microenvironment (73). TECs produce energy through aerobic glycolysis and fatty acid oxidation, rather than oxidative phosphorylation. This metabolic reprogramming enables the tumor ECsto check the production of reactive oxygen species (ROS), and also allows the production of ATP more rapidly than through oxidative metabolism (74). Alam et al. (2014) has identified suprabasin as a new marker for TECs. Suprabasin, an upstream component of the AKT pathway, was substantially expressed in TECs compared to normal ECs and linked favourably with TECs’ capacity for migration and tube formation (75). On the other hand, microarray and immunohistochemical studies revealed that biglycan is a specific marker of TEC and an autocrine angiogenic factor of TECs (76). Because of the aggressive behaviour of tumor endothelial cells and their particular molecular, cytogenetic, and metabolic characteristics, the tumor microenvironment can therefore selectively draw in immune suppressive cells. The role of TECs in increasing immunological suppression is discussed in the following sections of this review.

The success of immunotherapy depends on adequate immune cell infiltration, and low immune cell infiltration reduces the effectiveness of immunotherapy. Despite the fact that angiogenesis triggered by tumors is crucial for the growth of solid tumors, mounting evidences indicate that it also aids in immune evasion by fostering a highly immunosuppressive TME by increasing the proportion of T reg cells and MDSCs. Furthermore, by preventing dendritic cell (DC) maturation and T cell growth, VEGF impacts immunological responses in TME (77). Leukocytes are extravasated to tissues from blood arteries during typical inflammation. These leukocytes in circulation were halted and firmly adhered to the endothelium cells by a multi-step procedure. This mechanism calls for the endothelial adhesion molecules (EAM), E-selectin (rolling), ICAM1 and VCAM1 (firm rest), VE-cadherin and CD31 (trans-endothelial migration). In addition to EAMs, surface antigens (such as HLA molecules) must be upregulated, pro-thrombotic endothelial cell changes (such as the loss of the surface anticoagulant molecules thrombomodulin and heparan sulphate), cytokines (such as IL–6, IL–8, and MCP–1) production, and changes in vascular tone (such as the loss of vascular integrity and expression of vasodilators) are associated with inflammation (78). When inflammatory cytokines like TNF, IFN, and IL–1 are released, normal ECs are activated. This causes an up-regulation of adhesion molecules, which in turn triggers the extravasation of leukocytes. However, the continual secretion of pro-angiogenic molecules VEGF and bFGF in hypoxic microenvironments adversely affects this process. Even when TNF is present, they prevent ICAM1, VCAM1, and E-selectin from being overstimulated (79–81). The hypoxia inducing factors favour the heterogeneity as well as inflammation of tumor endothelial cell (82). In vitro and in vivo studies by Tellier et al. (2015) showed that ECs exposed to hypoxia expressed tumor-promoting pro-inflammatory cytokines and chemokine such as IL-6, IL-8, and CXCL1 (83). In addition, hypoxic tumor niche affects protein glycosylation and favours recruitment of immune suppressive cells to TME, thereby impacting tumor progression (84). Research shows that the tumor endothelium downregulates the expression of endothelial adhesion molecules (EAM) such as ICAM1/2, VCAM1, E-selectin, and CD34. This lessens immune cell infiltration or leucocyte extravasation in the TME, enabling the TME to adopt an immune evasion strategy and accelerate tumor growth (72, 79, 85). Hypoxic tumor microenvironment alters the tumor endothelium leading to its diversity and also induces its “stemness”. This induction of stemness is favourable for cancer stem cells, which can drive tumor initiation and progression (86). Moreover, breast cancer stem cells enhance its tumorigenic phenotype (87). Cancer stem cells and their role in tumor progression is a vast horizon and beyond the scope of this review. Stemness genes such stem cell antigen-1 (Sca-1), MDR-1, and aldehyde dehydrogenase (ALDH) have been found to have elevated expression in TECs. This tumour stromal stem-like cell population affects the TEC population in the TME (57). With higher VEGF expression, ovarian, oesophageal, and colorectal tumors showed worse survival rates and a higher chance of relapse. VEGF synthesis in tumors affects tumor behaviour by reducing T cell numbers in addition to promoting angiogenesis. In pre-clinical tumor models, it was found that using anti-VEGF antibodies increased the recruitment of T lymphocytes into the tumor microenvironment (88, 89). Increased nitric oxide (NO) in the TME has been found to upregulate VEGF in solid tumors and further regulate the expression of several adhesion molecules involved in the interaction between EC and leukocytes. FoxP3+ T regulatory (Treg) cells predominated and there was little CD8+ infiltration in solid tumors of both humans and mice that express specific Fas ligands in the tumor vasculature. Similarly, blockade of Fas-Fas L signalling increased intra-tumoral CD8+ T cells and subsequent reduction in tumor size (90). It is now understood that the tumor endothelium plays a crucial dual role in the development of the tumor by favouring a niche where inflammation is increased by upregulating EAMs and by negatively regulating the influx of leucocytes through endothelial anergy ( Figure 3 ). The role of hypoxia in attracting immune suppressive cells to the TME and its relationship with cancer endothelium are discussed in the forthcoming sections of this review.

Due to metastasis and disease recurrence, breast cancer is the cancer that claims the lives of more women than any other (136). Journey of tumor cells across the endothelial membrane is a key step in the process of tumor invasion and metastasis. Tumor cells cross the vascular membrane stimulated by several cytokines and growth factors such as Transforming Growth Factor-Beta (TGF-β) superfamily of proteins,Bone Morphogenetic Proteins (BMPs). This is called Endothelial-mesenchymal transition (EndMT). EndMT induced by TGF-β shows a decrease in the expression of endothelial markers such as VE-cadherin, claudin and zona-occludens 1 (ZO-1) and an elevation in EMT transcription factors such as Snail, Slug and ZEB-1 which marks metastasis. TGF-β promotes ECs to change into CAF-like cells, which results in the loss of endothelial adhesion molecules and remodelling of the endothelium cytoskeleton via the Rho and Rac-1 signalling pathway, which is the primary characteristic of EndMT (76, 137, 138). It has been found that hypoxia associated with inflammation or tissue damage can also cause EndEMT (139). Rokana, et al. (2021) have recently studied the role of ICAM in trans-endothelial migration in breast cancer and also assessed the therapeutic efficacy of anti-ICAM1 neutralizing antibody on breast tumor cell aggregation and trans-endothelial migration. This anti-ICAM treatment inhibited the cluster formation of TNBC cells in suspension. Clinical data also showed high levels of ICAM1 mRNA expression in breast tumors which might mediate distant metastasis. This makes ICAM as a potential therapeutic target in TNBC metastasis (140). S100P and Ezrin, two members of the S100 family of short calcium binding proteins, also encourage the trans-endothelial migration of triple negative breast cancer cells. Furthermore, S100P activity has been linked to a variety of malignancies, a poor prognosis, metastasis and recurrence, and a low rate of survival in TNBC patients (141). An investigation on the SDF-1/CXCR4 axis in a breast carcinoma model revealed their role of hypoxia. It was confirmed that in a hypoxic tumor niche activation of HIF leads to the transcription of an array of HIF target genes including SDF-1 and CXCR4 which contributes to tumor cell migration and adhesion to endothelial cells in breast cancer cells. Moreover, the study also illustrated that SDF-1 binding to CXCR4 stimulated tube formation in endothelial cells, which point towards its role in angiogenesis and trans-endothelial migration (142). Since metastasis and related poor survival is a hallmark of breast cancer, pathways targeting trans-endothelial migration could have a significant impact in clinical trials.

The hypoxic microenvironment and impaired immune response to cancer cells by innate immune cells continue to be major obstacles for immunotherapy. Recent advancements in nano-immunotherapy, however, might lessen immunosuppression brought on by hypoxia and enhance systemic antitumor immune responses to eradicate metastatic breast cancer cells. Similar to this, in situ O₂ generation, O₂ delivery, normalisation of tumor vasculature, and mitochondrial-respiration inhibition could be the alluring therapy to overcome hypoxia-driven immune suppression for preventing the growth and progression of metastatic breast cancer cell lines. Monoclonal antibodies, immune modulators, and biodegradable bio-nanomaterials could address the problems in a very satisfactory way when used in conjunction with such methodologies. Nowadays, there is a great deal of interest in using nanomaterials as treatments to target the downstream pathways that lead to hypoxia-driven immune suppression in breast cancer (143). In this sense, PD1 receptor antagonist delivered in nanoform has the potential to effectively address the problem of hypoxic endothelium-driven immunosuppression during the treatment of breast cancer. The specific ligand antagonist of the upstream hyper-expressive biomarkers, which is responsible for the immunosuppressive state caused by hypoxic endothelium around the breast cancer microenvironment, can be used to develop new strategies. This may be a new strategy to improve immune monitoring in the treatment of breast cancer, especially for managing disease progression. Additionally, by preventing hypoxia in TME, such an approach could activate the immune cells, strengthen immune surveillance, and destroy breast cancer cells. By disrupting the hypoxia-mediated cancer signaling pathway, they may specifically target the hypoxic TME (144). Through the generation of oxygen-derived free radicals, radiation destroys tumor cells by causing DNA fragmentation. Due to the absence of oxygen and a reduction in DNA repair processes, hypoxia confers resistance to radiation therapy and reduces the effectiveness of radiation. The main cause of chemotherapy resistance in hypoxic tumors is the fact that many of the commonly used medications need oxygen to release the deadly free radicals that kills the tumor cells (145). In addition, another study that looked at the connection between HIF-1α/CAIX and the response to epirubicin found a strong correlation between high HIF-1α expression and a subpar response to treatment (146). This gives a clear insight that hypoxic pathway is involved in significant events that have a direct impact on the efficacy of numerous treatment methods. In the earlier sections of the review, we addressed the role of pro-angiogenic factor stimulation that causes tumor-associated endothelial cells to become anergic, losing the capacity to react to inflammatory signals and rendering them unable to activate EAMs (79, 147). It was discovered that this anti-infiltration barrier helped tumors avoid being destroyed by the immune system. Consequently, it is thought that using angiogenesis inhibitors to encourage leukocyte infiltration into the tumor is a useful way to increase the effectiveness of ICIs (148, 149). Bevacizumab, a VEGFA neutralizing antibody, has been shown to increase the number and activation of DC (149–151) as well as the number of cytotoxic T cells (152, 153) and to reverse VEGF-induced T cell exhaustion (154). Whereas, Sutinib, a TKI of VEGFR and other kinases, was shown to decrease the number of MDSCs and T reg cell (155, 156). Xiaodong et al. (2020). identified a tumor endothelial specific marker CLEC14A, which specifically recruits T reg recruitment and subsequently enhance immune suppression in the TME. CLEC 14 A specific CART cells, exhibited substantial decrease in tumor growth through IFN-γ indicating their antitumor potential (157).

TNBCs exhibit a markedly increased HIF transcriptional activity and a subpar response to the existing therapeutic strategies (158). Therefore, it makes sense to speculate that a novel therapeutic approach to treat TNBCs might involve targeting hypoxia in the TME. Preclinical investigations indicate that the combination of cytotoxic chemotherapy with molecules that block HIFs is particularly promising. Therapeutic studies with Digoxin and acriflavine, two HIF-1α inhibitors, demonstrated reduced initial tumor development, vascularization, invasion, and metastasis in breast cancer animal models. Furthermore, digoxin prevents HIF–1α dependent transcriptional responses that encourage cancer stem cell (CSC) resistance to chemotherapy, which causes tumor regression in TNBC when combined with paclitaxel or gemcitabine (159). Numerous synthetic and natural substances have been shown to block the regulation of HIF-1 on downstream target genes by lowering HIF-1 mRNA levels, accelerating the protein’s breakdown, and preventing HIF-1 and HIF-1 dimerization (160, 161) ( Table 2 ). An effective anticancer therapy may include normalizing the tumor vasculature rather than destroying it. Anti-angiogenic drugs must be dosed carefully during vascular normalization in order to reverse the aberrant phenotype of the tumor vasculature and increase blood flow and oxygenation. Through vessel maturation and the alleviation of immunosuppression brought on by hypoxia and/or VEGF, it has been demonstrated that vascular normalization enhances immunological responses (16, 162). It indicates that restoring the structural and functional integrity of the tumour vasculature is a viable method for polarising TAMs to an anti-tumor phenotype (163). The hypoxic tumor endothelium is hyperglycolytic, thus, targeting tumor endothelial metabolism might offer a novel therapeutic strategy. Glycolysis is the energy source for endothelial sprouting in angiogenesis rather than oxidative phosphorylation for ATP production. Thus, blocking PFKFB3, a key molecule involved in endothelial glycolysis pathway, reduced vessel sprouting and angiogenesis (164). VEGF and PFKFB3, which are both implicated in the TEC’s glycolytic pathway, were downregulated when tumor-cell-specific cyclooxygenase (COX-2) was pharmacologically inhibited. The restoration of glucose metabolism in TECs and the reduction of tip cells, filopodia, and branching are affected by COX-2 inhibitor therapy-induced inhibition of PFKFB3-mediated endothelial cell motility (165). The role of anti-angiogenic drugs in regulating the tumor vasculature and reducing hypoxia-induced immune suppression has been shown in a number of preclinical investigations. These anti-angiogenic therapies have also been successful in overcoming endothelial anergy, which results in normalized production of endothelial adhesion molecules, which are necessary for leukocyte trans-endothelial migration into the TMC. Treatment that targets the VEGF pathway may result in increased immune infiltration and ICAM1 overexpression in renal cell carcinoma (166). Since TECs have higher glycolytic rates than typical proliferating ECs, reducing glycolysis by blocking PFKFB3 with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) may be able to suppress tumor growth. According to studies, 3PO therapy increased the effectiveness of chemotherapy, aided in the normalisation of tumor vessels, tightened the EC barrier to prevent cancer metastasis, and encouraged tumor vessel normalisation. In mouse tumor models, proliferating ECs were treated with the weak mitochondrial uncoupler Embelin, which resulted in reduced mitochondrial oxidative phosphorylation, inhibited tumor growth, and reduced microvessel density (164). The endothelium of tumors significantly differs from endothelium of healthy tissues in a number of aspects, including metabolic reprogramming, alternative morphology, cytogenetics, and molecular genetics (82). So, a viable strategy to restore normal tumor vasculature involves attacking tumor endothelium. The research carried out by our group has also shown a rise in the infiltration of effector T cells, which in mouse TNBC inhibit the pro-angiogenic CXCR2 receptor. Targeting CXCR2 or other pro-angiogenic expressed on tumor endothelium may produce breakthrough outcomes in accordance with the promising results. (Unpublished data). Similar results were seen in vitro when a novel synthetic quinoline derivative was administered to block the pro-angiogenic chemokine (Unpublished data). Targeting the molecules involved in the regulation of tumors would open up the potential of a wide range of therapeutic options, normalising vascular function, and improving immune surveillance. Future therapeutic implications may benefit from knowledge of the variations in chemokines or pro-angiogenic substances produced by NECs and TECs in a hypoxic environment, as well as the expression of these receptors in normal and malignant cells.

In the light of the foregoing discussion, it will be wise to more effectively employ therapeutic alternatives in BCs with treatment resistance. Understanding the control of selective immune cell trafficking via hypoxic tumour endothelium may also be necessary. Clinical studies have found that the early preventative methods for the development of new blood vessels have only limited effects. The establishment of resistant mechanisms and enhanced tumor hypoxia are the causes of this insufficient efficacy. The development of effective treatment combinations can be facilitated by an understanding of how tumors use hypoxic endothelium cells to evade the immune system. Hypoxia regulating molecules and Immune Checkpoint Inhibitors (ICIs) should be combined to improve the prognosis of patients with hypoxic breast cancer in the light of the role played by the hypoxic TME in immune evasion. Therefore, molecules linked to selective trafficking may offer brand-new prognostic criteria to justify the use of particular immunotherapy regimens in conjunction with vascular targeting therapeutics. Though it will take time to investigate and comprehend such issues, promising research on anti-angiogenic adjuvant immunotherapy techniques offers hope for the improvement of treatment ways to improve the outcomes of breast cancer patients. A concerted effort from all investigators is imperative to usher in an era free from the woes of life-threatening diseases such as the BCs.

SB and PP conceived the concept and contributed writing and editing of the review. All authors contributed to the article and approved the submitted version.