A cancer needs a good blood supply to provide itself with food and oxygen and to remove waste products. When it has reached 1 to 2 mm across, a tumour needs to grow its own blood vessels in order to continue to get bigger.

Angiogenesis means the growth of new blood vessels. So anti angiogenic drugs are treatments that stop tumours from growing their own blood vessels.

If the drug is able to stop a cancer from growing blood vessels, it might slow the growth of the cancer or sometimes shrink it. Some cancer cells make a protein called vascular endothelial growth factor VEGF. The VEGF protein attaches to receptors on cells that line the walls of blood vessels within the tumour.

The cells are called endothelial cells. This triggers the blood vessels to grow so the cancer can then grow. Some drugs block vascular endothelial growth factor VEGF from attaching to the receptors on the cells that line the blood vessels.

This stops the blood vessels from growing. An example of a drug that blocks VEGF is bevacizumab Avastin. Bevacizumab is also a monoclonal antibody. It is a treatment for several different types of cancer.

Other examples include:. Some drugs stop the VEGF receptors from sending growth signals into the blood vessel cells. These treatments are also called cancer growth blockers or tyrosine kinase inhibitors TKIs. Therefore, further preclinical and clinical studies are warranted to optimize anti-angiogenic therapy in the era of cancer immunotherapy to open the vascular normalization window within the TME and enhance anti-tumor immunity.

Immune cells orchestrate the whole process of tumor angiogenesis via both direct and indirect mechanisms Fig.

Numerous pro- or anti-angiogenic factors derived from immune cells directly influence tumor vessels and determine the endothelial phenotype and function 5 , 7 , 41 , Moreover, certain types of immune cells can communicate and polarize other types of immune cells to be either pro-angiogenic or anti-angiogenic, indirectly affecting the quantity and quality of tumor angiogenesis 41 , Immune cells directly influence the phenotypes and functions of tumor vessels through various cytokines.

Innate immune cells, such as mature dendritic cells mDCs and M1-like TAMs, produce cytokines IFN-α, IL, IL, or TNF and chemokines CXCL9, CXCL10, or CCL21 that suppress tumor angiogenesis.

However, immature DCs iDCs , myeloid-derived suppressor cells MDSCs , M2 TAMs and Tie2-expressing macrophages TEM significantly promote tumor angiogenesis by secreting VEGF, IL, Bv8, and MMP Moreover, T reg , T H 2, and T H 17 cells can also release pro-angiogenic factors such as VEGF, IL-4, IL-5, IL, and IL In addition to direct effects on tumor vasculature, immune cells regulate tumor vasculature indirectly by communicating and polarizing with each other.

mDC, CD8, and T H 1 cells can skew macrophage polarization away from the M2 to the M1 phenotype. However, MDSCs and T reg cells can reprogram TAMs from M1 to M2. Macrophages exhibit notable plasticity in the regulation of tumor angiogenesis.

They constitute functionally heterogeneous innate immune cells depending on the type of secreted cytokines and growth factors.

Notably, they modify their transcriptional program in response to stimuli from the TME along a continuous spectrum, with an M1- and M2-like phenotype at both extremes; M1-like TAMs suppress tumor angiogenesis, whereas M2-like TAMs promote tumor angiogenesis 41 , 42 , 44 , 45 , M1-like TAMs suppress sprouting angiogenesis and induce vessel maturation by secreting anti-angiogenic cytokines, such as interleukin IL and TNF-α 47 , Intriguingly, M1-like TAM-derived IL polarizes macrophages toward the M1 phenotype, thereby generating a positive feedback loop for the anti-angiogenic M1 phenotype.

Accordingly, immunotherapy with IL not only reduces microvessel density but also enhances M1 macrophage polarization in tumors 48 , 49 , These factors not only enhance the migration and proliferation of ECs but also further skew macrophage polarization away from M1 to the tumor-promoting M2 phenotype 44 , 47 , As M2-like TAMs are a more dominant population than M1-like TAMs in most advanced tumors, pharmacological depletion of macrophages with clodronate- liposome generally suppresses tumor angiogenesis and tumor growth in transplanted tumor models 53 , Another distinct subtype of macrophages that was defined relatively recently is TEMs, which also plays an important role in encouraging tumor angiogenesis 55 , 56 , When Tie-2 on the surface of TEMs binds to angiopoietin-2 secreted from endothelial and tumor cells, a strong angiogenic switch is turned on in the TME.

Consistently, tumors fail to sustain angiogenesis in the absence of Tie-2 signaling in macrophages In addition, selective depletion of Tie-2 expression in macrophages induces tumor vascular normalization and the regression of established tumors, supporting the critical role of TEMs during tumor angiogenesis 56 , DCs, another important innate immune component of the TME, can regulate tumor angiogenesis depending on their maturation status Mature DCs can be classified into two major subtypes, conventional DCs cDCs or plasmacytoid DCs pDCs 60 , Mature cDCs suppress tumor angiogenesis by secreting anti-angiogenic cytokines, namely, IL and IL, and angiostatic chemokines, including CXCL9, CXCL10, and CCL21 62 , 63 , In contrast, mature pDCs secrete interferon-α IFN-α , which inhibits the proliferation and motility of ECs and increases anti-angiogenic cytokines and chemokines in the tumor 65 , Unfortunately, in the TME, the most frequent subset of DCs is immature DCs iDCs because cancer cells can preferentially recruit iDCs from peripheral blood vessels by releasing a number of cytokines e.

MDSCs, a heterogeneous population of immature myeloid cells, can augment tumor angiogenesis via several mechanisms. MDSCs enhance angiogenesis by increasing IL and decreasing IL in the TME 43 , 45 , 46 , Furthermore, MDSCs can promote angiogenesis by producing Bv8 and MMP MDSC-derived Bv8 can directly promote neovessel formation via endocrine gland-derived VEGF1 EG-VEGF1 and VEGF2 EG-VEGF2 and can further accumulate MDSCs within the tumor 71 , 72 , Therefore, neutralizing antibodies against Bv8 significantly reduce tumor vascular density and the number of tumor-infiltrating MDSCs Simultaneously, MMP-9 can induce tumor angiogenesis by releasing biologically active VEGF from the extracellular matrix of the TME.

Accordingly, MMPdeficient MDSCs fail to induce tumor angiogenesis 46 , Third, unlike other immune cells, some MDSCs can differentiate into EC-like cells. These EC-like MDSCs express endothelial markers, such as CD31 and VEGFR2, and have the ability to integrate into the tumor vasculature 45 , 46 , Adaptive immune cells are also critical players in the orchestration of tumor angiogenesis by directly affecting EC biology and indirectly modulating myeloid cell phenotypes.

IFN-γ directly inhibits the proliferation and migration of human endothelial cells and secretes IFN-inducible protein 10 IP and monokine induced by IFN-γ MIG. These cytokines also react with CXCR3, restraining the proliferation of endothelial cells and tumor vascularization 74 , Furthermore, IFN-γ signaling downregulates VEGF-A but upregulates CXCL9, CXCL10, and CXCL11, which collectively stimulate vascular maturation by enhancing pericyte recruitment along ECs 74 , 77 , Another important aspect of IFN-γ in tumor angiogenesis is the reprogramming of TAMs from M2- to M1-like TAMs.

T H 1 cells also polarize M2-like TAMs to M1-like TAMs and induce DC maturation in the TME, which suppresses tumor angiogenesis 82 , T H 2 cells expressing IL-4, IL-5, and IL recruit M2-like TAMs through STAT-6 activation and promote tumor angiogenesis 41 , 50 , 77 , The expression of IL by T H 17 correlates with the infiltration of ECs and abnormal tumor vasculature 41 , 77 , 85 , Tumor-infiltrating Treg cells also play a critical role by sustaining angiogenesis directly through VEGF secretion and supporting endothelial cell recruitment and expansion 83 , Furthermore, Tregs promote angiogenesis indirectly by restraining the activity of T H 1 cells and by triggering the activation of M2-like macrophages In ovarian cancer, hypoxia results in CCL28 upregulation, leading to a robust increase in Treg infiltration, VEGF and blood vessels, whereas depletion of Tregs reduces intratumoral VEGF levels and tumor angiogenesis 18 , The interactions between tumor immunity and angiogenesis suggest that tumor vascular remodeling could enhance the efficacy of cancer immunotherapy.

Emerging preclinical evidence demonstrates the potential of combining immunotherapy with vascular-targeting treatment 24 , 37 , 75 , 88 , 89 , 90 , Allen et al. demonstrated that anti-angiogenic therapy with anti-VEGFR2 enhances the efficacy of anti-PD-L1 immunotherapy in pancreatic neuroendocrine tumor RT2-PNET , mammary carcinoma MMTV-PyMT , and glioblastoma NFppGBM models Furthermore, the combination of anti-angiogenic and immunotherapy increased pericyte coverage and normalized tumor vessels, promoting intratumoral infiltration of activated T cells.

In addition to vascular normalization, the vessel phenotype represents the characteristics of high endothelial venules HEVs , which are morphologically thickened with plump endothelial cells ECs and functionally more specialized in lymphocyte extravasation than other tumor ECs.

Notably, the LTβR signaling pathway is involved in the generation of intratumoral HEVs after combined treatment with anti-VEGFR2 and anti-PD-L1.

Therefore, these results suggest that anti-angiogenic therapy could improve the efficacy of cancer immunotherapy and overcome resistance to cancer immunotherapy via tumor vessel normalization and intratumoral HEV formation.

Shigeta et al. also reported consistent synergism of anti-VEGFR2 and anti-PD-L1 in hepatocellular carcinoma HCC They observed that anti-VEGFR2 therapy upregulates PD-L1 expression under hypoxic conditions, mediated in part by IFN-γ secreted by ECs.

Dual combination therapy has also been shown to improve overall survival OS and anti-cancer immunity with increased intratumoral accumulation of CTLs and M1-like TAMs.

Collectively, combination therapy with anti-VEGFR2 and anti-PD-1 reprograms the immune microenvironment via vessel normalization, further strengthening the anti-cancer immune response and overcoming resistance to cancer immunotherapy in HCC. Anti-angiogenic therapy can also overcome resistance to anti-PD-1 by abolishing the TOX-mediated T-cell exhaustion program in the TME Kim et al.

revealed that VEGF significantly upregulates the transcription factor TOX, which influences the phenotype and function of CTLs. The TOX-mediated transcriptional program resulted in severe T-cell exhaustion and upregulated inhibitory immune checkpoint receptors such as PD-1, TIM-3, LAG-3, and TIGIT and reduced the proliferation of cytokine production by CTLs.

Combination treatment with anti-VEGFR2 and anti-PD-1 enhanced the immunotherapeutic efficacy and T-cell reinvigoration. Collectively, combinatory treatment with anti-angiogenic agents and ICIs is a potential therapeutic option in anti-PDresistant cancer.

Schmittnaegel et al. demonstrated that combined blockade of VEGF-A and ANGPT2 by a bispecific antibody A2V enhanced the therapeutic activity compared with either anti-VEGF-A or anti-ANGPT2 monotherapy alone in both genetically engineered and transplant tumor models A2V effectively inhibited tumor angiogenesis but promoted vascular maturation in the TME.

This negative feedback mechanism was successfully overcome by combining A2V with anti-PD-1, leading to better immunotherapeutic efficacy. These results encourage further testing of combining ICIs with various anti-angiogenic targets other than VEGF in advanced cancers. Recently, a novel immunotherapeutic target, simulator of IFN genes STING , was reported to be involved in the regulation of the tumor vasculature and demonstrated synergism with anti-VEGFR2 and ICIs Yang et al.

revealed that intratumoral STING signaling activation suppresses tumor angiogenesis and induces vessel normalization through type I IFN signaling activation and the upregulation of genes related to vascular normalization and endothelial-lymphocyte interaction.

STING agonist combined with anti-VEGFR2 synergistically enhanced vascular normalization, leading to durable anti-cancer immunity. Therefore, these data suggest that combining novel therapeutics with the combination of anti-angiogenic agents and ICIs could help overcome resistance to anti-angiogenic and immunotherapy in refractory cancers.

On the other hand, immune checkpoint blockade, such as anti-CTLA-4 or anti-PD-1, increases vascular perfusion to improve therapeutic efficacy. Zheng et al. Notably, IVP can distinguish tumors that are sensitive to ICIs from those that are resistant. In addition, IVP was time-dependently induced by anti-CTLA-4 even before tumor regression was detectable.

Collectively, these findings indicate that IVP could be a prerequisite of ICI to improve anti-cancer immunity, thereby enabling it to be used as a predictive indicator for ICI efficacy. Preclinical studies continue to yield encouraging results regarding the synergistic effects of ICIs and anti-angiogenic agent combination therapy, which have led to clinical investigations to reproduce these results in patients with advanced cancer 92 , 93 , 94 , 95 , 96 , 97 , Several pivotal clinical trials have already demonstrated the superiority of combining anti-angiogenic agents and ICIs in various malignancies.

The most successful results of combination therapy have been reported in renal cell carcinoma RCC and hepatocellular carcinoma HCC. RCC is a highly immunogenic tumor that has been treated with high-dose IL-2 in some patients. Immunotherapy has recently been revisited and reevaluated when phase 3 clinical trials demonstrated that nivolumab anti-PD-1 treatment leads to longer OS with significantly lower toxicity.

In KEYNOTE, patients with previously untreated metastatic RCC were treated with either pembrolizumab anti-PD-1 and axitinib VEGFR1, 2, and 3 inhibitor combination therapy or sunitinib monotherapy, and significantly increased progression-free survival PFS was demonstrated in the combination group compared with the sunitinib group Although the incidence of hepatic toxicity was higher in the combination group, no relevant death event occurred.

Based on the significant efficacy and acceptable toxicity profile, combination therapy with pembrolizumab and axitinib was approved by the FDA for treatment-naïve patients with metastatic RCC. JAVELIN Renal NCT is a phase 3 clinical trial that evaluated the efficacy of avelumab anti-PD-L1 and axitinib combination therapy against sunitinib monotherapy in patients with metastatic RCC in a first-line setting Although the data are premature for OS analysis and require further follow-up, the median PFS of the combination group has already been reported to be In addition, the ORR and complete response rate were Based on this study, the FDA approved avelumab for use in combination with axitinib as first-line treatment for patients with advanced RCC.

In HCC, two highly anticipated phase III studies testing PD-1 inhibitor monotherapy failed to meet their primary endpoints, leading to doubts regarding the use of ICIs in this cancer.

However, a randomized phase III clinical trial, IMBRAVE NCT , demonstrated significant improvements in co-primary end points, PFS and OS, using the combination of atezolizumab anti-PD-L1 and bevacizumab anti-VEGF-A compared with sorafenib This was the first study to propose a new first-line treatment option that is superior to sorafenib, which has been the standard of care for a decade.

The FDA granted the Breakthrough Therapy designation based on these data, and the phase III IMBRAVE trial was initiated. At the ESMO Asia Congress, the median OS with the atezolizumab and bevacizumab combination was not reached until analysis when compared with In terms of patient-reported outcomes, the combination group exhibited delayed deterioration of quality of life compared with sorafenib.

The safety and efficacy of the combination of pembrolizumab and lenvatinib were evaluated in patients with unresectable HCC in KEYNOTE, a multicenter, open-label, single-arm phase Ib study This clinical trial also yielded a promising response rate during the early stage and was granted Breakthrough Therapy designation by the FDA, initiating LEPP, a phase 3 trial to evaluate pembrolizumab in combination with lenvatinib as a potential first-line treatment for patients with advanced HCC In non-squamous non-small cell lung cancer NSCLC , a phase 3 clinical trial Impower, NCT comparing atezolizumab anti-PD-L1 , bevacizumab anti-VEGF , carboplatin, and paclitaxel combination therapy ABCP group against bevacizumab, carboplatin, and paclitaxel combination therapy BCP group showed significantly extended PFS and OS in the ABCP group compared with the BCP group median PFS: 8.

The ORR was significantly higher in the ABCP group than in the BCP group ORR: Based on these results, atezolizumab was approved by the FDA for use in combination with bevacizumab, paclitaxel, and carboplatin as first-line treatment for patients with metastatic non-squamous NSCLC. Recently, the FDA granted accelerated approval for the use of a combination of pembrolizumab and lenvatinib in patients with advanced endometrial cancer who have experienced disease progression after systemic therapy.

In this trial, patients who had previously been treated for metastatic endometrial cancer were evaluated for their response to lenvatinib and pembrolizumab.

Interim analysis showed that the ORR was However, immune-mediated AEs, including endocrine, gastrointestinal, hepatic, skin, pulmonary, and renal events, occurred in In several years, these ongoing trials are expected to generate consistent results, which will evolve the therapeutic landscape of advanced cancers.

Years of research have demonstrated the potential of ICI monotherapy as well as its limitations, which have led to further attempts to overcome these limitations by combination immunotherapy. Of the potential candidates, the combination of ICI and anti-angiogenic agents continues to yield promising results in both preclinical and clinical studies, not only highlighting that it is one of the most effective combination immunotherapy regimens thus far but also changing the treatment landscape for RCC and HCC.

Nonetheless, several issues remain to optimize the efficacy of this combination therapy. First, predictive biomarkers must be developed to identify the subset of patients who will benefit from this combination treatment.

Third, whether the effects of this combination are synergistic or merely additive must be evaluated. Finally, the angiogenic phenotype differs according to organ; thus, more in-depth analyses must be performed to further our knowledge of the response to ICI treatment at the organ level. Ribas, A.

Cancer immunotherapy using checkpoint blockade. Science , — CAS PubMed PubMed Central Google Scholar. Fukumura, D. Enhancing cancer immunotherapy using antiangiogenics: opportunities and challenges. Khan, K. Improving immunotherapy outcomes with anti-angiogenic treatments and vice versa.

CAS PubMed Google Scholar. Yi, M. et al. Synergistic effect of immune checkpoint blockade and anti-angiogenesis in cancer treatment. Cancer 18 , 60 PubMed PubMed Central Google Scholar.

Huang, Y. Improving immune-vascular crosstalk for cancer immunotherapy. Rahma, O. The intersection between tumor angiogenesis and immune suppression. Cancer Res. De Palma, M. Microenvironmental regulation of tumour angiogenesis. Cancer 17 , — PubMed Google Scholar.

Xia, A. T Cell Dysfunction in Cancer Immunity and Immunotherapy. Barsoum, I. A mechanism of hypoxia-mediated escape from adaptive immunity in cancer cells. Chon, H. Tumor microenvironment remodeling by intratumoral oncolytic vaccinia virus enhances the efficacy of immune-checkpoint blockade.

Kim, C. Vascular RhoJ is an effective and selective target for tumor angiogenesis and vascular disruption. Cancer Cell 25 , — Park, J. Normalization of tumor vessels by Tie2 activation and Ang2 inhibition enhances drug delivery and produces a favorable tumor microenvironment.

Cancer Cell 30 , — Lee, J. Novel glycosylated VEGF decoy receptor fusion protein, VEGF-Grab, efficiently suppresses tumor angiogenesis and progression. Cancer Ther. Jain, R. Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia.

Cancer Cell 26 , — Kim, Y. Methylation-dependent regulation of HIF-1alpha stability restricts retinal and tumour angiogenesis. Schaaf, M. Defining the role of the tumor vasculature in antitumor immunity and immunotherapy. Cell Death Dis.

Ramjiawan, R. Anti-angiogenesis for cancer revisited: Is there a role for combinations with immunotherapy? Angiogenesis 20 , — Facciabene, A. Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and T reg cells. Nature , — Baluk, P.

Cellular abnormalities of blood vessels as targets in cancer. Lugano, R. Tumor angiogenesis: causes, consequences, challenges and opportunities.

Cell Mol. Life Sci. Article PubMed PubMed Central Google Scholar. Motz, G. Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors. Gabrilovich, D. Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells.

Oyama, T. Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-kappa B activation in hemopoietic progenitor cells. VEGF-A drives TOX-dependent T cell exhaustion in anti-PDresistant microsatellite stable colorectal cancers.

Khan, O. Gerald, D. Angiopoietin an attractive target for improved antiangiogenic tumor therapy. Goumans, M. TGF-beta signaling in vascular biology and dysfunction. Cell Res. Tian, M. COX-2 is an important mediator of angiogenesis and tumour growth.

COX-2 expression occurs in a wide range of preneoplastic and malignant conditions. The enzyme has been localised to neoplastic cells, endothelial cells, immune cells, and stromal fibroblasts within tumours. It mediates its pro-angiogenic effects primarily by three products of arachidonic acid metabolism - thromboxane A2, prostaglandin E2 and prostaglandin I2.

These products promote angiogenesis by a number of mechanisms including stimulation of VEGF, promotion of vascular sprouting and tube formation, increased survival of endothelial cells and activation of EGFR-mediated angiogenesis.

Studies have shown that selective inhibition of COX-2 activity will suppress angiogenesis in vitro and in vivo and therefore COX-2 inhibitors could be a useful adjunct to therapy. Expression of human epidermal growth factor receptor 2 HER-2 within tumour cells is closely associated with angiogenesis and VEGF expression.

This is thought to be mediated by transregulation of HER-2 by proteins called heregulins. These heregulins regulate the expression and secretion of VEGF in breast cancer cells. Trastuzumab is a monoclonal antibody that blocks HER Trastuzumab is currently available for patients with metastatic breast cancer if the tumour overexpresses HER It is not known how much of the anticancer effects of drugs aimed at molecular structures are due to their angiogenic effects.

Angiogenesis is a complex process and successful inhibition of angiogenesis may involve the combination of multiple drugs with differing modes of action.

Another strategy related to angiogenesis is the destruction of new vessels. This has led to the development of vascular targeting drugs Table 1. The full spectrum and aetiology of toxicities produced by the angiogenesis inhibitors has yet to be defined. The induction of venous and arterial thromboses, bleeding, hypertension and proteinuria by drugs, such as bevacizumab, is probably directly related to their effects on endothelial cells.

The gut perforation occasionally associated with bevacizumab may be related to induction of ischaemia. The teratogenic effects of thalidomide may have been due to its action on peripheral blood vessel development in the fetus.

However, it is unlikely that the common adverse effects of thalidomide such as somnolence, rash and neuropathy are related to its effect on angiogenesis.

The use of angiogenesis inhibitors is an exciting new area of cancer research. Their optimal use has yet to be defined. Ferrara N. Vascular endothelial growth factor: basic science and clinical progress [review].

Endocr Rev ; Sparano JA, Gray R, Giantonio B, O'Dwyer P, Comis RL. Evaluating antiangiogenesis agents in the clinic: the Eastern Cooperative Oncology Group portfolio of clinical trials. Clin Cancer Res ; Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, et al.

Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med ; Bilenker JH, Haller DG. Future directions with angiogenesis inhibitors in colorectal cancer [review]. Clin Colorectal Cancer ;4 Suppl 2:S Increased expression of angiogenic factors is associated with an improved prognosis for patients with cancer.

Sydney Cancer Centre and Department of Medicine, University of Sydney, Concord Hospital Clinical School, Sydney. Skip to main content. Home Angiogenesis inhibitors in cancer - mechanisms of action A A. ISSUE 1 FEBRUARY Experimental and Clinical Pharmacology Angiogenesis inhibitors in cancer - mechanisms of action Stephen J.

Clarke, Rohini Sharma Aust Prescr ; 1 February DOI: Clarke SJ, Sharma R. Angiogenesis inhibitors in cancer - mechanisms of action. Aust Prescr ; Article Authors. Subscribe to Australian Prescriber.

Summary Tumours need to develop a new blood supply to grow and metastasise. Introduction Small tumours are able to grow because they can obtain nutrients and oxygen by diffusion.

Angiogenesis The development of blood vessels is a complex equilibrium regulated by anti-and pro-angiogenic factors. Vascular endothelial growth factor Of the angiogenic factors secreted, VEGF is perhaps the most specific for endothelial cells. Angiogenesis inhibition in the treatment of cancer To stop angiogenesis requires treatment with anti-angiogenic factors, or drugs which reduce the production of pro-angiogenic factors, prevent them binding to their receptors or block their actions.

Endogenous anti-angiogenic factors Endostatin is the carboxy-terminal fragment of collagen XVII. Thalidomide There has been renewed interest in this potent teratogen since it has been shown to be both an immunomodulatory and anti-angiogenic drug. Bevacizumab Bevacizumab is derived from a monoclonal antibody to murine VEGF.

Previous studies using dual or multi-target antibodies which simultaneously inhibit several angiogenic signals exhibited an incremental anti-angiogenic efficacy in different tumor types Li et al. However, many processes and factors contributing to inefficacy and resistance to angiogenesis inhibitors, in particular those involving the tumor endothelium, remain ambiguous.

Nevertheless, the time window of vessel re-organization and normalization is not well understood in the clinical setting but could play a major role in the transmission of chemical agents directly to the tumor, thereby enhancing anti-cancer efficacy Johnson et al.

The interaction of tumor vasculature with immune cells has a severe impact on the responsiveness and immunodeficiency of the tumor. Vascular normalization due to VEGF-inhibiting therapy exhibited increased lymphocyte infiltration and T-cell activation which, combined with immune checkpoint inhibitors ICI , elicited an improved anti-tumor immunity in preclinical trials Allen et al.

Additionally, combinational therapy of anti-angiogenic agents and ICI resulted in the formation of HEVs, which enhances activation of circulating B- and T-cells by mediating migration into secondary lymphoid organs Ager and May, When surrounded by dense B- and T-cell rich areas, HEV can further adapt to tertiary lymphoid structures TLS thereby triggering potent anti-tumor immunity, which can significantly improve patient outcomes Martinet and Girard, We are confronted with a network of considerable aspects when it comes to anti-angiogenic therapy, many of which still require thorough investigation.

Further characterization of the TME and the associated endothelium can help improve anti-angiogenic therapies and optimize the proposed powerful synergic efficacy of combinational therapeutical approaches in NSCLC. Physiological angiogenesis has already been characterized in detail and previously reviewed elsewhere Góth et al.

The process of tumor angiogenesis, which occurs early during tumor progression, is similar to physiological vessel formation, but with differences in regulation and grade of activity Hanahan and Folkman, ; Raica et al. This activation results in increased proliferation, survival and migration, leading to distortion of the basement membrane as well as pericyte coverage in the tumor vasculature Hida et al.

Consequently, TECs exhibit dysregulated behavior and polarization resulting in leaky, hemorrhagic, and dysfunctional vessels. Thus, oxygen levels, nutrient availability and waste disposal is diminished, which has severe effects on the TME Colegio et al.

Furthermore, dysfunctional TECs severely impact lymphocyte adhesion, trafficking and migration to the local tissue, resulting in a highly immunosuppressive TME Fridman et al. Additionally, the tumor stroma, which consists of a mix of resident fibroblasts and pericytes as well as bone-marrow derived tumor infiltrating leukocytes e.

M2 polarized tumor associated macrophages can either directly activate angiogenesis by releasing VEGF, bFGF and PlGF or indirectly via the release of matrix-metalloproteinases MMPs , which in turn remodel the extracellular matrix for an enhanced endothelial migration Kessenbrock et al.

Fibroblasts, as well as myeloid derived suppressor cells MDSCs promote angiogenesis through expression of growth factors such as VEGF and bFGF Shi et al. CSF-1, a cytokine crucial for the survival and differentiation of monocytes and macrophages, mediates the recruitment of MDSCs into the tumor niche, which in turn increases angiogenesis due to growth factor release Shojaei et al.

By blocking the CSF-1 signaling in combination with anti-VEGFR2 therapy, tumor growth could be markedly decreased in murine lung carcinoma models Priceman et al. Mast cells comprise a major compartment of inflammatory cells present in the TME and exhibit important regulatory features regarding angiogenesis Ribatti and Crivellato, Their granules contain various proteases, cytokines and growth factors including pro-angiogenic molecules such as VEGF, bFGF, PDGF and the potent angiogenic factor tryptase, which is released upon activation of IgE or c-kit receptors Ribatti and Ranieri, Tryptase induces vascularization and vessel tube formation by stimulating proliferation of ECs and activation of MMPs Ribatti and Crivellato, In NSCLC the number of tryptase positive MCs linearly correlates with microvascular density, confirming the important role of this enzyme in regulating tumor angiogenesis Ibaraki et al.

Inhibition of c-kit and its ligand SCF could hamper mast cell infiltration into the TME, preventing degranulation and thereby producing a synergizing anti-angiogenic effect Huang et al. Current vessel-inhibiting therapies for treating advanced NSCLC mainly focus on repressing the process of vessel sprouting predominantly triggered by VEGF signaling.

In the past years, however, non-angiogenic processes in the TME have gained attention as they are suggested to significantly contribute to tumor progression while being resistant to traditional angiogenesis inhibitors.

In highly vascularized organs such as the lung, it was observed that cancer cells start to grow along existing vessels to preserve access to essential nutrients and gases without the need to form new vasculature.

This process is referred to as vessel co-option Pezzella et al. In contrast to the chaotic growth of angiogenic tumor vessels, co-opted vasculature remains well organized as deduced from normal tissues Adighibe et al. So far, vessel co-option is suggested to result, at least in part, of differential mitochondrial metabolism, but it may also involve reduced inflammation Donnem et al.

The ECs of co-opted vessels experience severe molecular changes during this process, for e. Thereupon, the tumor core becomes hypoxic, which consequently activates the angiogenic switch in tumor vessels Holash et al.

In vitro studies of glioma cells suggest that tumor cells that facilitate vessel co-option are dependent on the endoplasmic reticulum based stress sensing protein IRE1 Auf et al.

Furthermore the MMP-activating protein B2R was shown to serve as a chemoattractant during the migration of glioma cells towards blood vessels Montana and Sontheimer, Finally, CDC42, a protein involved in actin-dependent formation of cytoplasmatic extensions, together with CD44, a protein crucial for establishing cell-cell contact, enable the connection between tumor cells and vessel covering pericytes for vessel co-option Caspani et al.

So far, the molecular mechanism behind VM is not yet understood, however, it appears that VE-cadherin, the most prominent receptor on ECs, may play an important role. VM networks resemble embryonic vasculogenesis, referring to a highly aggressive tumor cell phenotype that converted to an embryonic-like, undifferentiated state to facilitate tube formation Maniotis et al.

Gene expression analysis of VM networks in aggressive melanoma identified genes correlated with various cellular phenotypes such as fibroblasts, ECs and epithelial cells Bittner et al. Tumors positive for VM show an increased expression of the ECM component laminin5γ2 and several MMPs, underlining the importance of ECM remodeling for initiating and promoting this non-angiogenic process Seftor et al.

Furthermore, VM is associated with poor prognosis as it is mainly observed in aggressive forms of melanoma and lung metastases Williamson et al. Taking the potent impact of these non-angiogenic processes in cancer progression into consideration, may help us explain the occurring resistance of lung tumors to VEGF-inhibitors Döme et al.

In summary, the pathological features of tumor-associated ECs and non-ECs which result in a complex cancer promoting TME are diverse, and consequently contribute to therapy failure of angiogenesis inhibitors as well as other therapy approaches in a remarkable fashion.

To better understand the biological mechanisms behind drug resistance or lack of clinical benefit, further investigation into the detailed characterization of the endothelial compartment in the TME are essential. Currently used anti-angiogenic agents have been developed and approved for clinical application after intense study of their molecular, cellular, and physiological mode of action using various experimental approaches.

In the following part we summarize currently available methods for investigating tumor angiogenesis as well as anti-angiogenic agents that have already been accepted for treating NSCLC. Experimental models remain the cornerstone for investigating tumor angiogenesis and the development of new anti-angiogenic therapies.

As vessel sprouting is a multistep process there is a wide array of assays which enable individual evaluation of different stages, and each possesses specific advantages and disadvantages Shahid et al. To unravel these complex processes, it is crucial to understand the analytical potential of each model.

In vitro methods represent the fundamental evaluation of tumor angiogenesis including basic functional analysis such as proliferation, migration, and tube formation.

The big advantages of in vitro assays are their simplicity, high reproducibility, and cost effectiveness, while the disadvantages include the incomplete representation of the cellular heterogeneity and prevailing conditions present in human organs.

Although findings from in vitro assays may never be conclusive alone, they serve as a preliminary projection of angiogenic processes upon treatment of choice and provide first insights into a testing hypothesis.

Ex vivo assays such as the thoracic aorta ring and retina angiogenesis methods represent the link between in vitro and in vivo analysis. The advantage of this method over in vitro assays is the preservation of original EC properties within the tissue that are normally modified due to isolation processes and repeated passaging.

The absence of blood flow and circulating EC progenitors or other factors constitute the main disadvantages of these methods. For more accurate information regarding angiogenic processes upon treatment in a biological system or to perform long-term studies, in vivo methods are necessary.

The most common systems to investigate angiogenesis in a living organism are the chicken chorioallantoic membrane CAM assay, matrigel plugs, and tumor xenograft models. CAM assays, which have already been in use for decades, utilize chorioallantoic membranes of fertilized chicken eggs to evaluate angiogenic processes.

While this method is cost effective, highly reproducible and the outcomes are easily visualized, it must be taken into consideration that vessel growth is evaluated during developmental stages, which can affect studies investigating mechanisms in mature vasculature.

Matrigel plug assays enable the use of an in vitro tool in an in vivo setting. Here, vascular growth is evaluated by injection of matrigel, a synthesized substrate resembling basement membrane matrix, into an animal model which allows easy stimulation, subsequent excision, and investigation of the plug with, for example, immunohistological stainings.

Compared with CAM assays, the matrigel plug can be used in more analytical methods and provides a fast and reliable representation of angiogenic processes in a biological system. Nevertheless, this method may require more replicates due to higher variability of results and is therefore more expensive.

Lastly, transplantation xenografts represent the most advanced method to investigate tumor angiogenesis in a living organism. Tumor cells, mostly of human origin, are injected into immunodeficient mice to induce formation of a cancer mass that can be further treated and monitored for changes regarding tumor angiogenesis.

This method most suitably reflects the pathological mechanism of vessel growth in vivo in the presence of blood circulation, as well as diverse environmental factors.

Furthermore, it enables the long-term study of diverse processes associated with angiogenesis that are observed in a biological system such as tissue invasion, distant metastasis formation as well as non-angiogenic processes like vessel co-option and VM, which are known to promote resistance mechanisms in various cancers.

Aside from the ethical aspect, a considerable disadvantage of this method is the incomplete or lacking representation of the immune system due to immunosuppression of the study organism. Examining which experimental assay is most suitable for investigating a chosen angiogenic process under certain conditions, necessitates extensive deliberation with the desired endpoint, required technical equipment, level of experimental throughput, cost, and ethics kept in mind.

Additionally, the complexity of angiogenesis cannot be unraveled using a single analytical method but the thought-out application of multiple overlapping analyses, ranging from cellular to physiological levels, are necessary to obtain robust findings worth testing in the clinical setting.

In , the first VEGFA-inhibiting antibody, bevacizumab, was approved for use in advanced colorectal cancer in combination with chemotherapy and was followed in in NSCLC Sandler et al. Since then, diverse anti-angiogenic antibodies or tyrosine kinase inhibitors TKIs have been developed, which block either VEGF-A binding to the receptor or directly inhibit VEGFR-2 to hamper vascularization in tumors.

VEGF-pathway inhibition has a broad anti-angiogenic effect in tumors: 1 it primarily inhibits vessel growth which induces regional cancer cell death and delays progression of the tumor rather than diminishing its size Escudier et al.

Angiogenesis inhibitors in combination with either chemotherapeutics, targeted therapies or ICI, in first or second-line therapies in NSCLC, have exhibited improved efficacy and feasible safety, which significantly improved response rates and prolonged progression free survival PFS in a large number of patients.

Despite the remarkable clinical benefits of these combinational approaches on response rate and PFS, the overall survival OS benefits were modest due to acquired drug resistance. It is important to mention that in most lung cancer studies anti-angiogenic therapy is administered until the onset of severe drug related adverse effects or disease progression.

So far, there is only preclinical evidence that discontinued angiogenesis inhibition results in TME reorganization and perhaps causes a rebound effect of tumor angiogenesis. In tumor and healthy mouse models, it could be shown that anti-VEGF therapy withdrawal resulted in rapid tissue revascularization and long lasting structural changes including vessel hyper-permeability and increased metastasis in the diseased cohort Yang et al.

The treatment-triggered hypoxia which induces angiogenesis especially during therapy-withdrawal is one possible explanation to this tumor promoting off-drug effect.

The benefit of continuous anti-angiogenic therapy beyond disease progression in the clinical setting was first analyzed in a phase 3b trail in which included advanced NSCLC patients Gridelli et al.

Here, bevacizumab was administered in addition to standard of care therapy beyond disease progression. While, the treatment continuation of bevacizumab yielded no substantial therapy benefit, improvements in efficacy, and no new safety signals were observed.

Based on these findings, the approach of continuous angiogenesis inhibition should be further investigated but may be recommended at a certain degree in the future.

Nevertheless, treatment decisions should be based on individual therapeutic efficacy, which needs to be tracked throughout the entire therapy. However, the absence of reliable biomarkers with predictive features for anti-angiogenic therapies hamper further therapy improvement, thus molecular screening for markers associated with tumor angiogenesis is currently of great value.

Table 1. As previously mentioned, there is a great need for biomarkers to predict and track anti-angiogenic therapy efficacy, to help overcome innate and acquired resistance as it is still the main obstacle that restrains clinical success Bergers and Hanahan, So far, predictive angiogenesis-associated biomarkers in NSCLC are lacking, highlighting the need for further investigation to improve this anti-tumor approach.

In a recent study, it was demonstrated that immunohistochemically confirmed TTF-1 expression in advanced non-squamous NSCLC samples, which is a known prognostic biomarker of lung adenocarcinomas, could be linked to therapy success of bevacizumab in combination with pemetrexed plus platinum derivatives Takeuchi et al.

TTF-1 positive tumors exhibited enhanced clinical benefits when bevacizumab was combined with the basic therapy whereas TTF-1 negative tumors did not benefit from this addition. Furthermore, despite the previous results of the IMpower study, where significant clinical benefits of bevacizumab in combination with ICI and chemotherapy were shown, regardless of PDL-L1 expression, a phase 1b study by Herbst et al.

observed contrary results. According to this, PD-L1 expression remains a predictive marker of ICI therapy or ICI therapy in combination with anti-angiogenesis agents in NSCLC. Qiu et al. recently examined the benefit of anti-angiogenic therapies bevacizumab, anlotinib or others with anti-PD-L1 agents nivolumab or pembrolizumab in a real-world study including 69 NSCLC patients.

Subgroup analyses in the cohort revealed that the response and PFS of this combinational therapy was significantly higher when it was administered as first-line therapy compared to other lines of treatment, and when the therapy was initiated within the first 6 months of diagnosis compared to later time points Qiu et al.

Additionally, patients with EGFR wildtype tumors exhibited significantly prolonged PFS after the combinational therapy compared to patients with EGFR mutated tumors.

Interestingly, no correlation between PDL-1 expression levels and the efficacy of this combinational therapy has been observed so far, however, follow up will be continued. In short, these study results can help to optimize the use of anti-angiogenic agents in combination with PD-L1 inhibitors, however, more factors need to be investigated to yield an optimal benefit.

Another potent multi-targeted anti-angiogenic TKI, anlotinib, has already shown profound benefits as third-line combinational therapy in advanced NSCLC Han et al.

A transcriptomics study of an anlotinib-resistant lung cancer cell line, indicated that CXCL2, a cytokine involved in wound healing and angiogenesis, was also involved in anlotinib-resistance Lu et al. In vitro assays demonstrated that exogenous CXCL2 could recover anti-angiogenic-induced inhibition of migration and invasion and prevent apoptosis of anlotinib-resistant cells.

Furthermore, in a retrospective analysis, anlotinib-induced decrease of the inflammatory cytokine CCL2 in serum correlated with prolonged PFS and OS Lu et al.

Nevertheless, resistance and poor response to anlotinib hinder drug efficacy. While the underlying mechanisms are still unknown, elevated serum-levels of two angiogenesis-related markers KLK5 and L1CAM were recently correlated with poor response to anlotinib Lu et al.

Easily available predictive biomarkers, e. Several studies suggested a potential prognostic value of VEGF in NSCLC but so far investigations into circulating VEGF levels have not yielded consistent results Rodríguez Garzotto et al.

In the E study, high VEGF levels in pretreatment plasma of patients with advanced stage NSCLC, who received combinational treatment of bevacizumab plus chemotherapy, correlated with increased overall response but had no predictive outcome on survival Dowlati et al.

Another study observed contrary results when baseline plasma biomarkers of non-squamous NSCLC patients undergoing similar therapy were evaluated Mok et al.

Here, baseline VEGFA levels in the plasma correlated with prolonged PFS and OS but showed no association with response rates to the therapy.

The predictive value of VEGF or other proangiogenic factors on anti-angiogenic drug response is a highly discussed matter revealing vastly variable results. This is partly due to analytical variability, including sample collection and handling, as well as the disagreements regarding the most suitable sample choice for evaluating circulating factors Rodríguez Garzotto et al.

For example, serum or platelet rich plasma may not adequately represent the physiological VEGF level as it has been shown that the clotting processes initiates VEGF release in platelets Webb et al. Moreover, the pathological situation can impact VEGF levels, as patients with more advanced tumors or several metastatic tumor sites exhibit a higher baseline level of plasma VEGFA, suggesting that VEGFA is linked to the tumor burden Mok et al.

Previously proposed correlations of circulating angiogenic factor levels with anti-angiogenic therapy efficacy in lung cancer seem to reflect tumor biology thus, have an important prognostic role rather than to be predictive Crohns et al. The observed trend of increasing circulating factors in response to angiogenesis inhibition on one hand was shown to depend considerably on the TME and may represent therapy-induced hypoxia Zaman et al.

On the other hand, high VEGFA levels could also be attributed to TP53 mutated lung tumors which correlated with improved efficacy of bevacizumab Schwaederlé et al.

A currently identified alternative biomarker for bevacizumab-based chemotherapy combinations in patients with advanced NSCLC is CXCL In the analyzed sera of 40 advanced staged NSCLC patients therapy-induced decrease of CXCL16 levels correlated with prolonged OS compared with patients exhibiting only moderate decrement Shibata et al.

However, confirming if any of these molecular markers indeed exhibit adequate predictive features necessitates further investigation. New aspects of processes which promote tumor angiogenesis, and a better understanding of the endothelium as driving force can help identify reliable biomarkers and overcome therapy failure in NSCLC.

There are several mechanisms on both the cellular and environmental levels which can promote vessel formation in human tumors, many of which are not yet been completely elucidated. Although angiogenesis may represent the most important part of tumor vascularization, other processes that result in perfusion of the tumor tissue should be investigated in more detail and considered when designing new anti-angiogenic approaches in NSCLC.

In the following part we summarize various levels of tumor vascularization that may represent new targets for vessel inhibition in NSCLC. All mentioned mechanisms are summarized in Figure 1.

Figure 1. Mechanisms of tumor vascularization in NSCLC. Tumor vascularization in lung cancer can be promoted by various processes which overlap during cancer progression. TECs exhibit upregulated metabolism to enable high angiogenic activity which includes processes involved in proliferation cholesterol synthesis and glycolysis and processes that enable migration via ECM remodeling collagen synthesis.

Potential targets involved in these pathways SQLE, PFKFB3, and ALDH18A1, respectively are considered to increase the angiogenic potential of TECs in NSCLC. Hypoxia and acidosis induced by high levels of lactate due to upregulated glycolysis constitute to a highly pro-angiogenic tumor environment.

Angiogenesis stimulating factors VEGF, bFGF, PDGF, HIF-1α, tryptase, and MMPs are released by both, cancer cells and stromal cells, including fibroblasts, pericytes, tumor associated macrophages and ECs.

Non-angiogenic processes constitute to tumor vascularization and are inaccessible for anti-angiogenic agents, thus contributing to therapy resistance. VM comprises the formation of tubular structures arising from cancer cells that gain endothelial like properties to maintain vascular supply during cancer progression.

Another mechanism of cancer cells to persist in circulation is to grow along existing vasculature, which is referred to as vessel co-option. In this figure we summarized the various mechanism of tumor vascularization that should be considered when targeting the inhibition of tumor vessels in NSCLC.

The endothelium is postulated to be a large contributor to the therapeutic efficacy of anti-angiogenic therapies, and therefore represents a possible source of therapy response or failure.

It is well known that the process of angiogenesis is comprised of different EC phenotypes which execute distinct functions. During the elongation of the sprouting vessel VEGF-sensitive tip ECs migrate into avascular tissue regions, thus leading the proliferating trailing stalk ECs, which built up the growing vessel.

Newly formed vasculature finally adapts a mature and quiescent phenotype referred to as phalanx ECs Carmeliet and Jain, ; Betz et al. The EC phenotypes involved are highly dynamic and can reprogram the gene expression to meet their current physiological requirements.

However, the tumor endothelium was not studied in depth and a recent single-cell RNA sequencing scRNA-Seq study identified even more EC phenotypes from both healthy and tumor tissue from lung cancer samples as already known, indicating a much more complex phenotypic heterogeneity of the tumor vasculature than initially presumed Goveia et al.

Interestingly, although phenotype proportions differed strongly between analyzed NSCLC patients, they collectively observed a low abundance of tip and proliferating TECs, which represent the main targets of traditional anti-angiogenic therapy.

Furthermore, they identified a so-far-unknown tumor exclusive phenotype of activated postcapillary vein EC that upregulated features known from HEVs in inflamed tissues such as immunomodulatory factors and ribosomal proteins.

The unexpected finding that activated and proliferating TECs only represent a minority of the pathological EC phenotypes found in NSCLC, allows us to reconsider currently used anti-angiogenic therapy as less of a vessel-inhibiting strategy, and more of a strategy to modulate the higher proportion of mature TECs into potent participants of tumor surveillance.

In order to develop new angiogenesis-inhibiting therapies, the molecular differences between physiological and pathological ECs will need to be elaborated. Genetically TEC and NEC phenotypes significantly differ in gene expression affecting diverse cellular mechanisms such as proliferation, migration, inflammation, and angiogenesis Figure 2.

Previous studies have shown that one key feature of TECs is a highly active metabolism, which permits pathological processes as increased proliferation and angiogenesis Cantelmo et al.

Hyperglycolytic TECs subsequently release high amounts of lactate into the environment, which in turn, further stimulates EC proliferation and angiogenesis Annan et al. It could be demonstrated that inhibition of PFKFB3 resulted in improved drug efficacy and decreased metastatic events in tumor mouse models Cantelmo et al.

Another study in xenograft NSCLC mouse models exhibited that PFKFB3 mRNA silencing in combination with docetaxel results in a chemoenhancing effect and increases anti-cancer efficacy compared with monotherapies alone Chowdhury et al. Furthermore, to sustain upregulated proliferative capacity, TECs exhibit elevated nucleotide biosynthesis including upstream pathways that are involved in serine and lipid synthesis Cantelmo et al.

In addition, Lambrechts et al. Interestingly, c-MYC expression induces angiogenesis in combination with HIF-1α and VEGF Lee and Wu, and recruits tryptase positive mast cells into the tumor niche Soucek et al. Figure 2. The multifaced picture of TECs in NSCLC.

TECs possess features that enable continuous angiogenic activity for progressing vascularization of the tumor. These features are ensured by genetical changes in the tumor endothelium that are triggered by diverse stimuli of the TME e.

The stroma, consisting of various cells, promote angiogenesis by directly releasing signaling molecules into the adjacent tissue, thereby stimulating TECs. Fibroblasts and myeloid derived suppressor cells MDSCs activate angiogenesis by releasing VEGF and bFGF into the TME.

Additionally, CSF-1 molecules, expressed by cancer cells, further recruit MDSCs into the tumor niche. Tumor associated macrophages TAMs can directly induce angiogenesis by releasing VEGF, bFGF, and PlGF, or indirectly by releasing matrix metalloproteinases MMPs which promote endothelial migration.

Mast cells secrete tryptase TRYPT into the TME which stimulates EC proliferation and enables ECM remodeling. Furthermore, to facilitate enhanced angiogenesis, TECs upregulate the surface expression of angiogenic receptors as well as increase metabolic activity including energy and amino acid metabolism and the biosynthesis of nucleotides.

In addition to the high angiogenic activity, TECs can directly suppress inflammatory responses by downregulation of inflammatory cytokines for immune cell recruitment CCL2, CCL8, and IL-6 , receptors required for immune cell homing ICAM or lymphocyte activation MHC I and MHC II which results in impaired immune cell trafficking and migration into the TME.

In summary the complex interaction of tumor-protecting environmental conditions and the pathological features of TECs lead to a pro-angiogenic and immune suppressive TME in NSCLC. Focusing on endothelial metabolism in cancer, a recent study could identify at least two metabolic signatures which are highly upregulated in angiogenic endothelium and TECs.

One for proliferation, which includes gene sets associated with biomass production e. These results educed two new possible metabolic targets to hamper tumor angiogenesis; aldehyde dehydrogenase 18 family member A1 ALDH18A1 , an enzyme essential for de novo biosynthesis of proline; and squalene epoxidase SQLE , the rate-limiting enzyme in cholesterol biosynthesis.

Silencing of ALDH18A as well as SQLE impaired EC proliferation, migration and vessel sprouting in in vitro assays. Summarized, targeting endothelial metabolism in cancer is an interesting therapeutic option that could possibly assist an anti-angiogenic approach for treating NSCLC.

Another key feature of TECs in lung cancer is the downregulation of inflammatory responses thus contributing to tumor-associated immune escape. Single-cell analysis of NSCLC samples identified the most downregulated genes of the tumor endothelium in connection to inflammation, which included CCL2, CCL18, and IL6, essential for immune cell recruitment; MHC I and II, essential for immune cell activation; and ICAM, required for immune cell homing Lambrechts et al.

As the endothelium represents the primary connection between the immune system and tumor cells, these results indicate the important role of TECs in immunomodulatory processes that hamper anti-tumor immunity.

Vessel normalization not only improves immune cell activation and infiltration, but is also suggested to enhance drug delivery to the tumor sites, thus improving its efficacy Allen et al. Additionally, combinational therapy of angiogenesis inhibitors and immunotherapy anti-PD-L1 in previous studies could elicit the formation of unique blood vessels in treated tumors that resemble HEVs typically found in lymphoid tissues, which implicated increased treatment efficacy Allen et al.

HEVs can mediate immune cell adhesion and migration into the tumor, which may be important for bypassing TEC-induced immune escape Ager and May, In the already discussed scRNA-Seq study by Goveia et al.

These remarkable observations indicate that TECs comprise the ability to transform into HEVs to promote immune cell infiltration into the tumor and induce a potent anti-tumor response. This extends the previous observations of favorable synergistic effects of immune therapy in combination with angiogenesis inhibitors in NSCLC, especially when it results in HEV formation.

Furthermore, direct induction of HEV formation could be a promising new strategy in anti-angiogenic approaches that may attain great clinical importance. However, currently there are no reliable biomarkers to track the process of vessel normalization or HEV formation in NSCLC which could help to predict and optimize this new treatment strategy.

As mentioned above, in some cases tumor vascularization can be facilitated by non-ECs which adapt certain properties to sustain access to the circulation, which may support anti-angiogenic drug resistance.

During tumor progression, processes that lead to vascularization of the malignant tissue can vary locally as well as temporarily and involve angiogenic as well as non-angiogenic mechanisms even in the same lesion Bridgeman et al. In lung tumors, where non-angiogenic tumor growth occurs most commonly, previous studies primarily located non-angiogenic processes in the tumor periphery, whereas angiogenesis is typically localized in the hypoxic tumor core Pezzella et al.

Here, we briefly discuss the impact of non-angiogenic processes in NSCLC on anti-angiogenic drug efficacy based on previous studies. VEGF-A inhibition using bevacizumab failed to inhibit VM in breast cancer cells in vitro , furthermore, sunitinib, a multi targeting anti-VEGFR inhibitor, even promoted VM in breast cancer mouse models Dey et al.

Additionally it could be demonstrated that VM in NSCLC depends on expression of Sema4D and its receptor plexinB1 which activate RhoA and downstream ROCK, comprising an already known angiogenesis-promoting process in tumors Basile et al.

Although the role of VM in NSCLC is not fully understood, previous observations suggest that it may contribute to anti-angiogenic therapy failure and may serve as an option to treat aggressive lung tumors. Vessel co-option on the other hand is a common phenomenon especially observed in lung metastases when tumor cells start to invade perivascular tissues Jensen, Anti-angiogenic therapy with sunitinib could induce a switch from angiogenic vessel formation to vessel co-option in a lung metastatic mouse model, which ultimately resulted in sunitinib resistance Bridgeman et al.

Unfortunately, regulative mechanisms of vessel co-option in human tumors remain unknown in large part, however, predicting the occurrence of either VM or vessel co-option could be a useful tactic to prevent anti-angiogenic drug resistance in some patients.

According to these and other results, it could be confirmed that non-angiogenic tumors contribute to anti-angiogenic therapy resistance which reveals the undoubted importance of targeting both angiogenic, but also non-angiogenic vessel growth to treat NSCLC Donnem et al.

Increasing knowledge of the physiological processes of tumor vascularization in addition to traditional angiogenesis has enlightened a variety of adaptive mechanisms which can promote anti-angiogenic therapy resistances.

This awareness fortifies the necessity for alternative anti-angiogenic agents besides traditional anti-VEGF therapy.

As previously examined, tumor angiogenesis depends on upregulated metabolic activity e. Cholesterol not only represents a fundamental structural component of cell membranes and serves as precursor for several steroid hormones, it is also crucial for membrane function and angiogenic signaling, making it a favorable target for tumor vessel inhibition Lyu et al.

Inhibition of intracellular cholesterol trafficking with anti-inflammatory drug chepharantine was shown to hamper angiogenesis and tumor growth in lung cancer xenograft mice while improving anti-tumor activity of standard chemotherapeutics Lyu et al. Another study has shown that pharmacological lowering of intracellular cholesterol levels with pitavastatin could reduce growth and migration and induced apoptosis in human lung tumor-associated ECs in vitro Hu et al.

In vivo experiments using lung cancer xenograft mice exhibited that pitavastatin-treatment could completely arrest tumor growth in these animals when combined with cisplatin and delayed tumor growth and impaired angiogenesis in cisplatin-resistant mouse models.

Another potential angiogenic target for cancer treatment is tie1. While the second tie receptor, tie2, is well characterized as a regulator during late stages of angiogenesis e. As tie1 is also upregulated in intratumoral vasculature, its deletion on ECs successfully produced a potent anti-angiogenic effect in different cancers Kaipainen et al.

In fact, EC-specific deletion of tie1 in lung carcinoma and melanoma mouse models resulted in delayed cancer growth, predominantly in late-stage tumors La Porta et al.

Furthermore, it inhibited neovessel sprouting and a reduced intratumoral vessel density, while the remaining mature vasculature became strongly normalized, which limited further metastatic formation. These findings, and the fact that tie1 expression is increased in angiogenic endothelium compared with resting vasculature, presents tie1 as a highly potent angiogenic target, especially in the treatment of advanced staged NSCLC.

Another considerable strategy of anti-angiogenic therapy could include targeting micro RNAs miRNAs as they represent a new paradigm in molecular cancer therapy.

The impact of miRNAs in post-transcriptional regulation has already been associated with pathways involved in cancer and vascular disease as summarized in Sun et al.

The following studies evaluated the potential role of specific angiogenesis-related miRNAs as targets in lung cancer. Hsu et al. observed that miRa, a micro RNA known to be hypoxia-associated, was overexpressed in exosomes of oxygen depleted CL lung cancer cells Hsu et al. Furthermore, these cancer-cell derived exosomes could induce angiogenesis via HIF-1α signaling in vitro when internalized by HUVECs.

Additionally, miRa transfection increased permeability and transendothelial migration of cancer cells in vitro by downregulation of the tight junction protein ZO-1 and stimulated neovascularization and tumor growth in vivo in CL xenograft mice, proposing it to be an appealing target for anti-angiogenic therapy.

Upregulation of miR in squamous lung cancer cells in vitro on the other hand could be associated with impaired VEGF expression and hampered migration and invasion, thereby facilitating a tumor-suppressive function.

Additionally, overexpression of miR in HUVECs was observed to inhibit tube formation and reduced the expression of VEGF, which hampered their angiogenesis activity in vitro Liu et al.

As it is an essential process during vessel growth, targeting ECM remodeling may also be an interesting approach to inhibit tumor angiogenesis in NSCLC. The most prominent enzymes involved in this process are matrix-metalloporoteinases MMPs which are inhibited under physiological conditions by tissue inhibitors of metalloproteinases TIMPs.

miRb could be identified as a promotor of MMP-2 activity and invasion of NSCLC cancer cells in vitro by downregulation of TIMP Additionally, it could be observed that miRb was significantly upregulated in tumor tissue of NSCLC patients with vascular cancer cell invasion Hirono et al.

According to these findings, targeting miRb could be a strategy to impede angiogenesis and cancer cell invasion in lung cancer. Uribesalgo et al. suggested targeting the apelin signaling pathway to inhibit tumor vessel formation in lung cancer Uribesalgo et al.

Apelin is a conserved peptide involved in developmental angiogenesis and is also upregulated in ECs within the TME. Previous studies could associate high apelin levels with a poor clinical outcome in patients with NSCLC Györffy et al.

In murine lung cancer models, apelin knockout reduced tumor burden and prolonged survival by inhibiting VEGF, TGF-β1, and TNF-α and simultaneously decreased MDSC infiltration in the TME Uribesalgo et al.

The combination of pharmacological inhibition of apelin with the anti-angiogenic drug sunitinib in lung cancer and mammary cancer mouse models, significantly delayed tumor growth and could almost double the survival, even in the KRAS driven or p53 mutated tumors, when compared with sunitinib treatment alone.

Finally, apelin loss also reduced vessel density and prevented sunitinib-induced hypoxia and poor vessel structure in the TME. Conclusively, apelin inhibition may provide a potent synergistic anti-tumor effect when combined with anti-angiogenic agents, while, and most importantly, avoiding therapy-induced hypoxia of the TME, thus decreasing the chance of metastases, and bypassing potential therapy resistances.

Single-target anti-angiogenic agents have already shown their limitations in clinical settings Jayson et al. Even in combination with other therapy approaches like standard chemotherapy or immune therapy, treatment success remains largely marginal.

Targeting several pro-angiogenic molecules with recombinant fusion proteins could therefore increase the anti-angiogenic effect of such therapies.

Zhang et al. When injected into lung cancer mouse models, autologous generated anti-peptibody antibodies inhibited tumor progression and angiogenesis and decreased expression of bFGF, VEGFA and PDGF in the tumor tissue. Targeting angiogenesis with fusion proteins exhibited potent anti-tumor efficacy in murine models and may represent a new approach for vessel inhibition in NSCLC, especially in combination with other therapy agents aimed at important angiogenic factors, previously discussed potential TEC specific markers or cellular mechanisms Table 2.

The instability of tumor vessels due to morphological abnormalities e. Although anti-angiogenic therapy can temporarily restore tissue perfusion and drug delivery by vascular normalization, treatment withdrawal often results in vessel hyper-permeability and can even induce a rebound effect of tumor angiogenesis Yang et al.

As continuous inhibition of angiogenesis remains difficult to implement for health or economic reasons, an alternative or more independent delivery system of anti-angiogenic agents could help to overcome these issues.

Nanomaterials have become an emerging field in cancer therapy in recent years, as their unique molecular properties make them suitable targeted drug delivery-systems. Physiochemically, these nanoparticles match the size of inter-endothelial junctions of blood vessels in the TME and therefore increase permeation and retention EPR resulting in a passive drug delivery Chauhan and Jain, Nanomaterials such as liposomes or nanotube carbon structures are used to deliver anti-angiogenic agents and improve drug specificity while reducing cytotoxic side effects, drug clearance and resistance mechanisms in the treatment of NSCLC Seshadri and Ramamurthi, In the past, studies using biodegradable polymers as nanocarriers to deliver chemotherapeutics and targeted drugs exhibited significant anti-tumor efficacy in vitro and in vivo.

For example, paclitaxel encapsulated aldehyde polyethylene glycol-polylactide PEG-PLGA conjugated to a VEGFR2-inhibiting peptide showed increased internalization in HUVECs in vitro as well as potent activity against breast cancer models in vivo Yu et al.

Although there are several peptide motifs that are suggested to target tumor endothelium such as RGD or NGR which can bind integrin heterodimers CD51 and CD61, or aminopeptidase N, respectively, their targeting with nanomaterial is not yet applied for treating NSCLC Sakurai et al.

Furthermore, non-angiogenic mechanisms such as VM or vessel co-option could also represent possible targets for nanomaterial-based therapy as the EPR effect of such molecules could help to overcome delivery and infiltration issues of traditional cancer therapeutics.

However, nanotherapeutics may provide a new potential anti-angiogenic therapeutical approach, but as already discussed, there is still a need for more specific biomarkers to exclusively target tumor vasculature in an organ specific manner. Taking this into consideration, chimeric antigen receptor CAR T-cell therapy, which serves as personalized immune therapy using autologous T-lymphocytes, engineered to target specific antigens present in a tumor, could be used to exclusively eliminate TECs without damaging healthy vasculature.

The therapy failure can, at least in part, be attributed to the impaired accessibility of the tumor mass due to dysfunctional vasculature and immunosuppressive conditions in the TME. Targeting tumor vessels directly with CAR T-cells could therefore be a good strategy to overcome these issues, which at best, can normalize the defective vasculature and improve drug efficacy in combinational therapy settings.

In a recent study Xie et al. Injected EIIIB-targeting CAR T-cells could delay tumor growth and improve survival in immunocompetent mouse models harboring aggressive melanoma, whereas colorectal cancer mouse models did not respond to the treatment.

Here, the expression levels of EIIIB in the different tissues had impact on the therapy outcome which again highlights the importance of organ specific vascular markers as well as the impact of organ specific angiogenic activity when targeting tumor vessel formation.

Other studies investigated the anti-angiogenic efficacy of TEM8-specific CAR T cells in solid cancer mouse models. TEM8 is one of the first discovered TEC markers and represents a promising target in anti-angiogenic therapy strategies St Croix et al.

In , a study reported that TEM8-specific CAR T-cells could improve survival and significantly decreased vascularization in triple negative breast cancer mouse models and induced tumor regression in mice with lung metastases Byrd et al. A more recent study, however, observed contrasting results where TEM8-sepcific CAR T-cells triggered high toxicity and induced inflammation in lung and spleen when injected into healthy mice Petrovic et al.

It is suggested that the engineered T-cells cross-reacted with other antigens or targeted TEM8 in healthy tissues, although it is normally expressed at a much lower quantity compared with pathological levels.

However, both processes resulted in severe toxicity in vivo and again emphasize the need for more adequate, highly specific tumor-vessel exclusive markers that can be targeted with either CAR T-cells or other previously discussed inhibiting molecules.

So far, the main obstacles of anti-angiogenic therapy in NSCLC are evading- or intrinsic resistance mechanisms which still remain elusive. We have discussed a wide array of possible therapies and therapy systems that could improve anti-angiogenic efficacy when combined with standard treatment.

The principal goal would be to expand the therapeutical effect of angiogenesis-inhibiting drugs on vessel normalization and render the tumor more vulnerable to additional agents such as chemotherapy or immunotherapy.

In a recent study, Hosaka et al. could show that dual angiogenesis inhibition could sensitize resistant off-target tumors to therapy.

Therefore they created mouse models of breast cancer or fibrosarcoma, both resistant to anti-VEGF and anti-PDGF treatment due to increased tumor associated expression of bFGF, a molecule which modulates the vasculature via pericyte recruitment in a PDGF-dependent process Hosaka et al.

Neither anti-VEGF nor anti-PDGF monotherapy had a significant anti-tumor effect on bFGF-positive tumors, but the combination of both agents produced a superior benefit, inhibiting cancer growth by suppressing proliferation and triggering apoptosis of tumor cells.

Interestingly, even the pan-blocking of FGF-receptors did not yield a comparable benefit. To explain this unexpected effect, angiogenesis has to be considered as an interacting network of various signaling pathways which cannot be disrupted by blocking a single molecule.

These findings demonstrate that the disruption of interacting angiogenic pathways by simultaneously targeting multiple angiogenic factors can provoke a highly potent anti-tumor effect which is able to circumvent mechanisms of therapy resistance, and thus should be considered as new approach to improve neovessel inhibition in cancer.

Angiogenesis is a main therapeutic concept in oncology, especially in NSCLC, where three approved agents are available in combination with chemotherapy or immunotherapy.

Increasing knowledge in angiogenic processes and non-angiogenic processes that contribute to tumor vascularization, provide precise targets for novel therapy strategies and pave the way for developing new anti-angiogenic treatment concepts that target e.

These therapeutic concepts need to be evaluated for synergistic effects as, in our view, modern anti-angiogenesis represents the concept of shaping the TME rather than being a direct anti-tumor therapy itself. However, these therapeutic strategies are very promising in preclinical setting and the translation into a clinical setting is not only warranted but highly desired.

Furthermore, a new horizon of targeted and functional TEC characterization was opened by scRNA-Seq studies, which proved that the tumor vasculature is highly heterogenous and differs from the normal adjacent vasculature more than primarily assumed in terms of metabolic activity, immune suppression and heterogeneity for example.

In addition, new synergistic effects of TECs in their role of immunomodulation were identified and induction of HEV formation for immune priming is suggested to be a new therapeutic strategy.

Next the organ specific context of the vasculature plays an important role and has to be further studied for better therapy allocation. In conclusion the concept and goal of anti-angiogenesis in NSCLC in the future can be reshaped by abolishing the traditional vessel priming concept and moving toward a side specific molding of the TME, using the tumor vasculature as a tool, like a trojan horse.

SD, HH, EN, AP, and DW developed the concept of the review. SD, HH, and EN drafted the review. DW and AP corrected and reviewed the review.

All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Adighibe, O. Is nonangiogenesis a novel pathway for cancer progression? A study using 3-dimensional tumour reconstructions. Cancer 94, — doi: PubMed Abstract CrossRef Full Text Google Scholar.

Ager, A. Understanding high endothelial venules: lessons for cancer immunology. Oncoimmunology 4:e Aguayo, A. Clinical relevance of Flt1 and Tie1 angiogenesis receptors expression in B-cell chronic lymphocytic leukemia CLL. Leukemia Res. CrossRef Full Text Google Scholar. Allen, E. Combined antiangiogenic and anti-PD-L1 therapy stimulates tumor immunity through HEV formation.

Alshangiti, A. Antiangiogenic therapies in non-small-cell lung cancer. Annan, D. Carbonic anhydrase 2 CAII supports tumor blood endothelial cell survival under lactic acidosis in the tumor microenvironment.

Cell Commun. Auf, G. Inositol-requiring enzyme 1alpha is a key regulator of angiogenesis and invasion in malignant glioma. Augustin, H. Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system. Cell Biol. Augustine, R. Therapeutic angiogenesis: from conventional approaches to recent nanotechnology-based interventions.

C Mater. Babina, I. Advances and challenges in targeting FGFR signalling in cancer. Cancer 17, — Basile, J. Semaphorin 4D provides a link between axon guidance processes and tumor-induced angiogenesis. Bergers, G. Tumorigenesis and the angiogenic switch. Cancer 3, — Modes of resistance to anti-angiogenic therapy.

Cancer 8, — Bertolini, F. The multifaceted circulating endothelial cell in cancer: towards marker and target identification. Cancer 6, — Betz, C. Cell behaviors and dynamics during angiogenesis. Development , — Bittner, M. Molecular classification of cutaneous malignant melanoma by gene expression profiling.

Nature , — de Bock, K. Role of PFKFB3-driven glycolysis in vessel sprouting. Cell , — Bridgeman, V. Vessel co-option is common in human lung metastases and mediates resistance to anti-angiogenic therapy in preclinical lung metastasis models.

Bruning, U. Impairment of Angiogenesis by Fatty Acid Synthase Inhibition Involves mTOR Malonylation. Cell Metab. Byrd, T. Cancer Res. Vascular reactions of mice to wounds and to normal and neoplastic transplants.

J Natl Cancer Inst — Article Google Scholar. Andrea TH, Frank L, Chad M, Hans-Peter G Identification and development of vascular disrupting agents: natural products that interfere with tumor growth. In: Natural products and cancer drug discovery, Cancer drug discovery and development.

Springer, New York, pp 17— Google Scholar. Baffert F, Le T, Sennino B, Thurston G, Kuo CJ, Hu-Lowe D, McDonald DM Cellular changes in normal blood capillaries undergoing regression after inhibition of VEGF signaling.

Am J Physiol Heart Circ Physiol 2 :H—H Baguley BC, Siemann DW Temporal aspects of the action of ASA vadimezan; DMXAA. Expert Opin Investig Drugs 19 11 — Article CAS PubMed PubMed Central Google Scholar. Baluk P, Hashizume H, McDonald DM Cellular abnormalities of blood vessels as targets in cancer.

Curr Opin Genet Dev 15 1 — SX 04 [pii]. Beauvais DM, Ell BJ, McWhorter AR, Rapraeger AC Syndecan-1 regulates alphavbeta3 and alphavbeta5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor.

J Exp Med 3 — Bosslet K, Straub R, Blumrich M, Czech J, Gerken M, Sperker B, Kroemer HK, Gesson JP, Koch M, Monneret C Elucidation of the mechanism enabling tumor selective prodrug monotherapy.

Cancer Res 58 6 — CAS PubMed Google Scholar. Brooks PC Cell adhesion molecules in angiogenesis. Cancer Metastasis Rev 15 2 — Bullitt E, Ewend MG, Aylward S, Lin W, Gerig G, Joshi S, Jung I, Muller K, Smith JK Abnormal vessel tortuosity as a marker of treatment response of malignant gliomas: preliminary report.

Technol Cancer Res Treat 3 6 — Article PubMed PubMed Central Google Scholar. Burge M, Francesconi AB, Kotasek D, Fida R, Smith G, Wilks A, Vasey PA, Lickliter JD Phase I, pharmacokinetic and pharmacodynamic evaluation of CYT, an orally-bioavailable cytotoxic and vascular-disrupting agent.

Investig New Drugs 31 1 — Article CAS Google Scholar. Burger JA, Peled A CXCR4 antagonists: targeting the microenvironment in leukemia and other cancers. Leukemia 23 1 — leu [pii]. Burrows FJ, Thorpe PE Eradication of large solid tumors in mice with an immunotoxin directed against tumor vasculature.

Proc Natl Acad Sci USA 90 19 — Byzova TV, Goldman CK, Pampori N, Thomas KA, Bett A, Shattil SJ, Plow EF A mechanism for modulation of cellular responses to VEGF: activation of the integrins.

Mol Cell 6 4 — doi:S 05 [pii]. Cesca M, Bizzaro F, Zucchetti M, Giavazzi R Tumor delivery of chemotherapy combined with inhibitors of angiogenesis and vascular targeting agents.

Front Oncol Chaplin DJ, Horsman MR, Siemann DW Current development status of small-molecule vascular disrupting agents. Curr Opin Investig Drugs 7 6 — Chen CS, Tan J, Tien J Mechanotransduction at cell-matrix and cell-cell contacts.

Annu Rev Biomed Eng — Chung F, Liu J, Ching LM, Baguley BC Consequences of increased vascular permeability induced by treatment of mice with 5,6-dimethylxanthenoneacetic acid DMXAA and thalidomide. Cancer Chemother Pharmacol 61 3 — Annu Rev Physiol — Article PubMed Google Scholar.

Dark GG, Hill SA, Prise VE, Tozer GM, Pettit GR, Chaplin DJ Combretastatin A-4, an agent that displays potent and selective toxicity toward tumor vasculature. Cancer Res 57 10 — De Bock K, De Smet F, Leite De Oliveira R, Anthonis K, Carmeliet P Endothelial oxygen sensors regulate tumor vessel abnormalization by instructing phalanx endothelial cells.

J Mol Med Berl 87 6 — Proc Natl Acad Sci USA 21 — DeMali KA, Wennerberg K, Burridge K Integrin signaling to the actin cytoskeleton. Curr Opin Cell Biol 15 5 — doi:S [pii].

Denekamp J, Hill SA, Hobson B Vascular occlusion and tumour cell death. Eur J Cancer Clin Oncol 19 2 — Denekamp J, Hobson B Endothelial-cell proliferation in experimental tumours.

Br J Cancer 46 5 — Dickson PV, Hamner JB, Sims TL, Fraga CH, Ng CY, Rajasekeran S, Hagedorn NL, McCarville MB, Stewart CF, Davidoff AM Bevacizumab-induced transient remodeling of the vasculature in neuroblastoma xenografts results in improved delivery and efficacy of systemically administered chemotherapy.

Clin Cancer Res 13 13 — Dome B, Timar J, Dobos J, Meszaros L, Raso E, Paku S, Kenessey I, Ostoros G, Magyar M, Ladanyi A, Bogos K, Tovari J Identification and clinical significance of circulating endothelial progenitor cells in human non-small cell lung cancer.

Cancer Res 66 14 — Drake CJ, Cheresh DA, Little CD An antagonist of integrin alpha v beta 3 prevents maturation of blood vessels during embryonic neovascularization. J Cell Sci Pt 7 — Eberhard A, Kahlert S, Goede V, Hemmerlein B, Plate KH, Augustin HG Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies.

Cancer Res 60 5 — Farace F, Massard C, Borghi E, Bidart JM, Soria JC Vascular disrupting therapy-induced mobilization of circulating endothelial progenitor cells. Ann Oncol 18 8 — Ferrara N Molecular and biological properties of vascular endothelial growth factor.

J Mol Med Berl 77 7 — Ferrara N Pathways mediating VEGF-independent tumor angiogenesis. Cytokine Growth Factor Rev 21 1 — Folkman J, Long DM Jr, Becker FF Growth and metastasis of tumor in organ culture.

Cancer — Franco M, Man S, Chen L, Emmenegger U, Shaked Y, Cheung AM, Brown AS, Hicklin DJ, Foster FS, Kerbel RS 1. Cancer Res 66 7 — Friedlander M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA, Cheresh DA Definition of two angiogenic pathways by distinct alpha v integrins. Science — Gan HK, Seruga B, Knox JJ Sunitinib in solid tumors.

Expert Opin Investig Drugs 18 6 — Gazit Y, Baish JW, Safabakhsh N, Leunig M, Baxter LT, Jain RK Fractal characteristics of tumor vascular architecture during tumor growth and regression. Microcirculation 4 4 — Grosios K, Loadman PM, Swaine DJ, Pettit GR, Bibby MC Combination chemotherapy with combretastatin A-4 phosphate and 5-fluorouracil in an experimental murine colon adenocarcinoma.

Anticancer Res 20 1A — Gutheil JC, Campbell TN, Pierce PR, Watkins JD, Huse WD, Bodkin DJ, Cheresh DA Targeted antiangiogenic therapy for cancer using Vitaxin: a humanized monoclonal antibody to the integrin alphavbeta3.

Clin Cancer Res 6 8 — Hamzah J, Jugold M, Kiessling F, Rigby P, Manzur M, Marti HH, Rabie T, Kaden S, Grone HJ, Hammerling GJ, Arnold B, Ganss R Vascular normalization in Rgs5-deficient tumours promotes immune destruction.

Nature — nature [pii]. Hanahan D, Weinberg RA Hallmarks of cancer: the next generation. Cell 5 — S 11 [pii]. Hartmann JT, Haap M, Kopp HG, Lipp HP Tyrosine kinase inhibitors — a review on pharmacology, metabolism and side effects.

Curr Drug Metab 10 5 — Hasani A, Leighl N Classification and toxicities of vascular disrupting agents. Clin Lung Cancer 12 1 — Hellberg C, Ostman A, Heldin CH PDGF and vessel maturation. Recent Results Cancer Res — Hey T, Fiedler E, Rudolph R, Fiedler M Artificial, non-antibody binding proteins for pharmaceutical and industrial applications.

Trends Biotechnol 23 10 — S 05 [pii]. Hicklin DJ, Ellis LM Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 23 5 — Hirsch E, Brancaccio M, Altruda F Tissue-specific KO of ECM proteins.

Methods Mol Biol — Huang FJ, You WK, Bonaldo P, Seyfried TN, Pasquale EB, Stallcup WB Pericyte deficiencies lead to aberrant tumor vascularizaton in the brain of the NG2 null mouse. Dev Biol 2 — S 10 [pii]. Huang X, Molema G, King S, Watkins L, Edgington TS, Thorpe PE Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature.

Hwang R, Varner J The role of integrins in tumor angiogenesis. Hematol Oncol Clin North Am 18 5 — S 04 [pii]. Hynes RO The emergence of integrins: a personal and historical perspective. Matrix Biol 23 6 — Ide AG, Baker NH, Warren SL Vascularization of the Brown-Pearce rabbit epithelioma transplant as seen in the transparent chamber.

Am J Radiol — Inai T, Mancuso M, Hashizume H, Baffert F, Haskell A, Baluk P, Hu-Lowe DD, Shalinsky DR, Thurston G, Yancopoulos GD, McDonald DM Inhibition of vascular endothelial growth factor VEGF signaling in cancer causes loss of endothelial fenestrations, regression of tumor vessels, and appearance of basement membrane ghosts.

Am J Pathol 1 — Innocenti F, Ramirez J, Obel J, Xiong J, Mirkov S, Chiu YL, Katz DA, Carr RA, Zhang W, Das S, Adjei A, Moyer AM, Chen PX, Krivoshik A, Medina D, Gordon GB, Ratain MJ, Sahelijo L, Weinshilboum RM, Fleming GF, Bhathena A Preclinical discovery of candidate genes to guide pharmacogenetics during phase I development: the example of the novel anticancer agent ABT Pharmacogenet Genomics 23 7 — Jain RK Determinants of tumor blood flow: a review.

Cancer Res 48 10 — Jain RK Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy. Nat Med 7 9 — nm [pii]. Jain RK Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy.

Mol Cancer Res 10 8 — MCR [pii]. Jordan MA, Wilson L Microtubules as a target for anticancer drugs. Nat Rev Cancer 4 4 — nrc [pii]. Jung YD, Ahmad SA, Liu W, Reinmuth N, Parikh A, Stoeltzing O, Fan F, Ellis LM The role of the microenvironment and intercellular cross-talk in tumor angiogenesis.

Semin Cancer Biol 12 2 — SX [pii]. Kalluri R Basement membranes: structure, assembly and role in tumour angiogenesis.

Nat Rev Cancer 3 6 — Kamba T, McDonald DM Mechanisms of adverse effects of anti-VEGF therapy for cancer. Br J Cancer 96 12 — Kashiwagi S, Tsukada K, Xu L, Miyazaki J, Kozin SV, Tyrrell JA, Sessa WC, Gerweck LE, Jain RK, Fukumura D Perivascular nitric oxide gradients normalize tumor vasculature.

Nat Med 14 3 — Kelly RJ, Darnell C, Rixe O Target inhibition in antiangiogenic therapy a wide spectrum of selectivity and specificity. Cancer J 16 6 — Kerbel R, Folkman J Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2 10 — Kienast Y, von Baumgarten L, Fuhrmann M, Klinkert WE, Goldbrunner R, Herms J, Winkler F Real-time imaging reveals the single steps of brain metastasis formation.

Nat Med 16 1 — Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, Ferrara N Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo.

Kim S, Harris M, Varner JA Regulation of integrin alpha vbeta 3-mediated endothelial cell migration and angiogenesis by integrin alpha5beta1 and protein kinase a.

J Biol Chem 43 — M [pii]. Kraling BM, Razon MJ, Boon LM, Zurakowski D, Seachord C, Darveau RP, Mulliken JB, Corless CL, Bischoff J E-selectin is present in proliferating endothelial cells in human hemangiomas. Am J Pathol 4 — CAS PubMed PubMed Central Google Scholar. Krause DS, Van Etten RA Tyrosine kinases as targets for cancer therapy.

N Engl J Med 2 — Lippert JW 3rd Vascular disrupting agents. Bioorg Med Chem 15 2 — S 06 [pii]. Lorusso PM, Boerner SA, Hunsberger S Clinical development of vascular disrupting agents: what lessons can we learn from ASA? J Clin Oncol 29 22 — Lyden D, Hattori K, Dias S, Costa C, Blaikie P, Butros L, Chadburn A, Heissig B, Marks W, Witte L, Wu Y, Hicklin D, Zhu Z, Hackett NR, Crystal RG, Moore MA, Hajjar KA, Manova K, Benezra R, Rafii S Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.

Nat Med 7 11 — Marneros AG, Olsen BR Physiological role of collagen XVIII and endostatin. FASEB J 19 7 — Masson V, de la Ballina LR, Munaut C, Wielockx B, Jost M, Maillard C, Blacher S, Bajou K, Itoh T, Itohara S, Werb Z, Libert C, Foidart JM, Noel A Contribution of host MMP-2 and MMP-9 to promote tumor vascularization and invasion of malignant keratinocytes.

FASEB J 19 2 — Matsumura T, Wolff K, Petzelbauer P Endothelial cell tube formation depends on cadherin 5 and CD31 interactions with filamentous actin. J Immunol 7 — Matsusaka S, Mishima Y, Suenaga M, Terui Y, Kuniyoshi R, Mizunuma N, Hatake K Circulating endothelial progenitors and CXCR4-positive circulating endothelial cells are predictive markers for bevacizumab.

Cancer 17 — Mazzone M, Dettori D, Leite de Oliveira R, Loges S, Schmidt T, Jonckx B, Tian YM, Lanahan AA, Pollard P, Ruiz de Almodovar C, De Smet F, Vinckier S, Aragones J, Debackere K, Luttun A, Wyns S, Jordan B, Pisacane A, Gallez B, Lampugnani MG, Dejana E, Simons M, Ratcliffe P, Maxwell P, Carmeliet P Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization.

McCarthy AD, Uemura T, Etcheverry SB, Cortizo AM Advanced glycation endproducts interefere with integrin-mediated osteoblastic attachment to a type-I collagen matrix. Int J Biochem Cell Biol 36 5 — S [pii]. Mitrus I, Sochanik A, Cichon T, Szala S Combination of combretastatin A4 phosphate and doxorubicin-containing liposomes affects growth of BF10 tumors.

Acta Biochim Pol 56 1 — doi [pii]. Moccia F, Zuccolo E, Poletto V, Cinelli M, Bonetti E, Guerra G, Rosti V Endothelial progenitor cells support tumour growth and metastatisation: implications for the resistance to anti-angiogenic therapy. Tumour Biol 36 9 — Blood 91 12 — Moore MA Putting the neo into neoangiogenesis.

J Clin Invest 3 — Morikawa S, Baluk P, Kaidoh T, Haskell A, Jain RK, McDonald DM Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors. Am J Pathol 3 — Mott JD, Werb Z Regulation of matrix biology by matrix metalloproteinases.

Curr Opin Cell Biol 16 5 — Mould AP, Humphries MJ Regulation of integrin function through conformational complexity: not simply a knee-jerk reaction? Nagy JA, Chang SH, Shih SC, Dvorak AM, Dvorak HF Heterogeneity of the tumor vasculature.

Semin Thromb Hemost 36 3 — Nelson CM, Chen CS VE-cadherin simultaneously stimulates and inhibits cell proliferation by altering cytoskeletal structure and tension.

J Cell Sci Pt 17 — Noel A, Maillard C, Rocks N, Jost M, Chabottaux V, Sounni NE, Maquoi E, Cataldo D, Foidart JM Membrane associated proteases and their inhibitors in tumour angiogenesis. J Clin Pathol 57 6 — Nowak K, Rafat N, Belle S, Weiss C, Hanusch C, Hohenberger P, Beck G Circulating endothelial progenitor cells are increased in human lung cancer and correlate with stage of disease.

Eur J Cardiothorac Surg 37 4 — Ono M, Kosaka N, Tominaga N, Yoshioka Y, Takeshita F, Takahashi RU, Yoshida M, Tsuda H, Tamura K, Ochiya T Exosomes from bone marrow mesenchymal stem cells contain a microRNA that promotes dormancy in metastatic breast cancer cells.

Sci Signal 7 :ra Article PubMed CAS Google Scholar. Blood 95 3 — Petitclerc E, Boutaud A, Prestayko A, Xu J, Sado Y, Ninomiya Y, Sarras MP Jr, Hudson BG, Brooks PC New functions for non-collagenous domains of human collagen type IV.

Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo.

In the following part we summarize various levels of tumor vascularization that may represent new targets for vessel inhibition in NSCLC.

All mentioned mechanisms are summarized in Figure 1. Figure 1. Mechanisms of tumor vascularization in NSCLC.

Tumor vascularization in lung cancer can be promoted by various processes which overlap during cancer progression. TECs exhibit upregulated metabolism to enable high angiogenic activity which includes processes involved in proliferation cholesterol synthesis and glycolysis and processes that enable migration via ECM remodeling collagen synthesis.

Potential targets involved in these pathways SQLE, PFKFB3, and ALDH18A1, respectively are considered to increase the angiogenic potential of TECs in NSCLC.

Hypoxia and acidosis induced by high levels of lactate due to upregulated glycolysis constitute to a highly pro-angiogenic tumor environment. Angiogenesis stimulating factors VEGF, bFGF, PDGF, HIF-1α, tryptase, and MMPs are released by both, cancer cells and stromal cells, including fibroblasts, pericytes, tumor associated macrophages and ECs.

Non-angiogenic processes constitute to tumor vascularization and are inaccessible for anti-angiogenic agents, thus contributing to therapy resistance.

VM comprises the formation of tubular structures arising from cancer cells that gain endothelial like properties to maintain vascular supply during cancer progression. Another mechanism of cancer cells to persist in circulation is to grow along existing vasculature, which is referred to as vessel co-option.

In this figure we summarized the various mechanism of tumor vascularization that should be considered when targeting the inhibition of tumor vessels in NSCLC. The endothelium is postulated to be a large contributor to the therapeutic efficacy of anti-angiogenic therapies, and therefore represents a possible source of therapy response or failure.

It is well known that the process of angiogenesis is comprised of different EC phenotypes which execute distinct functions. During the elongation of the sprouting vessel VEGF-sensitive tip ECs migrate into avascular tissue regions, thus leading the proliferating trailing stalk ECs, which built up the growing vessel.

Newly formed vasculature finally adapts a mature and quiescent phenotype referred to as phalanx ECs Carmeliet and Jain, ; Betz et al. The EC phenotypes involved are highly dynamic and can reprogram the gene expression to meet their current physiological requirements.

However, the tumor endothelium was not studied in depth and a recent single-cell RNA sequencing scRNA-Seq study identified even more EC phenotypes from both healthy and tumor tissue from lung cancer samples as already known, indicating a much more complex phenotypic heterogeneity of the tumor vasculature than initially presumed Goveia et al.

Interestingly, although phenotype proportions differed strongly between analyzed NSCLC patients, they collectively observed a low abundance of tip and proliferating TECs, which represent the main targets of traditional anti-angiogenic therapy.

Furthermore, they identified a so-far-unknown tumor exclusive phenotype of activated postcapillary vein EC that upregulated features known from HEVs in inflamed tissues such as immunomodulatory factors and ribosomal proteins. The unexpected finding that activated and proliferating TECs only represent a minority of the pathological EC phenotypes found in NSCLC, allows us to reconsider currently used anti-angiogenic therapy as less of a vessel-inhibiting strategy, and more of a strategy to modulate the higher proportion of mature TECs into potent participants of tumor surveillance.

In order to develop new angiogenesis-inhibiting therapies, the molecular differences between physiological and pathological ECs will need to be elaborated. Genetically TEC and NEC phenotypes significantly differ in gene expression affecting diverse cellular mechanisms such as proliferation, migration, inflammation, and angiogenesis Figure 2.

Previous studies have shown that one key feature of TECs is a highly active metabolism, which permits pathological processes as increased proliferation and angiogenesis Cantelmo et al. Hyperglycolytic TECs subsequently release high amounts of lactate into the environment, which in turn, further stimulates EC proliferation and angiogenesis Annan et al.

It could be demonstrated that inhibition of PFKFB3 resulted in improved drug efficacy and decreased metastatic events in tumor mouse models Cantelmo et al. Another study in xenograft NSCLC mouse models exhibited that PFKFB3 mRNA silencing in combination with docetaxel results in a chemoenhancing effect and increases anti-cancer efficacy compared with monotherapies alone Chowdhury et al.

Furthermore, to sustain upregulated proliferative capacity, TECs exhibit elevated nucleotide biosynthesis including upstream pathways that are involved in serine and lipid synthesis Cantelmo et al.

In addition, Lambrechts et al. Interestingly, c-MYC expression induces angiogenesis in combination with HIF-1α and VEGF Lee and Wu, and recruits tryptase positive mast cells into the tumor niche Soucek et al. Figure 2. The multifaced picture of TECs in NSCLC.

TECs possess features that enable continuous angiogenic activity for progressing vascularization of the tumor. These features are ensured by genetical changes in the tumor endothelium that are triggered by diverse stimuli of the TME e. The stroma, consisting of various cells, promote angiogenesis by directly releasing signaling molecules into the adjacent tissue, thereby stimulating TECs.

Fibroblasts and myeloid derived suppressor cells MDSCs activate angiogenesis by releasing VEGF and bFGF into the TME.

Additionally, CSF-1 molecules, expressed by cancer cells, further recruit MDSCs into the tumor niche. Tumor associated macrophages TAMs can directly induce angiogenesis by releasing VEGF, bFGF, and PlGF, or indirectly by releasing matrix metalloproteinases MMPs which promote endothelial migration.

Mast cells secrete tryptase TRYPT into the TME which stimulates EC proliferation and enables ECM remodeling. Furthermore, to facilitate enhanced angiogenesis, TECs upregulate the surface expression of angiogenic receptors as well as increase metabolic activity including energy and amino acid metabolism and the biosynthesis of nucleotides.

In addition to the high angiogenic activity, TECs can directly suppress inflammatory responses by downregulation of inflammatory cytokines for immune cell recruitment CCL2, CCL8, and IL-6 , receptors required for immune cell homing ICAM or lymphocyte activation MHC I and MHC II which results in impaired immune cell trafficking and migration into the TME.

In summary the complex interaction of tumor-protecting environmental conditions and the pathological features of TECs lead to a pro-angiogenic and immune suppressive TME in NSCLC.

Focusing on endothelial metabolism in cancer, a recent study could identify at least two metabolic signatures which are highly upregulated in angiogenic endothelium and TECs.

One for proliferation, which includes gene sets associated with biomass production e. These results educed two new possible metabolic targets to hamper tumor angiogenesis; aldehyde dehydrogenase 18 family member A1 ALDH18A1 , an enzyme essential for de novo biosynthesis of proline; and squalene epoxidase SQLE , the rate-limiting enzyme in cholesterol biosynthesis.

Silencing of ALDH18A as well as SQLE impaired EC proliferation, migration and vessel sprouting in in vitro assays. Summarized, targeting endothelial metabolism in cancer is an interesting therapeutic option that could possibly assist an anti-angiogenic approach for treating NSCLC.

Another key feature of TECs in lung cancer is the downregulation of inflammatory responses thus contributing to tumor-associated immune escape. Single-cell analysis of NSCLC samples identified the most downregulated genes of the tumor endothelium in connection to inflammation, which included CCL2, CCL18, and IL6, essential for immune cell recruitment; MHC I and II, essential for immune cell activation; and ICAM, required for immune cell homing Lambrechts et al.

As the endothelium represents the primary connection between the immune system and tumor cells, these results indicate the important role of TECs in immunomodulatory processes that hamper anti-tumor immunity.

Vessel normalization not only improves immune cell activation and infiltration, but is also suggested to enhance drug delivery to the tumor sites, thus improving its efficacy Allen et al. Additionally, combinational therapy of angiogenesis inhibitors and immunotherapy anti-PD-L1 in previous studies could elicit the formation of unique blood vessels in treated tumors that resemble HEVs typically found in lymphoid tissues, which implicated increased treatment efficacy Allen et al.

HEVs can mediate immune cell adhesion and migration into the tumor, which may be important for bypassing TEC-induced immune escape Ager and May, In the already discussed scRNA-Seq study by Goveia et al. These remarkable observations indicate that TECs comprise the ability to transform into HEVs to promote immune cell infiltration into the tumor and induce a potent anti-tumor response.

This extends the previous observations of favorable synergistic effects of immune therapy in combination with angiogenesis inhibitors in NSCLC, especially when it results in HEV formation.

Furthermore, direct induction of HEV formation could be a promising new strategy in anti-angiogenic approaches that may attain great clinical importance. However, currently there are no reliable biomarkers to track the process of vessel normalization or HEV formation in NSCLC which could help to predict and optimize this new treatment strategy.

As mentioned above, in some cases tumor vascularization can be facilitated by non-ECs which adapt certain properties to sustain access to the circulation, which may support anti-angiogenic drug resistance.

During tumor progression, processes that lead to vascularization of the malignant tissue can vary locally as well as temporarily and involve angiogenic as well as non-angiogenic mechanisms even in the same lesion Bridgeman et al.

In lung tumors, where non-angiogenic tumor growth occurs most commonly, previous studies primarily located non-angiogenic processes in the tumor periphery, whereas angiogenesis is typically localized in the hypoxic tumor core Pezzella et al. Here, we briefly discuss the impact of non-angiogenic processes in NSCLC on anti-angiogenic drug efficacy based on previous studies.

VEGF-A inhibition using bevacizumab failed to inhibit VM in breast cancer cells in vitro , furthermore, sunitinib, a multi targeting anti-VEGFR inhibitor, even promoted VM in breast cancer mouse models Dey et al. Additionally it could be demonstrated that VM in NSCLC depends on expression of Sema4D and its receptor plexinB1 which activate RhoA and downstream ROCK, comprising an already known angiogenesis-promoting process in tumors Basile et al.

Although the role of VM in NSCLC is not fully understood, previous observations suggest that it may contribute to anti-angiogenic therapy failure and may serve as an option to treat aggressive lung tumors.

Vessel co-option on the other hand is a common phenomenon especially observed in lung metastases when tumor cells start to invade perivascular tissues Jensen, Anti-angiogenic therapy with sunitinib could induce a switch from angiogenic vessel formation to vessel co-option in a lung metastatic mouse model, which ultimately resulted in sunitinib resistance Bridgeman et al.

Unfortunately, regulative mechanisms of vessel co-option in human tumors remain unknown in large part, however, predicting the occurrence of either VM or vessel co-option could be a useful tactic to prevent anti-angiogenic drug resistance in some patients. According to these and other results, it could be confirmed that non-angiogenic tumors contribute to anti-angiogenic therapy resistance which reveals the undoubted importance of targeting both angiogenic, but also non-angiogenic vessel growth to treat NSCLC Donnem et al.

Increasing knowledge of the physiological processes of tumor vascularization in addition to traditional angiogenesis has enlightened a variety of adaptive mechanisms which can promote anti-angiogenic therapy resistances.

This awareness fortifies the necessity for alternative anti-angiogenic agents besides traditional anti-VEGF therapy. As previously examined, tumor angiogenesis depends on upregulated metabolic activity e.

Cholesterol not only represents a fundamental structural component of cell membranes and serves as precursor for several steroid hormones, it is also crucial for membrane function and angiogenic signaling, making it a favorable target for tumor vessel inhibition Lyu et al.

Inhibition of intracellular cholesterol trafficking with anti-inflammatory drug chepharantine was shown to hamper angiogenesis and tumor growth in lung cancer xenograft mice while improving anti-tumor activity of standard chemotherapeutics Lyu et al. Another study has shown that pharmacological lowering of intracellular cholesterol levels with pitavastatin could reduce growth and migration and induced apoptosis in human lung tumor-associated ECs in vitro Hu et al.

In vivo experiments using lung cancer xenograft mice exhibited that pitavastatin-treatment could completely arrest tumor growth in these animals when combined with cisplatin and delayed tumor growth and impaired angiogenesis in cisplatin-resistant mouse models.

Another potential angiogenic target for cancer treatment is tie1. While the second tie receptor, tie2, is well characterized as a regulator during late stages of angiogenesis e.

As tie1 is also upregulated in intratumoral vasculature, its deletion on ECs successfully produced a potent anti-angiogenic effect in different cancers Kaipainen et al. In fact, EC-specific deletion of tie1 in lung carcinoma and melanoma mouse models resulted in delayed cancer growth, predominantly in late-stage tumors La Porta et al.

Furthermore, it inhibited neovessel sprouting and a reduced intratumoral vessel density, while the remaining mature vasculature became strongly normalized, which limited further metastatic formation.

These findings, and the fact that tie1 expression is increased in angiogenic endothelium compared with resting vasculature, presents tie1 as a highly potent angiogenic target, especially in the treatment of advanced staged NSCLC.

Another considerable strategy of anti-angiogenic therapy could include targeting micro RNAs miRNAs as they represent a new paradigm in molecular cancer therapy. The impact of miRNAs in post-transcriptional regulation has already been associated with pathways involved in cancer and vascular disease as summarized in Sun et al.

The following studies evaluated the potential role of specific angiogenesis-related miRNAs as targets in lung cancer. Hsu et al. observed that miRa, a micro RNA known to be hypoxia-associated, was overexpressed in exosomes of oxygen depleted CL lung cancer cells Hsu et al. Furthermore, these cancer-cell derived exosomes could induce angiogenesis via HIF-1α signaling in vitro when internalized by HUVECs.

Additionally, miRa transfection increased permeability and transendothelial migration of cancer cells in vitro by downregulation of the tight junction protein ZO-1 and stimulated neovascularization and tumor growth in vivo in CL xenograft mice, proposing it to be an appealing target for anti-angiogenic therapy.

Upregulation of miR in squamous lung cancer cells in vitro on the other hand could be associated with impaired VEGF expression and hampered migration and invasion, thereby facilitating a tumor-suppressive function.

Additionally, overexpression of miR in HUVECs was observed to inhibit tube formation and reduced the expression of VEGF, which hampered their angiogenesis activity in vitro Liu et al. As it is an essential process during vessel growth, targeting ECM remodeling may also be an interesting approach to inhibit tumor angiogenesis in NSCLC.

The most prominent enzymes involved in this process are matrix-metalloporoteinases MMPs which are inhibited under physiological conditions by tissue inhibitors of metalloproteinases TIMPs.

miRb could be identified as a promotor of MMP-2 activity and invasion of NSCLC cancer cells in vitro by downregulation of TIMP Additionally, it could be observed that miRb was significantly upregulated in tumor tissue of NSCLC patients with vascular cancer cell invasion Hirono et al.

According to these findings, targeting miRb could be a strategy to impede angiogenesis and cancer cell invasion in lung cancer. Uribesalgo et al.

suggested targeting the apelin signaling pathway to inhibit tumor vessel formation in lung cancer Uribesalgo et al. Apelin is a conserved peptide involved in developmental angiogenesis and is also upregulated in ECs within the TME.

Previous studies could associate high apelin levels with a poor clinical outcome in patients with NSCLC Györffy et al. In murine lung cancer models, apelin knockout reduced tumor burden and prolonged survival by inhibiting VEGF, TGF-β1, and TNF-α and simultaneously decreased MDSC infiltration in the TME Uribesalgo et al.

The combination of pharmacological inhibition of apelin with the anti-angiogenic drug sunitinib in lung cancer and mammary cancer mouse models, significantly delayed tumor growth and could almost double the survival, even in the KRAS driven or p53 mutated tumors, when compared with sunitinib treatment alone.

Finally, apelin loss also reduced vessel density and prevented sunitinib-induced hypoxia and poor vessel structure in the TME. Conclusively, apelin inhibition may provide a potent synergistic anti-tumor effect when combined with anti-angiogenic agents, while, and most importantly, avoiding therapy-induced hypoxia of the TME, thus decreasing the chance of metastases, and bypassing potential therapy resistances.

Single-target anti-angiogenic agents have already shown their limitations in clinical settings Jayson et al. Even in combination with other therapy approaches like standard chemotherapy or immune therapy, treatment success remains largely marginal.

Targeting several pro-angiogenic molecules with recombinant fusion proteins could therefore increase the anti-angiogenic effect of such therapies.

Zhang et al. When injected into lung cancer mouse models, autologous generated anti-peptibody antibodies inhibited tumor progression and angiogenesis and decreased expression of bFGF, VEGFA and PDGF in the tumor tissue. Targeting angiogenesis with fusion proteins exhibited potent anti-tumor efficacy in murine models and may represent a new approach for vessel inhibition in NSCLC, especially in combination with other therapy agents aimed at important angiogenic factors, previously discussed potential TEC specific markers or cellular mechanisms Table 2.

The instability of tumor vessels due to morphological abnormalities e. Although anti-angiogenic therapy can temporarily restore tissue perfusion and drug delivery by vascular normalization, treatment withdrawal often results in vessel hyper-permeability and can even induce a rebound effect of tumor angiogenesis Yang et al.

As continuous inhibition of angiogenesis remains difficult to implement for health or economic reasons, an alternative or more independent delivery system of anti-angiogenic agents could help to overcome these issues. Nanomaterials have become an emerging field in cancer therapy in recent years, as their unique molecular properties make them suitable targeted drug delivery-systems.

Physiochemically, these nanoparticles match the size of inter-endothelial junctions of blood vessels in the TME and therefore increase permeation and retention EPR resulting in a passive drug delivery Chauhan and Jain, Nanomaterials such as liposomes or nanotube carbon structures are used to deliver anti-angiogenic agents and improve drug specificity while reducing cytotoxic side effects, drug clearance and resistance mechanisms in the treatment of NSCLC Seshadri and Ramamurthi, In the past, studies using biodegradable polymers as nanocarriers to deliver chemotherapeutics and targeted drugs exhibited significant anti-tumor efficacy in vitro and in vivo.

For example, paclitaxel encapsulated aldehyde polyethylene glycol-polylactide PEG-PLGA conjugated to a VEGFR2-inhibiting peptide showed increased internalization in HUVECs in vitro as well as potent activity against breast cancer models in vivo Yu et al.

Although there are several peptide motifs that are suggested to target tumor endothelium such as RGD or NGR which can bind integrin heterodimers CD51 and CD61, or aminopeptidase N, respectively, their targeting with nanomaterial is not yet applied for treating NSCLC Sakurai et al.

Furthermore, non-angiogenic mechanisms such as VM or vessel co-option could also represent possible targets for nanomaterial-based therapy as the EPR effect of such molecules could help to overcome delivery and infiltration issues of traditional cancer therapeutics.

However, nanotherapeutics may provide a new potential anti-angiogenic therapeutical approach, but as already discussed, there is still a need for more specific biomarkers to exclusively target tumor vasculature in an organ specific manner.

Taking this into consideration, chimeric antigen receptor CAR T-cell therapy, which serves as personalized immune therapy using autologous T-lymphocytes, engineered to target specific antigens present in a tumor, could be used to exclusively eliminate TECs without damaging healthy vasculature.

The therapy failure can, at least in part, be attributed to the impaired accessibility of the tumor mass due to dysfunctional vasculature and immunosuppressive conditions in the TME. Targeting tumor vessels directly with CAR T-cells could therefore be a good strategy to overcome these issues, which at best, can normalize the defective vasculature and improve drug efficacy in combinational therapy settings.

In a recent study Xie et al. Injected EIIIB-targeting CAR T-cells could delay tumor growth and improve survival in immunocompetent mouse models harboring aggressive melanoma, whereas colorectal cancer mouse models did not respond to the treatment.

Here, the expression levels of EIIIB in the different tissues had impact on the therapy outcome which again highlights the importance of organ specific vascular markers as well as the impact of organ specific angiogenic activity when targeting tumor vessel formation.

Other studies investigated the anti-angiogenic efficacy of TEM8-specific CAR T cells in solid cancer mouse models. TEM8 is one of the first discovered TEC markers and represents a promising target in anti-angiogenic therapy strategies St Croix et al.

In , a study reported that TEM8-specific CAR T-cells could improve survival and significantly decreased vascularization in triple negative breast cancer mouse models and induced tumor regression in mice with lung metastases Byrd et al.

A more recent study, however, observed contrasting results where TEM8-sepcific CAR T-cells triggered high toxicity and induced inflammation in lung and spleen when injected into healthy mice Petrovic et al. It is suggested that the engineered T-cells cross-reacted with other antigens or targeted TEM8 in healthy tissues, although it is normally expressed at a much lower quantity compared with pathological levels.

However, both processes resulted in severe toxicity in vivo and again emphasize the need for more adequate, highly specific tumor-vessel exclusive markers that can be targeted with either CAR T-cells or other previously discussed inhibiting molecules.

So far, the main obstacles of anti-angiogenic therapy in NSCLC are evading- or intrinsic resistance mechanisms which still remain elusive.

We have discussed a wide array of possible therapies and therapy systems that could improve anti-angiogenic efficacy when combined with standard treatment. The principal goal would be to expand the therapeutical effect of angiogenesis-inhibiting drugs on vessel normalization and render the tumor more vulnerable to additional agents such as chemotherapy or immunotherapy.

In a recent study, Hosaka et al. could show that dual angiogenesis inhibition could sensitize resistant off-target tumors to therapy. Therefore they created mouse models of breast cancer or fibrosarcoma, both resistant to anti-VEGF and anti-PDGF treatment due to increased tumor associated expression of bFGF, a molecule which modulates the vasculature via pericyte recruitment in a PDGF-dependent process Hosaka et al.

Neither anti-VEGF nor anti-PDGF monotherapy had a significant anti-tumor effect on bFGF-positive tumors, but the combination of both agents produced a superior benefit, inhibiting cancer growth by suppressing proliferation and triggering apoptosis of tumor cells.

Interestingly, even the pan-blocking of FGF-receptors did not yield a comparable benefit. To explain this unexpected effect, angiogenesis has to be considered as an interacting network of various signaling pathways which cannot be disrupted by blocking a single molecule.

These findings demonstrate that the disruption of interacting angiogenic pathways by simultaneously targeting multiple angiogenic factors can provoke a highly potent anti-tumor effect which is able to circumvent mechanisms of therapy resistance, and thus should be considered as new approach to improve neovessel inhibition in cancer.

Angiogenesis is a main therapeutic concept in oncology, especially in NSCLC, where three approved agents are available in combination with chemotherapy or immunotherapy. Increasing knowledge in angiogenic processes and non-angiogenic processes that contribute to tumor vascularization, provide precise targets for novel therapy strategies and pave the way for developing new anti-angiogenic treatment concepts that target e.

These therapeutic concepts need to be evaluated for synergistic effects as, in our view, modern anti-angiogenesis represents the concept of shaping the TME rather than being a direct anti-tumor therapy itself.

However, these therapeutic strategies are very promising in preclinical setting and the translation into a clinical setting is not only warranted but highly desired. Furthermore, a new horizon of targeted and functional TEC characterization was opened by scRNA-Seq studies, which proved that the tumor vasculature is highly heterogenous and differs from the normal adjacent vasculature more than primarily assumed in terms of metabolic activity, immune suppression and heterogeneity for example.

In addition, new synergistic effects of TECs in their role of immunomodulation were identified and induction of HEV formation for immune priming is suggested to be a new therapeutic strategy.

Next the organ specific context of the vasculature plays an important role and has to be further studied for better therapy allocation. In conclusion the concept and goal of anti-angiogenesis in NSCLC in the future can be reshaped by abolishing the traditional vessel priming concept and moving toward a side specific molding of the TME, using the tumor vasculature as a tool, like a trojan horse.

SD, HH, EN, AP, and DW developed the concept of the review. SD, HH, and EN drafted the review. DW and AP corrected and reviewed the review. All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Adighibe, O. Is nonangiogenesis a novel pathway for cancer progression? A study using 3-dimensional tumour reconstructions. Cancer 94, — doi: PubMed Abstract CrossRef Full Text Google Scholar.

Ager, A. Understanding high endothelial venules: lessons for cancer immunology. Oncoimmunology 4:e Aguayo, A. Clinical relevance of Flt1 and Tie1 angiogenesis receptors expression in B-cell chronic lymphocytic leukemia CLL.

Leukemia Res. CrossRef Full Text Google Scholar. Allen, E. Combined antiangiogenic and anti-PD-L1 therapy stimulates tumor immunity through HEV formation.

Alshangiti, A. Antiangiogenic therapies in non-small-cell lung cancer. Annan, D. Carbonic anhydrase 2 CAII supports tumor blood endothelial cell survival under lactic acidosis in the tumor microenvironment.

Cell Commun. Auf, G. Inositol-requiring enzyme 1alpha is a key regulator of angiogenesis and invasion in malignant glioma. Augustin, H. Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system. Cell Biol. Augustine, R. Therapeutic angiogenesis: from conventional approaches to recent nanotechnology-based interventions.

C Mater. Babina, I. Advances and challenges in targeting FGFR signalling in cancer. Cancer 17, — Basile, J. Semaphorin 4D provides a link between axon guidance processes and tumor-induced angiogenesis.

Bergers, G. Tumorigenesis and the angiogenic switch. Cancer 3, — Modes of resistance to anti-angiogenic therapy. Cancer 8, — Bertolini, F. The multifaceted circulating endothelial cell in cancer: towards marker and target identification.

Cancer 6, — Betz, C. Cell behaviors and dynamics during angiogenesis. Development , — Bittner, M. Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature , — de Bock, K. Role of PFKFB3-driven glycolysis in vessel sprouting.

Cell , — Bridgeman, V. Vessel co-option is common in human lung metastases and mediates resistance to anti-angiogenic therapy in preclinical lung metastasis models.

Bruning, U. Impairment of Angiogenesis by Fatty Acid Synthase Inhibition Involves mTOR Malonylation. Cell Metab. Byrd, T. Cancer Res. Cantelmo, A. Inhibition of the Glycolytic Activator PFKFB3 in Endothelium Induces Tumor Vessel Normalization.

Impairs Metastasis, and Improves Chemotherapy. Cancer Cell 30, — Carlini, M. Mast cell phenotypes and microvessels in non-small cell lung cancer and its prognostic significance. Carmeliet, P. Molecular mechanisms and clinical applications of angiogenesis.

Caspani, E. Glioblastoma: a pathogenic crosstalk between tumor cells and pericytes. PLoS One 9:e Chauhan, V. Strategies for advancing cancer nanomedicine. Chowdhury, N. Coelho, A. Angiogenesis in NSCLC: is vessel co-option the trunk that sustains the branches?

Oncotarget 8, — Colegio, O. Functional polarization of tumour-associated macrophages by tumour-derived lactic acid. Crawford, Y. PDGF-C mediates the angiogenic and tumorigenic properties of fibroblasts associated with tumors refractory to anti-VEGF treatment.

Cancer Cell 15, 21— Crinò, L. Safety and efficacy of first-line bevacizumab-based therapy in advanced non-squamous non-small-cell lung cancer SAiL, MO : a phase 4 study.

Lancet Oncol. Crohns, M. Cytokines in bronchoalveolar lavage fluid and serum of lung cancer patients during radiotherapy - Association of interleukin-8 and VEGF with survival. Cytokine 50, 30— Delgado-Bellido, D. Vasculogenic mimicry signaling revisited: focus on non-vascular VE-cadherin.

Cancer Dey, N. Evading anti-angiogenic therapy: resistance to anti-angiogenic therapy in solid tumors. Google Scholar.

Döme, B. Alternative vascularization mechanisms in cancer: pathology and therapeutic implications. Donnem, T. Vessel co-option in primary human tumors and metastases: an obstacle to effective anti-angiogenic treatment?

Cancer Med. Non-angiogenic tumours and their influence on cancer biology. Cancer 18, — Dowlati, A. Cell adhesion molecules, vascular endothelial growth factor, and basic fibroblast growth factor in patients with non-small cell lung cancer treated with chemotherapy with or without bevacizumab—an Eastern Cooperative Oncology Group Study.

Escudier, B. Sorafenib in advanced clear-cell renal-cell carcinoma. New Eng. Fridman, W. The immune contexture in human tumours: impact on clinical outcome. Cancer 12, — Fujio, Y. Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner.

Garon, E. Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy REVEL : a multicentre, double-blind, randomised phase 3 trial. Lancet , — Gerber, H. Góth, M.

Physiological and pathological angiogenesis in the endocrine system. Microscopy Res. Goveia, J. An integrated gene expression landscape profiling approach to identify lung tumor endothelial cell heterogeneity and angiogenic candidates.

Cancer Cell 37, Gridelli, C. Safety and efficacy of bevacizumab plus standard-of-care treatment beyond disease progression in patients with advanced non-small cell lung cancer: the AvaALL randomized clinical trial. JAMA Oncol. Györffy, B. An online survival analysis tool to rapidly assess the effect of 22, genes on breast cancer prognosis using microarray data of 1, patients.

Breast Cancer Res Treatment , — Hall, R. Angiogenesis inhibition as a therapeutic strategy in non-small cell lung cancer NSCLC. Lung Cancer Res. Han, B. Effect of anlotinib as a third-line or further treatment on overall survival of patients with advanced non-small cell lung cancer: the alter phase 3 randomized clinical trial.

Anlotinib as a third-line therapy in patients with refractory advanced non-small-cell lung cancer: a multicentre, randomised phase II trial ALTER Cancer , — Hanahan, D. Patterns and Emerging Mechanisms of the Angiogenic Switch during Tumorigenesis. Cell 86, — Hallmarks of cancer: the next generation.

Herbst, R. Hida, K. Tumor angiogenesis—characteristics of tumor endothelial cells. Hirono, T. MicroRNAb functions as an oncomiRNA in non-small cell lung cancer by targeting tissue inhibitor of metalloproteinase Holash, J. New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF.

Oncogene 18, — Hosaka, K. Therapeutic paradigm of dual targeting VEGF and PDGF for effectively treating FGF-2 off-target tumors. Hsu, Y. Hypoxic lung cancer-secreted exosomal miRa increased angiogenesis and vascular permeability by targeting prolyl hydroxylase and tight junction protein ZO Oncogene 36, — Hu, T.

Anti Cancer Drugs 31, — Huang, B. SCF-mediated mast cell infiltration and activation exacerbate the inflammation and immunosuppression in tumor microenvironment. Blood , — Ibaraki, T.

The relationship of tryptase- and chymase-positive mast cells to angiogenesis in stage I non-small cell lung cancer. Janning, M. Anti-Angiogenics: their Value in Lung Cancer Therapy.

Jayson, G. Antiangiogenic therapy in oncology: current status and future directions. Jensen, L. When tumors are co- opting to resist anti-angiogenic treatment. Johnson, D. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer.

Clin Oncol 22, — Kaipainen, A. Enhanced expression of the tie receptor tyrosine kinase mesenger RNA in the vascular endothelium of metastatic melanomas. Kessenbrock, K. Matrix metalloproteinases: regulators of the tumor microenvironment.

Cell , 52— Kim, M. Opposing actions of angiopoietin-2 on Tie2 signaling and FOXO1 activation. Korhonen, E. Tie1 controls angiopoietin function in vascular remodeling and inflammation. Kut, C. Where is VEGF in the body? A meta-analysis of VEGF distribution in cancer. Cancer 97, — La Porta, S. Endothelial Tie1-mediated angiogenesis and vascular abnormalization promote tumor progression and metastasis.

Lambrechts, D. Accordingly, MMPdeficient MDSCs fail to induce tumor angiogenesis 46 , Third, unlike other immune cells, some MDSCs can differentiate into EC-like cells.

These EC-like MDSCs express endothelial markers, such as CD31 and VEGFR2, and have the ability to integrate into the tumor vasculature 45 , 46 , Adaptive immune cells are also critical players in the orchestration of tumor angiogenesis by directly affecting EC biology and indirectly modulating myeloid cell phenotypes.

IFN-γ directly inhibits the proliferation and migration of human endothelial cells and secretes IFN-inducible protein 10 IP and monokine induced by IFN-γ MIG. These cytokines also react with CXCR3, restraining the proliferation of endothelial cells and tumor vascularization 74 , Furthermore, IFN-γ signaling downregulates VEGF-A but upregulates CXCL9, CXCL10, and CXCL11, which collectively stimulate vascular maturation by enhancing pericyte recruitment along ECs 74 , 77 , Another important aspect of IFN-γ in tumor angiogenesis is the reprogramming of TAMs from M2- to M1-like TAMs.

T H 1 cells also polarize M2-like TAMs to M1-like TAMs and induce DC maturation in the TME, which suppresses tumor angiogenesis 82 , T H 2 cells expressing IL-4, IL-5, and IL recruit M2-like TAMs through STAT-6 activation and promote tumor angiogenesis 41 , 50 , 77 , The expression of IL by T H 17 correlates with the infiltration of ECs and abnormal tumor vasculature 41 , 77 , 85 , Tumor-infiltrating Treg cells also play a critical role by sustaining angiogenesis directly through VEGF secretion and supporting endothelial cell recruitment and expansion 83 , Furthermore, Tregs promote angiogenesis indirectly by restraining the activity of T H 1 cells and by triggering the activation of M2-like macrophages In ovarian cancer, hypoxia results in CCL28 upregulation, leading to a robust increase in Treg infiltration, VEGF and blood vessels, whereas depletion of Tregs reduces intratumoral VEGF levels and tumor angiogenesis 18 , The interactions between tumor immunity and angiogenesis suggest that tumor vascular remodeling could enhance the efficacy of cancer immunotherapy.

Emerging preclinical evidence demonstrates the potential of combining immunotherapy with vascular-targeting treatment 24 , 37 , 75 , 88 , 89 , 90 , Allen et al. demonstrated that anti-angiogenic therapy with anti-VEGFR2 enhances the efficacy of anti-PD-L1 immunotherapy in pancreatic neuroendocrine tumor RT2-PNET , mammary carcinoma MMTV-PyMT , and glioblastoma NFppGBM models Furthermore, the combination of anti-angiogenic and immunotherapy increased pericyte coverage and normalized tumor vessels, promoting intratumoral infiltration of activated T cells.

In addition to vascular normalization, the vessel phenotype represents the characteristics of high endothelial venules HEVs , which are morphologically thickened with plump endothelial cells ECs and functionally more specialized in lymphocyte extravasation than other tumor ECs.

Notably, the LTβR signaling pathway is involved in the generation of intratumoral HEVs after combined treatment with anti-VEGFR2 and anti-PD-L1. Therefore, these results suggest that anti-angiogenic therapy could improve the efficacy of cancer immunotherapy and overcome resistance to cancer immunotherapy via tumor vessel normalization and intratumoral HEV formation.

Shigeta et al. also reported consistent synergism of anti-VEGFR2 and anti-PD-L1 in hepatocellular carcinoma HCC They observed that anti-VEGFR2 therapy upregulates PD-L1 expression under hypoxic conditions, mediated in part by IFN-γ secreted by ECs. Dual combination therapy has also been shown to improve overall survival OS and anti-cancer immunity with increased intratumoral accumulation of CTLs and M1-like TAMs.

Collectively, combination therapy with anti-VEGFR2 and anti-PD-1 reprograms the immune microenvironment via vessel normalization, further strengthening the anti-cancer immune response and overcoming resistance to cancer immunotherapy in HCC.

Anti-angiogenic therapy can also overcome resistance to anti-PD-1 by abolishing the TOX-mediated T-cell exhaustion program in the TME Kim et al. revealed that VEGF significantly upregulates the transcription factor TOX, which influences the phenotype and function of CTLs. The TOX-mediated transcriptional program resulted in severe T-cell exhaustion and upregulated inhibitory immune checkpoint receptors such as PD-1, TIM-3, LAG-3, and TIGIT and reduced the proliferation of cytokine production by CTLs.

Combination treatment with anti-VEGFR2 and anti-PD-1 enhanced the immunotherapeutic efficacy and T-cell reinvigoration. Collectively, combinatory treatment with anti-angiogenic agents and ICIs is a potential therapeutic option in anti-PDresistant cancer.

Schmittnaegel et al. demonstrated that combined blockade of VEGF-A and ANGPT2 by a bispecific antibody A2V enhanced the therapeutic activity compared with either anti-VEGF-A or anti-ANGPT2 monotherapy alone in both genetically engineered and transplant tumor models A2V effectively inhibited tumor angiogenesis but promoted vascular maturation in the TME.

This negative feedback mechanism was successfully overcome by combining A2V with anti-PD-1, leading to better immunotherapeutic efficacy. These results encourage further testing of combining ICIs with various anti-angiogenic targets other than VEGF in advanced cancers.

Recently, a novel immunotherapeutic target, simulator of IFN genes STING , was reported to be involved in the regulation of the tumor vasculature and demonstrated synergism with anti-VEGFR2 and ICIs Yang et al. revealed that intratumoral STING signaling activation suppresses tumor angiogenesis and induces vessel normalization through type I IFN signaling activation and the upregulation of genes related to vascular normalization and endothelial-lymphocyte interaction.

STING agonist combined with anti-VEGFR2 synergistically enhanced vascular normalization, leading to durable anti-cancer immunity. Therefore, these data suggest that combining novel therapeutics with the combination of anti-angiogenic agents and ICIs could help overcome resistance to anti-angiogenic and immunotherapy in refractory cancers.

On the other hand, immune checkpoint blockade, such as anti-CTLA-4 or anti-PD-1, increases vascular perfusion to improve therapeutic efficacy.

Zheng et al. Notably, IVP can distinguish tumors that are sensitive to ICIs from those that are resistant. In addition, IVP was time-dependently induced by anti-CTLA-4 even before tumor regression was detectable. Collectively, these findings indicate that IVP could be a prerequisite of ICI to improve anti-cancer immunity, thereby enabling it to be used as a predictive indicator for ICI efficacy.

Preclinical studies continue to yield encouraging results regarding the synergistic effects of ICIs and anti-angiogenic agent combination therapy, which have led to clinical investigations to reproduce these results in patients with advanced cancer 92 , 93 , 94 , 95 , 96 , 97 , Several pivotal clinical trials have already demonstrated the superiority of combining anti-angiogenic agents and ICIs in various malignancies.

The most successful results of combination therapy have been reported in renal cell carcinoma RCC and hepatocellular carcinoma HCC. RCC is a highly immunogenic tumor that has been treated with high-dose IL-2 in some patients. Immunotherapy has recently been revisited and reevaluated when phase 3 clinical trials demonstrated that nivolumab anti-PD-1 treatment leads to longer OS with significantly lower toxicity.

In KEYNOTE, patients with previously untreated metastatic RCC were treated with either pembrolizumab anti-PD-1 and axitinib VEGFR1, 2, and 3 inhibitor combination therapy or sunitinib monotherapy, and significantly increased progression-free survival PFS was demonstrated in the combination group compared with the sunitinib group Although the incidence of hepatic toxicity was higher in the combination group, no relevant death event occurred.

Based on the significant efficacy and acceptable toxicity profile, combination therapy with pembrolizumab and axitinib was approved by the FDA for treatment-naïve patients with metastatic RCC.

JAVELIN Renal NCT is a phase 3 clinical trial that evaluated the efficacy of avelumab anti-PD-L1 and axitinib combination therapy against sunitinib monotherapy in patients with metastatic RCC in a first-line setting Although the data are premature for OS analysis and require further follow-up, the median PFS of the combination group has already been reported to be In addition, the ORR and complete response rate were Based on this study, the FDA approved avelumab for use in combination with axitinib as first-line treatment for patients with advanced RCC.

In HCC, two highly anticipated phase III studies testing PD-1 inhibitor monotherapy failed to meet their primary endpoints, leading to doubts regarding the use of ICIs in this cancer.

However, a randomized phase III clinical trial, IMBRAVE NCT , demonstrated significant improvements in co-primary end points, PFS and OS, using the combination of atezolizumab anti-PD-L1 and bevacizumab anti-VEGF-A compared with sorafenib This was the first study to propose a new first-line treatment option that is superior to sorafenib, which has been the standard of care for a decade.

The FDA granted the Breakthrough Therapy designation based on these data, and the phase III IMBRAVE trial was initiated. At the ESMO Asia Congress, the median OS with the atezolizumab and bevacizumab combination was not reached until analysis when compared with In terms of patient-reported outcomes, the combination group exhibited delayed deterioration of quality of life compared with sorafenib.

The safety and efficacy of the combination of pembrolizumab and lenvatinib were evaluated in patients with unresectable HCC in KEYNOTE, a multicenter, open-label, single-arm phase Ib study This clinical trial also yielded a promising response rate during the early stage and was granted Breakthrough Therapy designation by the FDA, initiating LEPP, a phase 3 trial to evaluate pembrolizumab in combination with lenvatinib as a potential first-line treatment for patients with advanced HCC In non-squamous non-small cell lung cancer NSCLC , a phase 3 clinical trial Impower, NCT comparing atezolizumab anti-PD-L1 , bevacizumab anti-VEGF , carboplatin, and paclitaxel combination therapy ABCP group against bevacizumab, carboplatin, and paclitaxel combination therapy BCP group showed significantly extended PFS and OS in the ABCP group compared with the BCP group median PFS: 8.

The ORR was significantly higher in the ABCP group than in the BCP group ORR: Based on these results, atezolizumab was approved by the FDA for use in combination with bevacizumab, paclitaxel, and carboplatin as first-line treatment for patients with metastatic non-squamous NSCLC.

Recently, the FDA granted accelerated approval for the use of a combination of pembrolizumab and lenvatinib in patients with advanced endometrial cancer who have experienced disease progression after systemic therapy.

In this trial, patients who had previously been treated for metastatic endometrial cancer were evaluated for their response to lenvatinib and pembrolizumab. Interim analysis showed that the ORR was However, immune-mediated AEs, including endocrine, gastrointestinal, hepatic, skin, pulmonary, and renal events, occurred in In several years, these ongoing trials are expected to generate consistent results, which will evolve the therapeutic landscape of advanced cancers.

Years of research have demonstrated the potential of ICI monotherapy as well as its limitations, which have led to further attempts to overcome these limitations by combination immunotherapy. Of the potential candidates, the combination of ICI and anti-angiogenic agents continues to yield promising results in both preclinical and clinical studies, not only highlighting that it is one of the most effective combination immunotherapy regimens thus far but also changing the treatment landscape for RCC and HCC.

Nonetheless, several issues remain to optimize the efficacy of this combination therapy. First, predictive biomarkers must be developed to identify the subset of patients who will benefit from this combination treatment.

Third, whether the effects of this combination are synergistic or merely additive must be evaluated. Finally, the angiogenic phenotype differs according to organ; thus, more in-depth analyses must be performed to further our knowledge of the response to ICI treatment at the organ level.

Ribas, A. Cancer immunotherapy using checkpoint blockade. Science , — CAS PubMed PubMed Central Google Scholar. Fukumura, D. Enhancing cancer immunotherapy using antiangiogenics: opportunities and challenges.

Khan, K. Improving immunotherapy outcomes with anti-angiogenic treatments and vice versa. CAS PubMed Google Scholar. Yi, M. et al. Synergistic effect of immune checkpoint blockade and anti-angiogenesis in cancer treatment.

Cancer 18 , 60 PubMed PubMed Central Google Scholar. Huang, Y. Improving immune-vascular crosstalk for cancer immunotherapy. Rahma, O. The intersection between tumor angiogenesis and immune suppression.

Cancer Res. De Palma, M. Microenvironmental regulation of tumour angiogenesis. Cancer 17 , — PubMed Google Scholar. Xia, A. T Cell Dysfunction in Cancer Immunity and Immunotherapy. Barsoum, I. A mechanism of hypoxia-mediated escape from adaptive immunity in cancer cells. Chon, H.

Tumor microenvironment remodeling by intratumoral oncolytic vaccinia virus enhances the efficacy of immune-checkpoint blockade.

Kim, C. Vascular RhoJ is an effective and selective target for tumor angiogenesis and vascular disruption. Cancer Cell 25 , — Park, J. Normalization of tumor vessels by Tie2 activation and Ang2 inhibition enhances drug delivery and produces a favorable tumor microenvironment. Cancer Cell 30 , — Lee, J.

Novel glycosylated VEGF decoy receptor fusion protein, VEGF-Grab, efficiently suppresses tumor angiogenesis and progression. Cancer Ther. Jain, R. Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia. Cancer Cell 26 , — Kim, Y. Methylation-dependent regulation of HIF-1alpha stability restricts retinal and tumour angiogenesis.

Schaaf, M. Defining the role of the tumor vasculature in antitumor immunity and immunotherapy. Cell Death Dis. Ramjiawan, R. Anti-angiogenesis for cancer revisited: Is there a role for combinations with immunotherapy?

Angiogenesis 20 , — Facciabene, A. Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and T reg cells. Nature , — Baluk, P. Cellular abnormalities of blood vessels as targets in cancer.

Lugano, R. Tumor angiogenesis: causes, consequences, challenges and opportunities. Cell Mol. Life Sci. Article PubMed PubMed Central Google Scholar. Motz, G. Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors. Gabrilovich, D. Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells.

Oyama, T. Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-kappa B activation in hemopoietic progenitor cells.

VEGF-A drives TOX-dependent T cell exhaustion in anti-PDresistant microsatellite stable colorectal cancers. Khan, O. Gerald, D. Angiopoietin an attractive target for improved antiangiogenic tumor therapy. Goumans, M. TGF-beta signaling in vascular biology and dysfunction. Cell Res. Tian, M.

Transforming growth factor-beta and the hallmarks of cancer. Cell Signal 23 , — De Falco, S. The discovery of placenta growth factor and its biological activity.

Odorisio, T. Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability. Cell Sci. Brodsky, S. Vascular density and VEGF expression in hepatic lesions. Gastrointestin Liver Dis.

Tumeh, P. Liver metastasis and treatment outcome with Anti-PD-1 monoclonal antibody in patients with melanoma and NSCLC. Cancer Immunol. Nishino, M. Immune-related response assessment during PD-1 inhibitor therapy in advanced non-small-cell lung cancer patients. Cancer 4 , 84 Paez-Ribes, M.

Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis.

Cancer Cell 15 , — Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science , 58—62 Martin, J.

Normalizing function of tumor vessels: progress, opportunities, and challenges. Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy. Natl Acad. USA , — Jung, K. Ly6Clo monocytes drive immunosuppression and confer resistance to anti-VEGFR2 cancer therapy.

Targeting CXCR4-dependent immunosuppressive Ly6C low monocytes improves antiangiogenic therapy in colorectal cancer. Rivera, L. Intratumoral myeloid cells regulate responsiveness and resistance to antiangiogenic therapy.

Cell Rep. Bruno, A. Orchestration of angiogenesis by immune cells. Biswas, S. Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Stockmann, C.

The impact of the immune system on tumor: angiogenesis and vascular remodeling. Intertwined regulation of angiogenesis and immunity by myeloid cells. Trends Immunol. Albini, A. Contribution to tumor angiogenesis from innate immune cells within the tumor microenvironment: implications for Immunotherapy.

Google Scholar. Murdoch, C. The role of myeloid cells in the promotion of tumour angiogenesis. Cancer 8 , — Qian, B. Macrophage diversity enhances tumor progression and metastasis. Cell , 39—51 Chen, P. Role of macrophage polarization in tumor angiogenesis and vessel normalization: implications for new anticancer therapies.

Watkins, S. IL rapidly alters the functional profile of tumor-associated and tumor-infiltrating macrophages in vitro and in vivo. Mantovani, A. Macrophage plasticity and polarization in tissue repair and remodelling. Hughes, R. Perivascular M2 macrophages stimulate tumor relapse after chemotherapy.

Cancer Res 75 , — Deletion of vascular endothelial growth factor in myeloid cells accelerates tumorigenesis. Reusser, N. Clodronate inhibits tumor angiogenesis in mouse models of ovarian cancer.

Cancer Biol. Zeisberger, S. Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach.

Cancer 95 , — Tumor-targeted interferon-alpha delivery by Tie2-expressing monocytes inhibits tumor growth and metastasis.

Cancer Cell 14 , — Tie2-expressing monocytes: regulation of tumor angiogenesis and therapeutic implications. Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors.

Cancer Cell 8 , — Mazzieri, R. Cancer Cell 19 , — Sozzani, S. Dendritic cell-endothelial cell cross-talk in angiogenesis. Colonna, M. Plasmacytoid dendritic cells in immunity.

Liu, K. Origin and development of dendritic cells. Piqueras, B. Upon viral exposure, myeloid and plasmacytoid dendritic cells produce 3 waves of distinct chemokines to recruit immune effectors. Blood , — Trinchieri, G.

Interleukin and the regulation of innate resistance and adaptive immunity. Curiel, T. Dendritic cell subsets differentially regulate angiogenesis in human ovarian cancer.

Asselin-Paturel, C. Production of type I interferons: plasmacytoid dendritic cells and beyond. Indraccolo, S. Differential effects of angiostatin, endostatin and interferon-alpha 1 gene transfer on in vivo growth of human breast cancer cells. Gene Ther. Okunishi, K. A novel role of hepatocyte growth factor as an immune regulator through suppressing dendritic cell function.

Conejo-Garcia, J. Tumor-infiltrating dendritic cell precursors recruited by a beta-defensin contribute to vasculogenesis under the influence of Vegf-A. Dikov, M. Vascular endothelial growth factor effects on nuclear factor-kappaB activation in hematopoietic progenitor cells.

Sinha, P. Cross-talk between myeloid-derived suppressor cells and macrophages subverts tumor immunity toward a type 2 response. LeCouter, J. Bv8 and endocrine gland-derived vascular endothelial growth factor stimulate hematopoiesis and hematopoietic cell mobilization.

Shojaei, F. Bv8 regulates myeloid-cell-dependent tumour angiogenesis. Yang, L. Cancer Cell 6 , — Kammertoens, T. Tumour ischaemia by interferon-gamma resembles physiological blood vessel regression.

Nature , 98— Yang, H. STING activation reprograms tumor vasculatures and synergizes with VEGFR2 blockade. Fathallah-Shaykh, H. Gene transfer of IFN-gamma into established brain tumors represses growth by antiangiogenesis. Bromley, S. Orchestrating the orchestrators: chemokines in control of T cell traffic.

Freedman, R. Clinical and biological effects of intraperitoneal injections of recombinant interferon-gamma and recombinant interleukin 2 with or without tumor-infiltrating lymphocytes in patients with ovarian or peritoneal carcinoma.

Baer, C. Suppression of microRNA activity amplifies IFN-gamma-induced macrophage activation and promotes anti-tumour immunity. Cell Biol. Tian, L. Mutual regulation of tumour vessel normalization and immunostimulatory reprogramming.

Balkwill, F. The tumor microenvironment at a glance. Heusinkveld, M. The parallel lives of angiogenesis and immunosuppression: cancer and other tales.

DeNardo, D. Cancer Cell 16 , 91— Liu, J. IL is associated with poor prognosis and promotes angiogenesis via stimulating VEGF production of cancer cells in colorectal carcinoma.

Both the median PFS and OS were longer in the pazopanib group compared with placebo arm PFS 4. The phase III AXIS study indicated that the median PFS was longer in the axitinib group compared to the sorafenib group 6. According to the update results of the AXIS trial, no difference of OS was found between these two arms [ 44 ].

The most commonly treatment-related side effects were diarrhea, hypertension, and fatigue, and the incidence of hypertension appears higher in axitinib than other TKIs [ 45 ]. It had demonstrated clinical effectiveness in patients with metastatic colorectal cancer mCRC who had progress after prior standard therapy and approved by the FDA in Two large randomized phase III trials, CORRECT and CONCUR, demonstrated that regorafenib prolonged the median OS and PFS in mCRC patients, and the drug-related adverse events were manageable [ 47 , 48 ].

Based on the results of phase III GRID clinical trial [ 49 ], the FDA expanded the indication of regorafenib to patients with advanced GIST following the failure of imatinib and sunitinib in In this study, regorafenib improved the PFS to 4.

Recently, a large phase III clinical trial RESORCE laid the foundation of the approved of regorafenib in HCC [ 50 ]. In RESORCE trial, patients who failed or tolerated sorafenib were enrolled, and the results demonstrated that regorafenib significantly prolonged OS 10·6 vs.

The most common AEs were hand-foot skin reaction, diarrhea, and fatigue. Based on the result of RESORCE trial, the FDA approved the regorafenib for the second line HCC [ 51 ].

The approval of cabozantinib by the FDA in November for metastatic medullary thyroid cancer MTC was based on a phase III trial EXAM [ 53 ]. In this study, patients with progressive metastatic MTC were randomly assigned to cabozantinib arm or placebo arm.

The study reached its primary end point by indicating an improvement of PFS in cabozantinib arm The most common AEs were diarrhea and palmar-plantar erthyrodysesthesia syndrome [ 54 ]. Cabozantinib also demonstrated its antitumor efficacy compared with everolimus in mRCC who had progressed after VEGFR-targeted therapy.

The pivotal phase III METEOR trial had leaded the approval by the FDA for advanced mRCC as second-line treatment [ 55 , 56 ]. According to the result, the median PFS was 7.

More importantly, the final updated results show a significant improvement in OS with cabozantinib More importantly, cabozantinib was expected to become a novel systemic therapy for patients with metastatic hepatocellular carcinoma mHCC based on the positive result of CELESTIAL trial [ 57 ].

The study showed that treatment with cabozantinib resulted in longer OS Encouragingly, cabozantinib garnered FDA approval for mHCC patients after the failure of sorafenib in January In the same year, nintedanib combined with docetaxel therapy was approved for non-squamous non-small cell lung cancer NSCLC patients as a second-line treatment by the European Medicines Agency EMA but not by the FDA [ 62 ].

In a pivotal randomized, double-blind phase III trial LUME-Lung 1 , PFS was improved in the docetaxel plus nintedanib group in relative to placebo plus docetaxel 3.

AEs were more common in the docetaxel plus nintedanib group compared with the docetaxel plus placebo group with diarrhea, decreased neutrophils, and fatigue. Subsequently, Hanna et al.

conducted a phase III trial, LUME-Lung 2 study, comparing the efficacy of nintedanib plus pemetrexed in patients with NSCLC exclusively [ 64 ].

Although the clinical trial was stopped prematurely, a PFS advantage was observed in the nintedanib plus pemetrexed group in the subsequent analysis.

However, the subgroup analysis in adenocarcinoma histology showed no discrepancy between two arms, which was inconsistent with the findings in LUME-Lung 1 study. The real value of nintedanib in the second-line therapy of advanced NSCLC remained unclear. It was first approved for patients with advanced RAI refractory DTC by the FDA based on the results of the SELECT study [ 66 , 67 ].

In this study, lenvatinib significantly prolonged the PFS Considering the dramatic efficacy and manageable side effects, the FDA expedited the approval of lenvatinib for RAI-refractory DTCs [ 65 ].

Encouragingly, it also showed anti-neoplastic activity in other solid tumors, including advanced RCC and HCC. Lenvatinib gained the FDA approval for the treatment of metastatic RCC based on the positive result of a phase II clinical trial [ 68 ].

The study was designed to assess whether the combination of lenvatinib plus everolimus was superior compared to the single agents. The primary end point of PFS in the combination group was significantly longer than everolimus alone Based on these results, lenvatinib in combination with everolimus was recommended as a second-line systemic therapy in mRCC.

Lenvatinib garnered the FDA approval for HCC in based on a series of clinical trials, among which a global phase III trial REFLECT was the most important [ 69 ].

This study aimed to assess the efficacy of lenvatinib vs. sorafenib as a first-line treatment for patients with unresectable HCC. The result was notable that the OS in lenvatinib group was non-inferior to the sorafenib group Lenvatinib also showed a greater ORR compared with the sorafenib group The past decade has also witnessed the great progress in the development of anti-tumor drugs developed by Chinese researchers.

Apatinib can simultaneously suppress the kinase activities of VEGFR-2, c-Kit, and c-Src and is approved by the CFDA for the treatment of advanced gastric cancer GC in October [ 71 , 72 ].

The efficacy and safety profile of apatinib in patients with metastatic gastric or gastroesophageal junction adenocarcinoma who had failed at least two lines of chemotherapy was evaluated in a series of clinical trials.

A phase III randomized clinical trial, conducted by Li and collaborators, has indicated its important role in three or more lines for GC patients [ 73 ]. The primary end points of OS and PFS were significantly prolonged by apatinib OS 6. Though the ORR showed no difference between two groups, the disease control rate DCR favored apatinib over placebo treatment.

The major treatment-related grade 3—4 AEs in apatinib arm included hand-foot syndrome, proteinuria, and hypertension [ 73 ]. The ALTER trial demonstrated 3. The results showed that both OS 9. Anlotinib also produced significant ORR and DCR benefits vs. placebo and had a manageable safety profile [ 76 , 77 ].

It was approved by the CFDA as a third-line or further therapy for advanced NSCLC patients in Until now, apatinib and anlotinib have not gained the approval of the FDA, but both of them were identified as orphan drugs in the USA.

In the phase II clinical trials, fruquintinib showed a significant PFS benefit in patients with treatment-refractory mCRC [ 79 ].

Then, a randomized, double bind, phase III FRESCO trial conducted by Li et al. laid the foundation for the approval of this drug on patients with mCRC by the CFDA in [ 80 ]. In this study, mCRC patients who had progressed after at least two lines of chemotherapy were allocated to receive either fruquintinib or placebo.

The primary end point median of OS was significantly longer in the fruquintinib group compared to placebo 9. Moreover, higher ORR and DCR were observed in patients receiving fruquintinib with a manageable safety profile.

Additionally, a phase I clinical trial is ongoing in the USA, exploring the efficacy and safety in non-Chinese populations [ 81 ]. While the approved anti-angiogenic TKIs are trying to expand their indication in other cancer types, numerous new anti-angiogenic TKIs are also being extensively explored.

Three representative TKI drugs with potential to be approved in the near future are presented. Motesanib was considered as a potent anti-tumor drug in Asian advanced NSCLC patients based on the subgroup analysis of MONET1 trial [ 83 ]. However, the results of later phase III trial MONETA were disappointing with no advantage in patients receiving motesanib plus paclitaxel and carboplatin over placebo plus paclitaxel and carboplatin [ 84 ].

Nevertheless, two phase II trials have indicated remarkable anticancer activity of motesanib among patients with advanced thyroid cancer [ 85 , 86 ]. Recently, Lubner et al. examined the efficacy of motesanib in low-grade NETs in a phase II trial [ 87 ].

The study reached its primary objective with a 4-month PFS of All in all, motesanib is as potential as a systemic targeted therapy for NETs, but its niche in the treatment of NETs still needs further study.

Though, cediranib had failed phase III clinical trials in NSCLC [ 89 ], mCRC [ 90 , 91 ], and recurrent glioblastoma [ 92 ], it showed new hope in recurrent ovarian cancer. The ICON6 trial evaluated the efficacy and safety of cediranib plus platinum-based chemotherapy and as continued maintenance treatment in patients with relapsed platinum-sensitive ovarian cancer [ 93 ].

Unfortunately, the ICON6 trail was prematurely terminated on account of the depressing results in other cancer types. Most common side effects of grade 3—4 in arm C were neutropenia, fatigue, and hypertension during the chemotherapy phase and diarrhea, fatigue, and neutropenia during maintenance treatment.

A phase I study NCT observed an acceptable safety profile and encouraging antitumor activity in patients with advanced solid tumors, particularly in NETs [ 95 ]. At present, one phase II study NCT and two phase III studies NCT, NCT conducted on advanced NETs are ongoing [ 96 ].

Immunotherapy has been changing the paradigm of oncology treatment in the recent years [ 97 , 98 , 99 ]. Whether the combination of TKIs and immunotherapy can create synthetic effect is a hot topic.

The emerging evidences suggest that anti-angiogenic therapy may not only inhibit neo-vascular formation, but also regulate the immune microenvironment [ ].

This provided a theoretic basis for the combination of TKIs and immunotherapy. Subsequently, hundreds of clinical trials were designed to access the efficacy of combining TKIs with immune checkpoint blockade.

A phase Ib study JAVELIN Renal conducted by Choueiri et al. interrogated the combination therapy of axitinib plus avelumab a PD-L1 mAb in advanced RCC patients [ ]. These encouraging results supported the further study of these drug combinations. Now, the phase III JAVELIN Renal trial finished [ ].

The result showed that in patients with mRCC, the axitinib plus avelumab group showed a remarkable improvement in median PFS compared with sunitinib The combination of axitinib and avelumab would be a promising strategy for patients with mHCC based on the positive result of JAVELIN Renal Other combinations such as lenvatinib plus pembrolizumab or SHR plus apatinib in patients with HCC were also ongoing [ ].

The combination of immunotherapy with TKIs has demonstrated promising outcome in a certain type of carcinomas, but further optimized combinations are required and caution must be taken to avoid severe toxicity. The development of anti-angiogenic agents has attracted great attention.

Bevacizumab, the first clinically approved anti-VEGF targeted agents, provides a first proof of principle of anti-angiogenic treatment in cancer. Though monotherapy with bevacizumab is largely inefficient, it really exerts therapeutic efficacy in various types of carcinoma when in combination with chemotherapy [ ].

Because tumor angiogenesis is regulated by multiple pathways, many interconnected pathways can compensate the effect of single inhibition of VEGF signaling. It seems that multi-targeted TKIs hold a therapeutic advantage over monoclonal antibody as they can block multiple angiogenic signaling pathways simultaneously.

Indeed, TKIs have shown their efficacy in many types of cancers, mainly RCC and HCC. Although all anti-angiogenic receptor TKIs share the same mechanism of action and the similar spectrum of targeted kinases, they are different in their pharmacokinetics and substance-specific AEs.

The one possible explanation may be that the subtle difference on chemical structure leads to the variable affinity and potency to targets. Another possibility is that those TKIs may act on some unidentified targets beyond known kinases.

With more and more anti-cancer agents available, it is a challenge for the oncologist to make an optimal choice in the sequence of treatment. For instance, 12 drugs have been approved for patients with HCC, including 6 anti-angiogenic TKIs until [ ]. Though, the international guidelines have reached a global consensus for the choice of drugs in different lines.

The optimal strategy and the sequence of drugs as well as the right time of the incorporation of other therapeutic methods such as surgery, radiology has not yet been resolved. Tolerance of receptor TKIs should also be taken into account. Another challenge for anti-angiogenesis TKIs is the lack of robust biomarkers to identify patients with cancer who will benefit from anti-angiogenic therapy.

Unlike RTK inhibitor, larotrectinib is special for cancer with tropomyosin receptor kinases TRK fusion-positive and has demonstrated significant efficiency in patients with different tumor histology [ ].

One of the main problems in identifying such a biomarker for anti-angiogenic therapy may come from the complex feedback loops and cross talk between signaling pathways. Currently, some biomarkers have been proposed, such as VEGF, VEGFR-2, FGF-2, or IL-8, but none of them have yet been validated for routine clinical use [ ].

Recently, a cohort study conducted by Liu et al. indicated a positive correlation between the anti-angiogenesis-related AEs and prolonged OS [ ]. It means that side effects, such as high blood pressure, hypothyroidism, or hand-foot syndrome, may associate with the anti-tumor efficacy.

Similarly, Rini et al. As there are no molecular biomarkers available for clinical use, those side effects might be helpful for clinical decision.

The future of TKIs could be their positioning besides metastatic setting, such as in adjuvant therapy and neoadjuvant treatment.

There were three well-known phase III clinical trials that explored the use of TKIs in RCC in adjuvant setting, namely ASSURE adjuvant sunitinib vs. sorafenib vs.

placebo , PROTECT pazopanib vs. placebo , and S-TRAC sunitinib vs. placebo [ , , ]. Only S-TRAC study showed a significant improvement by sunitinib in disease-free survival in high-risk RCC after nephrectomy [ ].

Based on the result of S-TRAC trial, sunitinib was approved by the FDA as an adjuvant therapy for RCC patents in Unfortunately, adjuvant sorafenib for HCC patients reached a negative result [ ]. The utilization of TKIs before surgery has also been studied. A phase II trial explored the safety and efficacy of the use of pazopanib prior to cytoreductive nephrectomy RCC patients, suggesting the safety, and clinical benefit could be expected [ ].

The precision role of anti-angiogenic TKI in adjuvant and neoadjuvant therapy needs further investigation. It is noted that the indication of these receptor TKIs are mainly restricted to highly vascular tumor, like RCC, HCC, NSCLC, and CRC.

Their efficacy in other types of cancers needs further exploration [ 30 ]. In most case, anti-angiogenesis treatment increases the PFS of patients, while the increase in OS is unsatisfactory.

Great breakthrough in immunotherapy brings new possibility for the combination of TKIs, and positive results in a certain type of carcinoma attract broad attention [ ]. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.

Article CAS PubMed Google Scholar. Gimbrone MA Jr, Leapman SB, Cotran RS, Folkman J. Tumor dormancy in vivo by prevention of neovascularization.

J Exp Med. Article PubMed PubMed Central Google Scholar. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. Ribatti D, Nico B, Crivellato E, Roccaro AM, Vacca A. The history of the angiogenic switch concept. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors.

Fagiani E, Christofori G. Angiopoietins in angiogenesis. Cancer Lett. Zheng X, Liu Q, Yi M, Qin S, Wu K. The regulation of cytokine signaling by retinal determination gene network pathway in cancer.

Onco Targets Ther. Seki T, Hosaka K, Lim S, Fischer C, Honek J, Yang Y, et al. Endothelial PDGF-CC regulates angiogenesis-dependent thermogenesis in beige fat.

Nat Commun. Article CAS PubMed PubMed Central Google Scholar. Antiangiogenesis in cancer therapy--endostatin and its mechanisms of action. Exp Cell Res.

O'Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma.

Ferrara N, Hillan KJ, Gerber HP, Novotny W. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov. Poole RM, Vaidya A.

Ramucirumab: first global approval. Zhao Y, Adjei AA. Targeting angiogenesis in cancer therapy: moving beyond vascular endothelial growth factor. Berndt N, Karim RM, Schonbrunn E. Advances of small molecule targeting of kinases. Curr Opin Chem Biol.

Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science New York, NY. Article CAS Google Scholar. Ling Y, Xie Q, Zhang Z, Zhang H. Protein kinase inhibitors for acute leukemia.

Biomark Res. Gotink KJ, Verheul HM. Anti-angiogenic tyrosine kinase inhibitors: what is their mechanism of action? Liu Q, Yu S, Zhao W, Qin S, Chu Q, Wu K. EGFR-TKIs resistance via EGFR-independent signaling pathways.

Mol Cancer. Article PubMed PubMed Central CAS Google Scholar. Cowan-Jacob SW. Structural biology of protein tyrosine kinases. Cell Mol Life Sci. Schlessinger J. Cell signaling by receptor tyrosine kinases.

Hubbard SR. Structural analysis of receptor tyrosine kinases. Prog Biophys Mol Biol. Kerbel RS. Tumor angiogenesis. N Engl J Med. Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis.

J Clin Oncol. Andrae J, Gallini R, Betsholtz C. Role of platelet-derived growth factors in physiology and medicine. Genes Dev. Beenken A, Mohammadi M.

The FGF family: biology, pathophysiology and therapy. Skouras VS, Maragkos C, Grapsa D, Syrigos KN. Targeting neovasculature with multitargeted antiangiogenesis tyrosine kinase inhibitors in non-small cell lung cancer.

Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Siebels M, et al. Sorafenib in advanced clear-cell renal-cell carcinoma. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al.

Sorafenib in advanced hepatocellular carcinoma. Brose MS, Nutting CM, Jarzab B, Elisei R, Siena S, Bastholt L, et al. Sorafenib in radioactive iodine-refractory, locally advanced or metastatic differentiated thyroid cancer: a randomised, double-blind, phase 3 trial.

Poddubskaya EV, Baranova MP, Allina DO, Smirnov PY, Albert EA, Kirilchev AP, et al. Personalized prescription of tyrosine kinase inhibitors in unresectable metastatic cholangiocarcinoma. Exp Hematol Oncol. Motzer RJ, Escudier B, Gannon A, Figlin RA. Curr Opin Genet Dev 15 1 — SX 04 [pii]. Beauvais DM, Ell BJ, McWhorter AR, Rapraeger AC Syndecan-1 regulates alphavbeta3 and alphavbeta5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor.

J Exp Med 3 — Bosslet K, Straub R, Blumrich M, Czech J, Gerken M, Sperker B, Kroemer HK, Gesson JP, Koch M, Monneret C Elucidation of the mechanism enabling tumor selective prodrug monotherapy.

Cancer Res 58 6 — CAS PubMed Google Scholar. Brooks PC Cell adhesion molecules in angiogenesis. Cancer Metastasis Rev 15 2 — Bullitt E, Ewend MG, Aylward S, Lin W, Gerig G, Joshi S, Jung I, Muller K, Smith JK Abnormal vessel tortuosity as a marker of treatment response of malignant gliomas: preliminary report.

Technol Cancer Res Treat 3 6 — Article PubMed PubMed Central Google Scholar. Burge M, Francesconi AB, Kotasek D, Fida R, Smith G, Wilks A, Vasey PA, Lickliter JD Phase I, pharmacokinetic and pharmacodynamic evaluation of CYT, an orally-bioavailable cytotoxic and vascular-disrupting agent.

Investig New Drugs 31 1 — Article CAS Google Scholar. Burger JA, Peled A CXCR4 antagonists: targeting the microenvironment in leukemia and other cancers. Leukemia 23 1 — leu [pii]. Burrows FJ, Thorpe PE Eradication of large solid tumors in mice with an immunotoxin directed against tumor vasculature.

Proc Natl Acad Sci USA 90 19 — Byzova TV, Goldman CK, Pampori N, Thomas KA, Bett A, Shattil SJ, Plow EF A mechanism for modulation of cellular responses to VEGF: activation of the integrins.

Mol Cell 6 4 — doi:S 05 [pii]. Cesca M, Bizzaro F, Zucchetti M, Giavazzi R Tumor delivery of chemotherapy combined with inhibitors of angiogenesis and vascular targeting agents.

Front Oncol Chaplin DJ, Horsman MR, Siemann DW Current development status of small-molecule vascular disrupting agents. Curr Opin Investig Drugs 7 6 — Chen CS, Tan J, Tien J Mechanotransduction at cell-matrix and cell-cell contacts.

Annu Rev Biomed Eng — Chung F, Liu J, Ching LM, Baguley BC Consequences of increased vascular permeability induced by treatment of mice with 5,6-dimethylxanthenoneacetic acid DMXAA and thalidomide. Cancer Chemother Pharmacol 61 3 — Annu Rev Physiol — Article PubMed Google Scholar.

Dark GG, Hill SA, Prise VE, Tozer GM, Pettit GR, Chaplin DJ Combretastatin A-4, an agent that displays potent and selective toxicity toward tumor vasculature.

Cancer Res 57 10 — De Bock K, De Smet F, Leite De Oliveira R, Anthonis K, Carmeliet P Endothelial oxygen sensors regulate tumor vessel abnormalization by instructing phalanx endothelial cells. J Mol Med Berl 87 6 — Proc Natl Acad Sci USA 21 — DeMali KA, Wennerberg K, Burridge K Integrin signaling to the actin cytoskeleton.

Curr Opin Cell Biol 15 5 — doi:S [pii]. Denekamp J, Hill SA, Hobson B Vascular occlusion and tumour cell death. Eur J Cancer Clin Oncol 19 2 — Denekamp J, Hobson B Endothelial-cell proliferation in experimental tumours.

Br J Cancer 46 5 — Dickson PV, Hamner JB, Sims TL, Fraga CH, Ng CY, Rajasekeran S, Hagedorn NL, McCarville MB, Stewart CF, Davidoff AM Bevacizumab-induced transient remodeling of the vasculature in neuroblastoma xenografts results in improved delivery and efficacy of systemically administered chemotherapy.

Clin Cancer Res 13 13 — Dome B, Timar J, Dobos J, Meszaros L, Raso E, Paku S, Kenessey I, Ostoros G, Magyar M, Ladanyi A, Bogos K, Tovari J Identification and clinical significance of circulating endothelial progenitor cells in human non-small cell lung cancer.

Cancer Res 66 14 — Drake CJ, Cheresh DA, Little CD An antagonist of integrin alpha v beta 3 prevents maturation of blood vessels during embryonic neovascularization. J Cell Sci Pt 7 — Eberhard A, Kahlert S, Goede V, Hemmerlein B, Plate KH, Augustin HG Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies.

Cancer Res 60 5 — Farace F, Massard C, Borghi E, Bidart JM, Soria JC Vascular disrupting therapy-induced mobilization of circulating endothelial progenitor cells.

Ann Oncol 18 8 — Ferrara N Molecular and biological properties of vascular endothelial growth factor. J Mol Med Berl 77 7 — Ferrara N Pathways mediating VEGF-independent tumor angiogenesis.

Cytokine Growth Factor Rev 21 1 — Folkman J, Long DM Jr, Becker FF Growth and metastasis of tumor in organ culture. Cancer — Franco M, Man S, Chen L, Emmenegger U, Shaked Y, Cheung AM, Brown AS, Hicklin DJ, Foster FS, Kerbel RS 1.

Cancer Res 66 7 — Friedlander M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA, Cheresh DA Definition of two angiogenic pathways by distinct alpha v integrins. Science — Gan HK, Seruga B, Knox JJ Sunitinib in solid tumors.

Expert Opin Investig Drugs 18 6 — Gazit Y, Baish JW, Safabakhsh N, Leunig M, Baxter LT, Jain RK Fractal characteristics of tumor vascular architecture during tumor growth and regression. Microcirculation 4 4 — Grosios K, Loadman PM, Swaine DJ, Pettit GR, Bibby MC Combination chemotherapy with combretastatin A-4 phosphate and 5-fluorouracil in an experimental murine colon adenocarcinoma.

Anticancer Res 20 1A — Gutheil JC, Campbell TN, Pierce PR, Watkins JD, Huse WD, Bodkin DJ, Cheresh DA Targeted antiangiogenic therapy for cancer using Vitaxin: a humanized monoclonal antibody to the integrin alphavbeta3.

Clin Cancer Res 6 8 — Hamzah J, Jugold M, Kiessling F, Rigby P, Manzur M, Marti HH, Rabie T, Kaden S, Grone HJ, Hammerling GJ, Arnold B, Ganss R Vascular normalization in Rgs5-deficient tumours promotes immune destruction. Nature — nature [pii].

Hanahan D, Weinberg RA Hallmarks of cancer: the next generation. Cell 5 — S 11 [pii]. Hartmann JT, Haap M, Kopp HG, Lipp HP Tyrosine kinase inhibitors — a review on pharmacology, metabolism and side effects. Curr Drug Metab 10 5 — Hasani A, Leighl N Classification and toxicities of vascular disrupting agents.

Clin Lung Cancer 12 1 — Hellberg C, Ostman A, Heldin CH PDGF and vessel maturation. Recent Results Cancer Res — Hey T, Fiedler E, Rudolph R, Fiedler M Artificial, non-antibody binding proteins for pharmaceutical and industrial applications. Trends Biotechnol 23 10 — S 05 [pii].

Hicklin DJ, Ellis LM Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 23 5 — Hirsch E, Brancaccio M, Altruda F Tissue-specific KO of ECM proteins.

Methods Mol Biol — Huang FJ, You WK, Bonaldo P, Seyfried TN, Pasquale EB, Stallcup WB Pericyte deficiencies lead to aberrant tumor vascularizaton in the brain of the NG2 null mouse. Dev Biol 2 — S 10 [pii]. Huang X, Molema G, King S, Watkins L, Edgington TS, Thorpe PE Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature.

Hwang R, Varner J The role of integrins in tumor angiogenesis. Hematol Oncol Clin North Am 18 5 — S 04 [pii]. Hynes RO The emergence of integrins: a personal and historical perspective.

Matrix Biol 23 6 — Ide AG, Baker NH, Warren SL Vascularization of the Brown-Pearce rabbit epithelioma transplant as seen in the transparent chamber. Am J Radiol — Inai T, Mancuso M, Hashizume H, Baffert F, Haskell A, Baluk P, Hu-Lowe DD, Shalinsky DR, Thurston G, Yancopoulos GD, McDonald DM Inhibition of vascular endothelial growth factor VEGF signaling in cancer causes loss of endothelial fenestrations, regression of tumor vessels, and appearance of basement membrane ghosts.

Am J Pathol 1 — Innocenti F, Ramirez J, Obel J, Xiong J, Mirkov S, Chiu YL, Katz DA, Carr RA, Zhang W, Das S, Adjei A, Moyer AM, Chen PX, Krivoshik A, Medina D, Gordon GB, Ratain MJ, Sahelijo L, Weinshilboum RM, Fleming GF, Bhathena A Preclinical discovery of candidate genes to guide pharmacogenetics during phase I development: the example of the novel anticancer agent ABT Pharmacogenet Genomics 23 7 — Jain RK Determinants of tumor blood flow: a review.

Cancer Res 48 10 — Jain RK Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy.

Nat Med 7 9 — nm [pii]. Jain RK Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Mol Cancer Res 10 8 — MCR [pii]. Jordan MA, Wilson L Microtubules as a target for anticancer drugs.

Nat Rev Cancer 4 4 — nrc [pii]. Jung YD, Ahmad SA, Liu W, Reinmuth N, Parikh A, Stoeltzing O, Fan F, Ellis LM The role of the microenvironment and intercellular cross-talk in tumor angiogenesis.

Semin Cancer Biol 12 2 — SX [pii]. Kalluri R Basement membranes: structure, assembly and role in tumour angiogenesis. Nat Rev Cancer 3 6 — Kamba T, McDonald DM Mechanisms of adverse effects of anti-VEGF therapy for cancer.

Br J Cancer 96 12 — Kashiwagi S, Tsukada K, Xu L, Miyazaki J, Kozin SV, Tyrrell JA, Sessa WC, Gerweck LE, Jain RK, Fukumura D Perivascular nitric oxide gradients normalize tumor vasculature.

Nat Med 14 3 — Kelly RJ, Darnell C, Rixe O Target inhibition in antiangiogenic therapy a wide spectrum of selectivity and specificity. Cancer J 16 6 — Kerbel R, Folkman J Clinical translation of angiogenesis inhibitors.

Nat Rev Cancer 2 10 — Kienast Y, von Baumgarten L, Fuhrmann M, Klinkert WE, Goldbrunner R, Herms J, Winkler F Real-time imaging reveals the single steps of brain metastasis formation. Nat Med 16 1 — Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, Ferrara N Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo.

Because angiogenesis inhibitors work by slowing or stopping tumor growth without killing cancer cells, they are given over a long period.

The U. Food and Drug Administration FDA has approved a number of angiogenesis inhibitors to treat cancer. Most of these are targeted therapies that were developed specifically to target VEGF, its receptor, or other specific molecules involved in angiogenesis.

Approved angiogenesis inhibitors include:. Side effects of treatment with VEGF-targeting angiogenesis inhibitors can include hemorrhage , clots in the arteries with resultant stroke or heart attack , hypertension , impaired wound healing, reversible posterior leukoencephalopathy syndrome a brain disorder , and protein in the urine.

Gastrointestinal perforation and fistulas also appear to be rare side effects of some angiogenesis inhibitors. Antiangiogenesis agents that target the VEGF receptor have additional side effects, including fatigue, diarrhea, biochemical hypothyroidism , hand-foot syndrome , cardiac failure, and hair changes.

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