Abrus agglutinin is a potent anti‐proliferative and anti‐angiogenic agent in human breast cancer

Abrus agglutinin (AGG), a plant lectin isolated from the seeds of Abrus precatorius, has documented antitumor and immunostimulatory effects in murine models. To examine possible antitumor activity against breast cancer, we established human breast tumor xenografts in athymic nude mice and intraperitoneally administered AGG. AGG inhibited tumor growth and angiogenesis as confirmed by monitoring the expression of Ki‐67 and CD‐31, respectively. In addition, TUNEL positive cells increased in breast tumors treated with AGG suggesting that AGG mediates anti‐tumorigenic activity through induction of apoptosis and inhibition of angiogenesis. On a molecular level, AGG caused extrinsic apoptosis through ROS generation that was AKT‐dependent in breast cancer cells, without affecting primary mammary epithelial cells, suggesting potential cancer specificity of this natural compound. In addition, using HUVECs, AGG inhibited expression of the pro‐angiogenic factor IGFBP‐2 in an AKT‐dependent manner, reducing angiogenic phenotypes both in vitro and in vivo. Overall, the present results establish that AGG promotes both apoptosis and anti‐angiogenic activities in human breast tumor cells, which might be exploited for treatment of breast and other cancers.     

Study on the tumor‐induced angiogenesis using mathematical models

We studied angiogenesis using mathematical models describing the dynamics of tip cells. We reviewed the basic ideas of angiogenesis models and its numerical simulation technique to produce realistic computer graphics images of sprouting angiogenesis. We examined the classical model of Anderson‐Chaplain using fundamental concepts of mass transport and chemical reaction with ECM degradation included. We then constructed two types of numerical schemes, model‐faithful and model‐driven ones, where new techniques of numerical simulation are introduced, such as transient probability, particle velocity, and Boolean variables.     

Targeting of tumor growth and angiogenesis underlies the enhanced antitumor activity of lenvatinib in combination with everolimus

The combination of lenvatinib, a multiple receptor tyrosine kinase inhibitor, plus everolimus, a mammalian target of rapamycin (mTOR) inhibitor, significantly improved clinical outcomes versus everolimus monotherapy in a phase II clinical study of metastatic renal cell carcinoma (RCC). We investigated potential mechanisms underlying the antitumor activity of the combination treatment in preclinical RCC models. Lenvatinib plus everolimus showed greater antitumor activity than either monotherapy in three human RCC xenograft mouse models (A‐498, Caki‐1, and Caki‐2). In particular, the combination led to tumor regression in the A‐498 and Caki‐1 models. In the A‐498 model, everolimus showed antiproliferative activity, whereas lenvatinib showed anti‐angiogenic effects. The anti‐angiogenic activity was potentiated by the lenvatinib plus everolimus combination in Caki‐1 xenografts, in which fibroblast growth factor (FGF)‐driven angiogenesis may contribute to tumor growth. The combination showed mostly additive activity in vascular endothelial growth factor (VEGF)‐activated, and synergistic activity against FGF‐activated endothelial cells, in cell proliferation and tube formation assays, as well as strongly suppressed mTOR‐S6K‐S6 signaling. Enhanced antitumor activities of the combination versus each monotherapy were also observed in mice bearing human pancreatic KP‐1 xenografts overexpressing VEGF or FGF. Our results indicated that simultaneous targeting of tumor cell growth and angiogenesis by lenvatinib plus everolimus resulted in enhanced antitumor activity. The enhanced inhibition of both VEGF and FGF signaling pathways by the combination underlies its superior anti‐angiogenic activity in human RCC xenograft models.     

Subgroup analysis of Japanese patients in a phase 3 study of lenvatinib in radioiodine‐refractory differentiated thyroid cancer

Lenvatinib significantly prolonged progression‐free survival (PFS) versus placebo in patients with radioiodine‐refractory differentiated thyroid cancer (RR‐DTC) in the phase 3 Study of (E7080) Lenvatinib in Differentiated Cancer of the Thyroid (SELECT) trial. This subanalysis evaluated the efficacy and safety of lenvatinib in Japanese patients who participated in SELECT. Outcomes for Japanese patients (lenvatinib, n = 30; placebo, n = 10) were assessed in relationship to the SELECT population (lenvatinib, n = 261; placebo, n = 131). The primary endpoint was PFS; secondary endpoints included overall survival, overall response rate, and safety. Lenvatinib PFS benefit was shown in Japanese patients (median PFS: lenvatinib, 16.5 months; placebo, 3.7 months), although significance was not reached, presumably due to sample size (hazard ratio, 0.39; 95% confidence interval, 0.10–1.57; P = 0.067). Overall response rates were 63.3% and 0% for lenvatinib and placebo, respectively. No significant difference was found in overall survival. The lenvatinib safety profile was similar between the Japanese and overall SELECT population, except for higher incidences of hypertension (any grade: Japanese, 87%; overall, 68%; grade ≥3: Japanese, 80%; overall, 42%), palmar–plantar erythrodysesthesia syndrome (any grade: Japanese, 70%; overall, 32%; grade ≥3: Japanese, 3%; overall, 3%), and proteinuria (any grade: Japanese, 63%; overall, 31%; grade ≥3: Japanese, 20%; overall, 10%). Japanese patients had more dose reductions (Japanese, 90%; overall, 67.8%), but fewer discontinuations due to adverse events (Japanese, 3.3%; overall, 14.2%). There was no difference in lenvatinib exposure between the Japanese and overall SELECT populations after adjusting for body weight. In Japanese patients with radioiodine‐refractory differentiated thyroid cancer, lenvatinib showed similar clinical outcomes to the overall SELECT population. Some differences in adverse event frequencies and dose modifications were observed. Clinical trial registration no.: NCT01321554.     

Tyrosine kinase inhibitors rechallenge in solid tumors: a review of literature and a case description with lenvatinib in thyroid cancer

Introduction: In the last decade tyrosine kinase inhibitors (TKIs) have been employed for a wide range of hematological and solid tumors and today they represent a valid therapeutic option for different neoplasms. Among them, both sorafenib and lenvatinib were approved for the treatment of radioactive iodine (RAI) refractory differentiated thyroid carcinoma (DTC). Unfortunately, in some cases the efficacy of TKIs is limited by the onset of drug resistance after the initial response.

Areas covered: We report the case of a patient with a RAI refractory advanced DTC, treated with lenvatinib after surgery, multiple RAI administrations, traditional chemotherapy, and sorafenib. During treatment with lenvatinib, a noticeable response was detected by sequential computed tomography scans but, after 27 months, tumor progression became evident and led to lenvatinib interruption. In absence of any active treatment, a further disease progression was documented, and lenvatinib was re-administered obtaining a new objective response. Starting from this case report, we review available reports about the rechallenge with TKIs in solid tumors, discussing the possible mechanisms underlying the efficacy of this approach.

Expert commentary: Rechallenge with TKIs in solid tumors could be a therapeutic option in subjects with advanced and metastatic DTC who experience a progressive disease after initial response to lenvatinib.     

Potent antitumor activity of cabozantinib,a c‐MET and VEGFR2 inhibitor,in a colorectal cancer patient‐derived tumor explant model

Antiangiogenic therapy is commonly used for the treatment of colorectal cancer (CRC). Although patients derive some clinical benefit, treatment resistance inevitably occurs. The MET signaling pathway has been proposed to be a major contributor of resistance to antiangiogenic therapy. MET is upregulated in response to vascular endothelial growth factor pathway inhibition and plays an essential role in tumorigenesis and progression of tumors. In this study, we set out to determine the efficacy of cabozantinib in a preclinical CRC patient‐derived tumor xenograft model. We demonstrate potent inhibitory effects on tumor growth in 80% of tumors treated. The greatest antitumor effects were observed in tumors that possess a mutation in the PIK3CA gene. The underlying antitumor mechanisms of cabozantinib consisted of inhibition of angiogenesis and Akt activation and significantly decreased expression of genes involved in the PI3K pathway. These findings support further evaluation of cabozantinib in patients with CRC. PIK3CA mutation as a predictive biomarker of sensitivity is intriguing and warrants further elucidation. A clinical trial of cabozantinib in refractory metastatic CRC is being activated.     

Formononetin,a novel FGFR2 inhibitor,potently inhibits angiogenesis and tumor growth in preclinical models

Most anti-angiogenic therapies currently being evaluated in clinical trials target vascular endothelial growth factor (VEGF) pathway, however, the tumor vasculature can acquire resistance to VEGF-targeted therapy by shifting to other angiogenesis mechanisms. Therefore, other potential therapeutic agents that block non-VEGF angiogenic pathways need to be evaluated. Here we identified formononetin as a novel agent with potential anti-angiogenic and anti-cancer activities. Formononetin demonstrated inhibition of endothelial cell proliferation, migration, and tube formation in response to basic fibroblast growth factor 2 (FGF2). In ex vivo and in vivo angiogenesis assays, formononetin suppressed FGF2-induced microvessel sprouting of rat aortic rings and angiogenesis. To understand the underlying molecular basis, we examined the effects of formononetin on different molecular components in treated endothelial cell, and found that formononetin suppressed FGF2-triggered activation of FGFR2 and protein kinase B (Akt) signaling. Moreover, formononetin directly inhibited proliferation and blocked the oncogenic signaling pathways in breast cancer cell. In vivo, using xenograft models of breast cancer, formononetin showed growth-inhibitory activity associated with inhibition of tumor angiogenesis. Moreover, formononetin enhanced the effect of VEGFR2 inhibitor sunitinib on tumor growth inhibition. Taken together, our results indicate that formononetin targets the FGFR2-mediated Akt signaling pathway, leading to the suppression of tumor growth and angiogenesis.     

The novel mitochondria‐targeted antioxidant SkQ1 modulates angiogenesis and inflammatory micromilieu in a murine orthotopic model of pancreatic cancer

Our understanding in the last few years about reactive oxygen species (ROS) has changed from being harmful substances to crucial intra‐ and extracellular messengers as well as important regulators controlling a wide spectrum of signaling pathways, including those in cancer immunology. Therefore, these multiple essential roles of ROS and especially of mitochondria‐derived ROS in malignant transformation and cancer progression make them a promising target for anticancer therapy. Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in the world. A link between ROS, antioxidants and the PDAC development and progression has been recently established. Therefore, usage of specific highly efficient antioxidants could bring an option for treatment and/or prevention of PDAC. 10‐(6′‐plastoquinonyl) decyltriphenylphosphonium (SkQ1) is a new antioxidant with the highest mitochondrion membrane penetrating ability and potent antioxidant capability. In this work, we investigated an impact of SkQ1 on tumor angiogenesis, immune micromilieu, and oncological parameters in the orthotopic Panc02 murine model of PDAC. We showed that in this model SkQ1 treatment leads to the elevation of pro‐angiogenic factors and to building of mainly an anti‐inflammatory cytokine milieu. On the cellular level we showed an increase in a percentage of memory T cells and a decrease in frequency on natural killer T (NKT) cells. At the same time, SkQ1 was ineffective in the improvement of oncological parameters of PDAC‐bearing mice. New studies are needed to clarify the absence of therapeutic and/or prophylactic benefits of the antioxidant.     

Combined anti‐PDGFRα and PDGFRβ targeting in non‐small cell lung cancer

Activation of the platelet‐derived growth factor (PDGF)‐receptors is critically involved into various stromal cell functions including recruitment of stromal cells and vascular endothelial growth factor (VEGF) induction in tumor and perivascular cells. To evaluate the effects of combined PDGFRα and ‐β inhibition in a non‐small cell lung cancer model, we stably transfected A549 lung cancer cells with the PDGF‐A mutant PDGF‐0. PDGF‐0 has been generated by substituting amino acids in the binding region of PDGF‐A with the corresponding VEGF‐A region, leading to a decreased receptor‐binding affinity and activation. Compared with control vector transfected cells, transfection with PDGF‐0 had no impact on monolayer growth and apoptosis in vitro, but significantly impaired the number of colony formation in soft agar. After subcutaneous injections, all mice developed tumors within 5 days. While control vector transfected A549 cells were characterized by constant tumor growth, PDGF‐0 transfected A549 revealed a reduced tumor mass (p < 0.001) with no further growth beyond 14 days (2 months observation time) and complete regressions in 7 of 13 cases. Immunohistochemical analyses revealed that PDGF‐0 transfected tumors demonstrated decreased recruitment of periendothelial cells, while the tumor invasion zone was similar to control vector transfectants. Similarly, conditioned medium from PDGF‐0 transfected cells induced significantly less migration of smooth muscle cells and fibroblasts in vitro. Interestingly, in PDGF‐0 transfectants, neither total vessel count nor VEGF expression were significantly altered. These studies demonstrate that combined inhibition of PDGFRα and ‐β results in markedly decreased tumor growth in vivo because of impaired recruitment of periendothelial cells. © 2008 Wiley‐Liss, Inc.     

CXC‐chemokine/CXCR2 biological axis promotes angiogenesis in vitro and in vivo in pancreatic cancer

Angiogenesis is essential for tumor growth and metastasis. Although ELR⁺‐CXC‐chemokines and their corresponding receptor, CXC‐receptor 2 (CXCR2), are known mediators of angiogenesis, little is known about their role in pancreatic cancer (PaCa). The aim of our study was to determine the role of ELR⁺‐CXC‐chemokine/CXCR2 biological axis in promoting PaCa angiogenesis. We prospectively collected secretin‐stimulated exocrine pancreatic secretions (SSEPS) from normal individuals (NP) and PaCa patients. We showed that summed concentrations of ELR⁺‐CXC‐chemokines in SSEPS from PaCa patients were significantly higher than in those from NP (p = 0.002). We measured ELR⁺‐CXC‐chemokine levels in supernatants from multiple PaCa cell lines and confirmed that BxPC‐3, Colo‐357 and Panc‐28 had significantly higher expression compared with an immortalized human pancreatic ductal epithelial (HPDE) cell line. After confirming lack of autocrine effects of ELR⁺‐CXC‐chemokines on PaCa cells (due to absence of CXCR2 expression), we investigated paracrine effects of these chemokines on human umbilical vein endothelial cells (HUVEC). Both recombinant ELR⁺‐CXC‐chemokines and co‐culturing with BxPC‐3 significantly enhanced proliferation, invasion, and tube formation of HUVEC (p < 0.05). These biological effects were significantly inhibited by treatment with a neutralizing antibody against CXCR2 (anti‐CXCR2 Ab) (p < 0.05). Finally, anti‐CXCR2 Ab significantly reduced tumor volume (p < 0.05), Ki‐67 proliferation index (p = 0.043) and Factor VIII⁺ microvessel density (p = 0.004) in an orthotopic nude mouse PaCa model. Our results show that ELR⁺‐CXC‐chemokines promote PaCa tumor‐associated angiogenesis through CXCR2, suggesting that CXCR2 is an anti‐angiogenic target in PaCa. © 2009 UICC     

Clodronate inhibits tumor angiogenesis in mouse models of ovarian cancer

Purpose Bisphosphonates have been shown to inhibit and deplete macrophages. The effects of bisphosphonates on other cell types in the tumor microenvironment have been insufficiently studied. Here, we sought to determine the effects of bisphosphonates on ovarian cancer angiogenesis and growth via their effect on the microenvironment, including macrophage, endothelial and tumor cell populations.Experimental Design Using in vitro and in vivo models, we examined the effects of clodronate on angiogenesis and macrophage density, and the overall effect of clodronate on tumor size and metastasis.Results Clodronate inhibited the secretion of pro-angiogenic cytokines by endothelial cells and macrophages, and decreased endothelial migration and capillary tube formation. In treated mice, clodronate significantly decreased tumor size, number of tumor nodules, number of tumor-associated macrophages and tumor capillary density.Conclusions Clodronate is a potent inhibitor of tumor angiogenesis. These results highlight clodronate as a potential therapeutic for cancer.     

Interleukin 8 mediates bcl‐xL‐induced enhancement of human melanoma cell dissemination and angiogenesis in a zebrafish xenograft model

The protein bcl‐xL is able to enhance the secretion of the proinflammatory chemokine interleukin 8 (CXCL8) in human melanoma lines. In this study, we investigate whether the bcl‐xL/CXCL8 axis is important for promoting melanoma angiogenesis and aggressiveness in vivo, using angiogenesis and xenotransplantation assays in zebrafish embryos. When injected into wild‐type embryos, bcl‐xL‐overexpressing melanoma cells showed enhanced dissemination and angiogenic activity compared with control cells. Human CXCL8 protein elicited a strong proangiogenic activity in zebrafish embryos and zebrafish Cxcr2 receptor was identified as the mediator of CXCL8 proangiogenic activity using a morpholino‐mediated gene knockdown. However, human CXCL8 failed to induce neutrophil recruitment in contrast to its zebrafish homolog. Interestingly, the greater aggressiveness of bcl‐xL‐overexpressing melanoma cells was mediated by an autocrine effect of CXCL8 on its CXCR2 receptor, as confirmed by an shRNA approach. Finally, correlation studies of gene expression and survival analyses using microarray and RNA‐seq public databases of human melanoma biopsies revealed that bcl‐xL expression significantly correlated with the expression of CXCL8 and other markers of melanoma progression. More importantly, a high level of co‐expression of bcl‐xL and CXCL8 was associated with poor prognosis in melanoma patients. In conclusion, these data demonstrate the existence of an autocrine CXCL8/CXCR2 signaling pathway in the bcl‐xL‐induced melanoma aggressiveness, encouraging the development of novel therapeutic approaches for high bcl‐xL‐expressing melanoma.     

INSL3 has tumor‐promoting activity in thyroid cancer

The functional role of INSL3 and its receptor RXFP2 in carcinogenesis is largely unknown. We have previously demonstrated (pro‐)cathepsin‐L as a target of INSL3 in human thyroid cancer cells facilitating penetration of tumor cells through elastin matrices. We demonstrate the expression of RXFP2 in human thyroid tissues and in mouse follicular thyroid epithelial cells using Cre‐recombinase transgene driven by Rxfp2 promoter. Recombinant and secreted INSL3 increased the motility of thyroid carcinoma (TC) cells in an autocrine/paracrine manner. This effect required the presence of RXFP2. We identified S100A4 as a novel INSL3 target molecule and showed that S100A4 facilitated INSL3‐induced enhanced motility. Stable transfectants of the human follicular TC cell line FTC‐133 expressing and secreting bioactive human INSL3 displayed enhanced anchorage‐independent growth in soft agar assays. Xenotransplant experiments in nude mice showed that INSL3, but not EGFP‐mock transfectants, developed fast‐growing and highly vascularized xenografts. We used human umbilical vein endothelial cells in capillary tube formation assays to demonstrate increased 2‐dimensional tube formations induced by recombinant human INSL3 and human S100A4 comparable to the effect of vascular endothelial growth factor used as positive control. We conclude that INSL3 is a powerful and multifunctional promoter of tumor growth and angiogenesis in human thyroid cancer cell xenografts. INSL3 actions involve RXFP2 activation and the secretion of S100A4 and (pro‐)cathepsin‐L.     

Myoferlin plays a key role in VEGFA secretion and impacts tumor‐associated angiogenesis in human pancreas cancer

Pancreatic ductal adenocarcinoma is one of the most deadly forms of cancers with no satisfactory treatment to date. Recent studies have identified myoferlin, a ferlin family member, in human pancreas adenocarcinoma where its expression was associated to a bad prognosis. However, the function of myoferlin in pancreas adenocarcinoma has not been reported. In other cell types, myoferlin is involved in several key plasma membrane processes such as fusion, repair, endocytosis and tyrosine kinase receptor activity. In this study, we showed that myoferlin silencing in BxPC‐3 human pancreatic cancer cells resulted in the inhibition of cell proliferation in vitro and in a significant reduction of the tumor volume in chick chorioallantoic membrane assay. In addition to be smaller, the tumors formed by the myoferlin‐silenced cells showed a marked absence of functional blood vessels. We further demonstrated that this effect was due, at least in part, to an inhibition of VEGFA secretion by BxPC‐3 myoferlin‐silenced cells. Using immunofluorescence and electron microscopy, we linked the decreased VEGFA secretion to an impairment of VEGFA exocytosis. The clinical relevance of our results was further strengthened by a significant correlation between myoferlin expression in a series of human pancreatic malignant lesions and their angiogenic status evaluated by the determination of the blood vessel density.     

Selective osteopontin knockdown exerts anti‐tumoral activity in a human glioblastoma model

Osteopontin (OPN), a member of the SIBLING (Small Integrin‐Binding LIgand N‐linked Glycoprotein) family, is overexpressed in human glioblastoma. Higher levels of OPN expression correlate with increased tumor grade and enhanced migratory capacity of tumor cells. Based on these observations, we explored the possibility that knocking down OPN expression in glioblastoma cells could exert an anti‐tumoral activity using an avian in vivo glioblastoma model that mimics closely human gliobastoma. Human U87‐MG glioma cells transfected with specific anti‐OPN small interfering RNAs (siRNAs) were grafted onto the chicken chorio‐allantoic membrane (CAM). OPN‐deficient U87‐MG cells gave rise to tumors that were significantly smaller than tumors formed from untransfected cells (paired t‐test, p < 0.05). Accordingly, the amount of proliferating cells in OPN‐deficient tumors showed a six‐fold reduction when compared to control tumors. However, OPN inhibition did not affect significantly tumor‐associated angiogenesis. In vitro, OPN‐silenced U87‐MG and U373‐MG cells showed decreased motility and migration. This is the first demonstration that OPN inhibition blocks glioma tumor growth, making this invasion‐related protein an attractive target for glioma therapy.     

B cells promote tumor progression in a mouse model of HPV‐mediated cervical cancer

Enhancing anti‐tumor immunity and preventing tumor escape are efficient strategies to increase the efficacy of therapeutic cancer vaccines. However, the treatment of advanced tumors remains difficult, mainly due to the immunosuppressive tumor microenvironment. Regulatory T cells and myeloid‐derived suppressor cells have been extensively studied, and their role in suppressing tumor immunity is now well established. In contrast, the role of B lymphocytes in tumor immunity remains unclear because B cells can promote tumor immunity or display regulatory functions to control excessive inflammation, mainly through IL‐10 secretion. Here, in a mouse model of HPV‐related cancer, we demonstrate that B cells accumulated in the draining lymph node of tumor‐bearing mice, due to a prolonged survival, and showed a decreased expression of MHC class II and CD86 molecules and an increased expression of Ly6A/E, PD‐L1 and CD39, suggesting potential immunoregulatory properties. However, B cells from tumor‐bearing mice did not show an increased ability to secrete IL‐10 and a deficiency in IL‐10 production did not impair tumor growth. In contrast, in B cell‐deficient μMT mice, tumor rejection occurred due to a strong T cell‐dependent anti‐tumor response. Genetic analysis based on single nucleotide polymorphisms identified genetic variants associated with tumor rejection in μMT mice, which could potentially affect reactive oxygen species production and NK cell activity. Our results demonstrate that B cells play a detrimental role in anti‐tumor immunity and suggest that targeting B cells could enhance the anti‐tumor response and improve the efficacy of therapeutic cancer vaccines.     

Cleavage of galectin‐3 by matrix metalloproteases induces angiogenesis in breast cancer

Galectin‐3 cleavage is related to progression of human breast and prostate cancer and is partly responsible for tumor growth, angiogenesis and apoptosis resistance in mouse models. A functional polymorphism in galectin‐3 gene, determining its susceptibility to cleavage by matrix metalloproteinases (MMPs)‐2/‐9 is related to racial disparity in breast cancer incidence in Asian and Caucasian women. The purpose of our study is to evaluate (i) if cleavage of galectin‐3 could be related to angiogenesis during the progression of human breast cancer, (ii) the role of cleaved galectin‐3 in induction of angiogenesis and (iii) determination of the galectin‐3 domain responsible for induction of angiogenic response. Galectin‐3 null breast cancer cells BT‐459 were transfected with either cleavable full‐length galectin‐3 or its fragmented peptides. Chemotaxis, chemoinvasion, heterotypic aggregation, epithelial‐endothelial cell interactions and angiogenesis were compared to noncleavable galectin‐3. BT‐549‐H⁶⁴ cells harboring cleavable galectin‐3 exhibited increased chemotaxis, invasion and interactions with endothelial cells resulting in angiogenesis and 3D morphogenesis compared to BT‐549‐P⁶⁴ cells harboring noncleavable galectin‐3. BT‐549‐H⁶⁴ cells induced increased migration and phosphorylation of focal adhesion kinase in migrating endothelial cells. Endothelial cells cocultured with BT‐549 cells transfected with galectin‐3 peptides indicate that amino acids 1–62 and 33–250 stimulate migration and morphogenesis of endothelial cells. Immunohistochemical analysis of blood vessel density and galectin‐3 cleavage in a breast cancer progression tissue array support the in vitro findings. We conclude that the cleavage of the N terminus of galectin‐3 followed by its release in the tumor microenvironment in part leads to breast cancer angiogenesis and progression.