Numerical simulation of solid tumor angiogenesis with Endostatin treatment:a combined analysis of inhibiting effect of anti-angiogenic factor and micro mechanical environment of extracellular matrix

To investigate the influence of anti-angiogenesis drug Endostatin on solid tumor angiogenesis, a mathematical model of tumor angiogenesis was developed with combined influences of local extra-cellular matrix mechanical environment, and the inhibiting effects of Angiostatin and Endostatin. Simulation results show that Angiostatin and Endostatin can effectively inhibit the process of tumor angiogenesis, and decrease the number of blood vessels in the tumor. The present model could be used as a valid theoretical method in the investigation of anti-angiogenic therapy of tumors.

THE CLINICAL SIGNIFICANCE OF TUMOR ANGIOGENESIS AND INVASIVENESS-RELATED GENE EXPRESSIONS IN GASTRIC CANCER

Objective: To investigate the correlation among tumor angiogenesis, expressions of p53, nm23-I1, CD44v6, c-erbB-2 proteins and biological behavior and clinical outcome of gastric cancer. Methods: The intratumoral microvessel density (MVD) and expressions of p53, nm23-H1, CD44v6, c-erbB-2 were analyzed semiquantitively by immunohistochemical staining (S-P) of 59 paraffin-embedded gastric tumor specimens that were radically resected at the Department of surgery, Beijing Institute for Cancer Research, between January 1990 and December 1992. The median follow-up period was 75 month (range: 60~96 months). The significdance of these indicators was analyzed retrospectively. Results: MVD for tumors with lymph node metastasis and vascular invasion was significantly higher than those without (P=0.0168 and 0.0176, respectively). The levels of p53, CD44v6, c-erbB-2 expression were significantly higher in the groups of lymph node metastasis, serosal infiltration and vascular invasion than in those without. All differences reached the statistically significant levels (P<0.01~<0.05). The low expression of nm23-H1 was negatively correlated with lymph node metastasis, serosal infiltration and vascular invasion (P<0.01; <0.05 and <0.01, respectively). Univariate analysis showed that the overall survival of patients with higher MVD, or overexpressions of p53, CD44v6, c-erbB-2, or low expression of nm23-H1 were significantly worse than those with opposite conditions (P=0.0214, 0.0062, 0.0045, 0.0159, and 0.0162, respectively). Multivariate analysis showed that expression of p53 in this series was an independent prognostic indicator. Conclusion: The data suggested that the above-mentioned factors might be helpful in evaluating the metastatic potential of gastric cancer and making more effective assessment of prognosis for individual patient. Further study with larger samples and prospective investigation of these results would be worthwhile.

Exploration on Effect and Mechanism of Suiqing Pill (髓清丸) on Tumor Angiogenesis in Nude Mouse B16 Melanoma Model

Objective: To study the effect of Suiqing Pill (SQP, 髓清丸),a TCM compound drug that can activate blood circulation and get rid of blood stasis, on angiogenesis in tumor and its possible mechanism. Method: BALB/c nude mice were inoculated with melanoma cell line B16 at right armpit subcutaneously to establish cancer spontaneous metastasis model. Levels of microvessel density (MVD), vascular endothelial growth factor (VEGF) and protein expression of matrix metalloproteinase-2 (MMP-2) in tumor tissues were observed and compared. Results: Strong expression of anti-Factor Ⅷ (FⅧ) related antigen and plentiful tumor angiogenesis were seen in model control animals, while in the high-dose and low-dose SQP treated model mice, MVD was inhibited by 33. 5% and 22. 6% respectively ( P<0. 01, P<0. 05). The strong positive protein expression of VEGF and MMP-2 in model control also reduced in the SQP treated groups. Conclusion: SQP could inhibit tumor angiogenesis, protein expression of VEGF and MMP-2 in nude mice B16 melanoma models.

Numerical simulation of tumor angiogenesis under the effect of Endostatin:considering mechanical environment in matrix and inhibiting effect of anti-angiogenic factor

To investigate tumor angiogenesis under the influence of Endostatin,mathematical modeling and numerical simulation of tumor angiogenesis are performed,with the mechanical environment in matrix,the inhibiting effects of Angiostatin and Endostatin into consideration.The

Construction of curcumin-loaded micelles and evaluation of the anti-tumor effect based on angiogenesis

Objective:Inhibition of tumor angiogenesis has become a new targeted tumor therapy.In this study,we established a micellar carrier with a tumor neovascularization-targeting effect modified by the neovascularization-targeting peptide NGR.Methods:The targeted polymer poly(ethylene glycol)-b-poly(lactide-co-glycolide)(PEG-PLGA)modified with Asn–Gly–Arg(NGR)peptide was prepared and characterized by 1H nuclear magnetic resonance and Fourier-transform infrared spectrometry.NGR-PEG-PLGA was used to construct curcumin(Cur)-loaded micelles by the solvent evaporation method.The physicochemical properties of the micelles were also investigated.Additionally,we evaluated the antitumor efficacy of the polymer micelles(PM)using in vitro cytology experiments and in vivo animal studies.Results:The particle size of Cur-NGR-PM was 139.70±2.51 nm,and the drug-loading capacity was 14.37±0.06%.In vitro cytological evaluation showed that NGR-modified micelles showed higher cellular uptake through receptor-mediated endocytosis pathways than did unmodified micelles,leading to the apoptosis of tumor cells.Then,in vivo antitumor experiments showed that the modified micelles significantly inhibited tumor growth and were safe.Conclusions:NGR-modified micelles significantly optimized the therapeutic efficacy of Cur.This strategy offers a viable avenue for cancer treatment.

Impaired tumor angiogenesis and VEGF- induced pathway in endothelial CD146 knockout mice

CD146 is a newly identified endothelial biomarker that has been implicated in angiogenesis. Though in vitro angio- genic function of CD146 has been extensively reported, in vivo evidence is still lacking. To address this issue, we generated endothelial-specific CD146 knockout （CD146 EC-Ko） mice using the Tg（Tek-cre） system. Surprisingly, these mice did not exhibit any apparent morphological defects in the development of normal retinal vasculature. To evaluate the role of CD146 in pathological angiogenesis, a xenograft tumor model was used. We found that both tumor volume and vascular density were significantly lower in CD146Ec-KO mice when compared to WT littermates. Additionally, the ability for sprouting, migration and tube formation in response to VEGF treatment was impaired in endothelial cells （ECs）of CD146Ec-Ko mice. Mechanistic studies further confirmed that VEGF- induced VEGFR-2 phosphorylation and AKT/p38 MAPKs/ NF-KB activation were inhibited in these CD146-null ECs, which might present the underlying cause for the observed inhibition of tumor angiogenesis in CD146Ec-Ko mice. These results suggest that CD146 plays a redundant role in physiological angiogenic processes, but becomes essential during pathological angiogenesis as observed in tumorigenesis.

CANSTATIN, A ENDOGENOUS INHIBITOR OF ANGIOGENESIS AND TUMOR GROWTH

Canstatin is a novel inhibitor of angiogenesis and tumor growth, derived from the C-terminal globular non-collageneous (NCl) domain of the a2 chain of type IV collagen. It inhibits endothelial cell proliferation and migration in a dose-dependent manner, and induces endothelial cell apoptosis. In vivo experiments show that canstatin significantly inhibits solid tumor growth. The canstatin mediated inhibition of tumor is related to apoptosis. Canstatin- induced apoptosis is associated with phosphatidylinositol 3-kinase/Akt inhibition and is dependend upon signaling events transduced trough membrane death receptor.

Regulation of tumor angiogenesis by the microtubule-binding protein CLIP-170

Angiogenesis,the expansion of preexisting blood vessels,is a complex process required for tumor growth and metastasis.Although current antiangiogenic strategies have shown promising results in several cancer types,identification of additional antiangiogenic targets is required to improve the therapeutic response.Herein,we show that the microtubule-binding protein CLIP-170(cytoplasmic linker protein of 170 kDa)is highly expressed in breast tumor samples and correlates positively with blood vessel density.Depletion of CLIP-170 signifi cantly impaired vascular endothelial tube formation and sprouting in vitro and inhibited breast tumor growth in mice by decreasing tumor vascularization.Our data further show that CLIP-170 is important for the migration but not the proliferation of vascular endothelial cells.In addition,CLIP-170 promotes the polarization of endothelial cells in response to the angiogenic stimulus.These fi ndings thus demonstrate a critical role for CLIP-170 in tumor angiogenesis and suggest its potential as a novel antiangiogenic target.

Two-dimensional discrete mathematical model of tumor-induced angiogenesis

A 2D discrete mathematical model of a nine-point finite difference scheme is built to simulate tumor-induced angiogenesis. Nine motion directions of an individual endothelial cell and two parent vessels are extended in the present model. The process of tumor-induced angiogenesis is performed by coupling random motility, chemotaxis, and haptotaxis of endothelial cell in different mechanical environments inside and outside the tumor. The results show that nearly realistic tumor microvascular networks with neoplastic pathophysiological characteristics can be generated from the present model. Moreover, the theoretical capillary networks generated in numerical simulations of the discrete model may provide useful information for further clinical research.

An experimental research into endostatin microbubble combined with focused ultrasound for anti-tumor angiogenesis in colon cancer

Objective:to evaluate the therapeutic effect of targeted endostatin-loaded microbubbles,combined with improved,focused,directional ultrasound radiation for inhibition of subcutaneous translocation in situ colon tumor angiogenesis in colon cancer.Methods:65 BALB/c nude mice with subcutaneous translocation in situ colon tumors were randomly divided into five groups.Group A was the control group,without any treatments.In Group B,the mouse was treated with ultrasonic radiation.In Group C,the mouse was treated with ultrasonic radiation combined with empty SonoVue microbubbles.In Group D,the mouse was treated with ultrasonic radiation combined with empty Targestar-SA microbubbles.In Group E,the mouse was treated with ultrasonic radiation combined with endostatin microbubbles.The tumor size was measured before and 1,14,and 28 days after irradiation.The peak intensity(PI),regional blood volume(RBV)and regional blood flow(RBF)were recorded using contrast-enhanced ultrasound.The tumor tissue was removed for pathological examination;the tumor necrosis area and microvascular density(MVD)were evaluated by immunohistochemistry.Results:Tumors in Groups C,D and E were significantly smaller than in Groups A and B at 28 days after irradiation,with Group E the smallest.PI,RBF and RBV of Groups C,D,and E were significantly decreased 28 days after radiation with Group E the lowest,and significantly lower than Groups A and B(all P<0.05).The tumor tissue necrosis area of Group E was clearly greater while MVD was obviously lower than the other groups(all P<0.01)at 28 days after treatment.Conclusion:The targeted endostatin microbubbles,combined with focused,directional ultrasound radiation can damage tumor microvasculature of subcutaneous colon translocation in situ colon cancer,as well as inhibit the tumor angiogenesis.

Glucocorticoids suppress tumor angiogenesis and growth of prostate cancer

Hormone-refractory prostate cancer ( HRPC) sometimes is responsive to treatment with glucocorticoids, such as prednisolone, hydrocortisone and dexamethasone, but the underlying mechanisms are not well established. In a recent paper (Clin Cancer Res, 2006, 12:3003-3009), Yano et al. Hypothesized and confirmed that the therapeutic effect of glucocorticoids on HRPC is attributed to inhibition of angiogenesis. A prostate cancer cell line DU145 that expresses glucocorticoid receptor was used to study the effect of dexamethasone (Dex) on the expres-

An isoflavone derivative potently inhibits the angiogenesis and progression of triple-negative breast cancer by targeting the MTA2/SerRS/VEGFA pathway

Objective:Angiogenesis plays a vital role in tumor growth and metastasis.Here,we aimed to find novel efficient antiangiogenic molecules targeting vascular endothelial growth factor A(VEGFA)at the transcriptional level to treat triple-negative breast cancer(TNBC).Methods:We used a cell-based seryl tRNA synthetase(SerRS)promoter-driven dual-luciferase reporter system to screen an in-house library of 384 naturally occurring small molecules and their derivatives to find candidate molecules that could upregulate the expression of SerRS,a potent transcriptional repressor of VEGFA.The levels of SerRS and VEGFA were examined by quantitative RT-PCR(qRT-PCR),western blotting,and/or ELISAs in TNBC cells after candidate molecule administration.Zebrafish,the Matrigel plug angiogenesis assay in mice,the TNBC allograft,and xenograft mouse models were used to evaluate thein vivoanti-angiogenic and anti-cancer activities.Furthermore,the potential direct targets of the candidates were identified by proteomics and biochemical studies.Results:We found the most active compound was 3-(4-methoxyphenyl)quinolin-4(1H)-one(MEQ),an isoflavone derivative.In TNBC cells,MEQ treatment resulted in increased SerRS mRNA(P<0.001)and protein levels and downregulated VEGFA production.Both the vascular development of zebrafish and Matrigel plug angiogenesis in mice were inhibited by MEQ.MEQ also suppressed the angiogenesis in TNBC allografts and xenografts in mice,resulting in inhibited tumor growth and prolonged overall survival(P<0.05).Finally,we found that MEQ regulated SerRS transcription by interacting with MTA2(Metastasis Associated 1 Family Member 2).Conclusions:Our findings suggested that the MTA2/SerRS/VEGFA axis is a drug-treatable anti-angiogenic target,and MEQ is a promising anti-tumor molecule that merits further investigation for clinical applications.

Tea polyphenols inhibit the growth and angiogenesis of breast cancer xenografts in a mouse model

Objective:To investigate the anti-angiogenic effect of tea polyphenols(TPS)on breast cancer and normal tissues in a mouse model.Methods:Breast cancer was successfully implanted into 48 BALB/c mice,which were then randomly divided into a TP oral gavage group,a TP local injection group,a ginsenoside Rg3 group,and a model control group according to a random number table.The tumor inhibitory rates of each group were calculated,while microvessel density(MVD)and the expression of vascular endothelial growth factor(VEGF),basic fibroblast growth factor(bFGF),and tissue inhibitor of metalloproteinase(TIMP-2)were detected by immunohistochemistry.Results:TPs could inhibit the growth of breast cancer xenografts in the mouse model.The tumor inhibition rates of the TP oral gavage and TP local injection groups were 37.43%and 40.94%,respectively.Compared with the model control group,MVD and VEGF and bFGF expression was downregulated(all P<.05),whereas TIMP-2 expression was elevated in the TP oral gavage and TP local injection groups(P=.015 and P=.032).TPs showed no significant effect on MVD and VEGF and TIMP-2 expression in the heart,brain,and kidney of the mouse model.Conclusion:TPs can restrict the growth of breast cancer by specifically inhibiting the angiogenesis of breast tumor tissue while having little effect on the normal tissue of important organs including the heart,brain,and kidney.

Research progress on the role of cancer-associated fibroblasts in tumorigenesis and development

Tumor microenvironment plays a very important role in the growth,invasion and metastasis of tumor cells.The tumor interstitial microenvironment is an important part of the tumor microenvironment,which includes two parts:the non-cellular and cellular components of the tumor interstitium,specifically including the extracellular matrix,blood vessels,and interstitial cells.Among them,activated interstitial fibroblasts,namely cancer-associated fibroblasts(CAFs),are the main components of tumor interstitial cells,which are most closely related to tumor interstitial fibrosis and tumor progress,and are expected to become a new target for cancer treatment.

Dynamic contrast-enhanced magnetic resonance imaging of prostate cancer: A review of current methods and applications

In many areas of oncology, dynamic contrast-enhanced magnetic resonance imaging(DCE-MRI) has proven to be a clinically useful, non-invasive functional imaging technique to quantify tumor vasculature and tumor perfusion characteristics. Tumor angiogenesis is an essential process for tumor growth, proliferation, and metastasis. Malignant lesions demonstrate rapid extravasation of contrast from the intravascular space to the capillary bed due to leaky capillaries associated with tumor neovascularity. DCE-MRI has the potential to provide information regarding blood flow, areas of hypoperfusion, and variations in endothelial permeability and microvessel density to aid treatment selection, enable frequent monitoring during treatment and assess response to targeted therapy following treatment. This review will discuss the current status of DCE-MRI in cancer imaging, with a focus on its use in imaging prostate malignancies as well as weaknesses that limit its widespread clinical use. The latest techniques for quantification of DCE-MRI parameters will be reviewed and compared.

Vaccination with a Recombinant Chicken FGFR-1 Bypasses Immunological Tolerance against Self-FGFR-1 in Mice

The possibility that a recombinant protein vaccine based on xenogeneic homologous FGFR-1 of chicken induces production of autoantibodies against self-FGFR-1 in BALB/c mice was examined by using ELISA, Western blot analysis and ELISPOT assay respectively. Autoantibodies against mouse FGFR-1 were identified by Western blot analysis and ELISA. Compared with the two control groups, the number of APBCs, which were detected by ELISPOT assay, was significantly in- creased in the spleens of mice immunized with cFR1 （P〈0.05）. IgG1 and IgG2b, which were detected by ELISA, were the major subclasses and were substantially increased in response to chicken FGFR-1 when compared with control group. The recombinant chicken FGFR-1 protein used as a vaccine can induce autoantibodies against self-FGFR-1 in mice and provide a basis for the active immunotherapy of tumor angiogenesis.

VEGF-C/VEGFR-3/iNOS Signaling in Osteosarcoma MG63 Cells Mediates Stimulatory Effects on Human Umbilical Vein Endothelial Cell Proliferation

Objectives To assess the effects of vascular endothelial growth factor-C(VEGF-C)/vascular endothelial growth factor receptor-3(VEGFR-3)signaling on nitric oxide(NO)production and inducible nitric oxide synthase(iNOS)expression in human osteosarcoma MG63 cells and the subsequent impact on the proliferation of human umbilical vein endothelial cells(HUVECs).Methods MG63 cells were treated with VEGF-C alone(VEGF-C group),VEGF-C+iNOS inhibitor aminoguanidine(AG;AG group),and VEGF-C+VEGFR-3 inhibitor MAZ51(MAZ51 group);untreated MG63 cells were used as controls.NO production was evaluated by a colorimetric method involving nitrate reductase.Meanwhile,mRNA and protein levels of iNOS were examined by reverse transcription polymerase chain reaction(RT-PCR)and Western blot.To explore the effect of VEGF-C/VEGFR-3/iNOS signaling of MG63 cells on proliferation of HUVECs,we set up six groups:HUVECs,HUVECs+MG63,HUVECs+VEGF-C,HUVECs+MG63+VEGF-C,HUVECs+MG63+VEGF-C+AG,and HUVECs+MG63+VEGF-C+MAZ51 groups.The proliferation of HUVEC cells was assessed by Cell Counting Kit-8(CCK-8),5-ethynyl-2'-deoxyuridine(EdU)incorporation assay,and proliferating cell nuclear antigen(PCNA)expression quantitation.Results VEGF-C treatment enhanced iNOS expression at both gene and protein levels(mRNA:LSD-t=4.152,P<0.01;protein:LSD-t=3.486,P<0.01)and increased NO release of MG63 cells(LSD-t=3.774,P<0.01);treatment with either AG or MAZ51 decreased these effects(mRNA:LSD-t=9.183,P<0.001;LSD-t=8.639,P<0.001;protein:LSD-t=5.170,P<0.001;LSD-t=7.25S,P<0.001;NO production:LSD-t=10.326,P<0.001;LSD-t=l0.540,P<0.001).Interestingly,co-incubation of HUVECs with MG63 cells and/or VEGF-C significantly promoted HUVEC proliferation(EdU:LSD-t=5.374,P<0.001;LSD-t=2.984,P<0.05;LSD-t=8.526,P<0.001;PCNA:LSD-t=9.267,P<0.001;LSD-t=5.515,P<0.001;LSD-t=14.873,P<0.001).The proliferation effects of HUVEC induced by MG63 cells and VEGF-C attenuated by the treatment of AG(EdU:LSD-t=10.770,P<0.001;PCNA:LSD-t=19.94O,P<0.001)or MAZ51(EdU:LSD-t=6.950,P<0.001;PCNA:LSD-t=14.001,P<0.001).Conclusion In human osteosarcoma MG63 cells,activation of VEGFR-3 by VEGF-C promotes iNOS expression and NO production,which subsequendy induces HUVEC proliferation.

A Hybrid Mathematical Model of Tumor-Induced Angiogenesis with Blood Perfusion

Angiogenesis, the growth of new blood vessel from existing ones, is a pivotal stage in cancer development,and is an important target for cancer therapy. We develop a hybrid mathematical model to understand the mechanisms behind tumor-induced angiogenesis. This model describes uptake of Tumor Angiogenic Factor（TAF）at extracellular level, uses partial differential equation to describe the evolution of endothelial cell density including TAF induced proliferation, chemotaxis to TAF, and haptotaxis to extracellular matrix. In addition we also consider the phenomenon of blood perfusion in the micro-vessels. The model produces sprout formation with realistic morphological and dynamical features, including the so-called brush border effect, the dendritic branching and fusing of the capillary sprouts forming a vessel network. The model also demonstrates the effects of individual mechanisms in tumor angiogenesis： Chemotaxis to TAF is the key driving mechanisms for the extension of sprout cell; endothelial proliferation is not absolutely necessary for sprout extension; haptotaxis to Extra Cellular Matrix（ECM） gradient provides additional guidance to sprout extension, suggesting potential targets for anti-angiogenic therapies.

Photoacoustic mesoscopy:pushing toward the depth limit in the high-resolution optical imaging for biomedical applications and clinical potentials

Photoacoustic mesoscopy(PAMe) offers high-sensitivity in vivo imaging based on the rich optical contrast in biological tissues,with sub-100-micron resolutions at a few millimeters depth. By benefiting from low ultrasonic scattering,this emerging technology has pushed the penetration depth beyond the optical diffuse limit unprecedented for high-resolution optical methods.Here,we review ed the state-of-art implementations of PAMe and their achievements in biological and primary clinical applications. With the high-frequency focused ultrasonic detector,the high-resolution optical visualization can be achieved by utilizing various PAMe systems. These capabilities of PAMe have made it well applicable for understanding the biological mechanisms,exploring the pathological features and analyzing the characteristics of human skin. Future improvements and prospects of PAMe are also mentioned,suggesting its great potential tow ards the corresponding emerging biomedical and clinical applications.

Bear Bile Powder Inhibits Angiogenesis In Vivo and In Vitro

Objective： To evaluate the effect of bear bile powder （BBP） on angiogenesis, and investigate the underlying molecular mechanisms. Methods： A chick embryo chorioallantoic membrane （CAM） assay was used to evaluate the angiogensis in vivo. Human umbilical vein endothelial cells （HUVECs） were treated with 0, 0.25, 0.5, 0.75, and 1.0 mg/mL of BBP for 24, 48 and 72 h, respectively. The 3-（4, 5-dimethylthiazol-2-yl）-2,5- diphenyltetrazolium bromide assay was performed to determine the viability of HUVECs. Ceil cycle progression of HUVECs was examined by fluorescence-activated cell sorting （FACS） analysis with propidium iodide staining. HUVEC migration was determined by wound healing method. An ECMatrix gel system was used to evaluate the tube formation of HUVECs. The mRNA and protein expression of vascular endothelial growth factor （VEGF）-A in both HUVECs and HepG2 human cells were examined by reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay, respectively. ]Results： Compared with the untreated group, BBP inhibited angiogenesis in vivo in the CAM model （P〈0.01）. In addition, treatment with 0.25-1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability by 14%-27%, 29%-69% and 33%-91%, compared with the untreated control cells （P〈0.01）. Additionally, BBP inhibited the proliferation of HUVECs via blocking the cell cycle G1 to S progression, compared with the S phase of untreated cells 48.05% ± 5.00%, 0.25-0.75 mg/mL BBP reduced S phase to 40.38% ± 5.30%, 36.54 ±4.50% and 32.13 ± 3.50%, respectively （P〈0.05）. Moreover, BBP inhibited the migration and tube formation of HUVECs, compared with the tube length of untreated cells 100%± 12%, 0.25-0.75 mg/mL BBP reduced the tube length to 62% ± 9%, 43% ± 5% and 17% ± 3%, respectively （P〈0.01）. Furthermore, BBP treatment down-regulated the mRNA and protein expression levels of VEGF-A in both HepG2 cells and HUVECs. Conclusion： BBP could inhibit the angiogenesis by reducing VEGF-A expression, which may, in part, explain its anti-tumor activity.