Tumor Vessel Maturation Research Articles

VEGF165b Mutant Promotes the Apoptosis of Murine Breast Cancer Cells Induced by Paclitaxel by Inducing Tumor Vessel Maturation.

The anti-angiogenic agent vascular endothelial growth factor 165b (VEGF165b) mutant (mVEGF165b), which was developed by our laboratory, has superior antitumor activity to that of native VEGF165b; however, its mechanism of action and druggability need further exploration. Using the commercial anti-angiogenic drug bevacizumab as a positive control, the mechanism and developability of mVEGF165b were evaluated and explored. The Cell Counting Kit-8 assay was performed to evaluate the effects of mVEGF165b and bevacizumab alone on the proliferation of human umbilical vein endothelial cells (HUVECs). Meanwhile, the inhibitory effects of mVEGF165b and bevacizumab combined with paclitaxel in a mouse model of breast cancer were assessed. Immunohistochemistry was used to detect their effects on tumor vascular maturation, and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to detect the apoptosis of tumor cells. In vitro cell experiments confirmed that mVEGF165b inhibited the proliferation of HUVECs with an efficacy equivalent to that of bevacizumab. mVEGF165b and bevacizumab combined with paclitaxel significantly delayed the growth of breast cancer in mice. Immunohistochemistry and the TUNEL assay showed that mVEGF165b and bevacizumab combined with paclitaxel-induced higher vascular maturity and more apoptosis than paclitaxel alone. mVEGF165b showed similar efficacy and mechanism of action as bevacizumab, indicating its potential to be developed into a safe and effective anti-angiogenic drug.

SOD3 Expression in Tumor Stroma Provides the Tumor Vessel Maturity in Oral Squamous Cell Carcinoma.

Tumor angiogenesis is one of the hallmarks of solid tumor development. The progressive tumor cells produce the angiogenic factors and promote tumor angiogenesis. However, how the tumor stromal cells influence tumor vascularization is still unclear. In the present study, we evaluated the effects of oral squamous cell carcinoma (OSCC) stromal cells on tumor vascularization. The tumor stromal cells were isolated from two OSCC patients with different subtypes: low invasive verrucous squamous carcinoma (VSCC) and highly invasive squamous cell carcinoma (SCC) and co-xenografted with the human OSCC cell line (HSC-2) on nude mice. In comparison, the CD34+ vessels in HSC-2+VSCC were larger than in HSC-2+SCC. Interestingly, the vessels in the HSC-2+VSCC expressed vascular endothelial cadherin (VE-cadherin), indicating well-formed vascularization. Our microarray data revealed that the expression of extracellular superoxide dismutase, SOD3 mRNA is higher in VSCC stromal cells than in SCC stromal cells. Moreover, we observed that SOD3 colocalized with VE-cadherin on endothelial cells of low invasive stroma xenograft. These data suggested that SOD3 expression in stromal cells may potentially regulate tumor vascularization in OSCC. Thus, our study suggests the potential interest in SOD3-related vascular integrity for a better OSCC therapeutic strategy.

Combination Treatment of TRPV4 Agonist with Cisplatin Promotes Vessel Normalization in an Animal Model of Oral Squamous Cell Carcinoma.

Background and Objectives: Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy in the world. Transient receptor potential vanilloid 4 (TRPV4) channel has been shown to be involved in angiogenesis in multiple types of tumors. However, not much is known about TRPV4′s involvement in OSCC. Thus, in this study, we investigate the effect of administering a TRPV4 agonist on angiogenesis in OSCC. Materials and Methods: Thirty-six Sprague Dawley (SD) rats were used in this study. 4-nitroquinoline 1-oxide (4NQO) was used to induce OSCC. Cisplatin (an anticancer drug), and GSK1016790A (an agonist for TRPV4) was used in this study. Immunohistochemistry was employed to examine the TRPV4 expression. An RT2 Profiler PCR Array was performed for gene expression analysis of TRPV4, vascular growth factors that correspond directly with angiogenesis, such as angiopoietin (Ang-1 and Ang-2), and tyrosine kinase (Tie-1 and Tie-2) receptors. Tumor vessel maturity was assessed by microvessel density and microvessel-pericyte-coverage index. Results: RT2 profiler PCR array showed significant elevated levels of Ang-1 (2.1-fold change; p < 0.05) and Tie-2 (4.5-fold change; p < 0.05) in OSCC following the administration of a combination of GSK1016790A and cisplatin. Additionally, the combination treatment significantly reduced the microvessel density (p < 0.01) and significantly increased the percentage of microvessels covered with pericytes (p < 0.01) in OSCC. Furthermore, tumor size was significantly reduced (p < 0.05) in rats that received cisplatin alone. The combination treatment also greatly reduced the tumor size; however, the data were not statistically significant. Conclusions: The findings suggest that combining a TRPV4 agonist with cisplatin for treatment of OSCC promote vessels normalization via modulation of Ang-1/Tie-2 pathway.

Value of dynamic MRI in monitoring the microenvironmental changes of anti-vascular therapy in a xenograft model

Objective: To explore the feasibility of dynamic-enhanced magnetic resonance imaging (DCE-MRI) and blood oxygen level-dependent MRI (BOLD-MRI) in assessing the hemodynamics and tumor aggressiveness during treatment. Methods: The colon cancer xenograft model was established in BALB/C nude mice with HCT116 cell line. Sixteen nude mice were randomly divided into treatment and control groups (aged 6 to 8 weeks, weighted 15 to 18 g, Certificate No. 11400700325797), which were treated with bevacizumab and saline by intraperitoneal injection on the 1st, 4th, 7th, 10th and 13th day. DCE-MRI and BOLD-MRI were performed before and on the 3th, 6th, 9th, 12th, and 15th day after treatment. The vascular maturity and microenvironment hypoxia were confirmed by pathology. Results: The tumor volume of treatment group was significantly smaller than that of control group after 15 days ((712±43) vs (1 051±112) mm(3),P<0.01).The measurements of K(trans) were (0.135±0.005),(0.147±0.006),(0.175±0.009),(0.161±0.006), (0.140±0.005),(0.116±0.008)/min (F=81.386, P<0.01); K(ep) were (0.788±0.030),(0.804±0.036),(0.983±0.059), (1.105±0.091),(0.840±0.047),(0.786±0.041)/min(F=45.901,P<0.01);Ve were (0.652±0.006), (0.559±0.026), (0.466±0.016), (0.286±0.027), (0.363±0.020), (0.246±0.033) (F=384.290, P<0.01) and R2* values were (24.813±0.961), (24.675±1.070), (21.425±1.371), (17.850±0.885), (24.613±0.640), (27.013±0.734)/s (F=89.323, P<0.01) showed different trends with time in the treatment group, and the differences were statistically significant. The K(trans) values and tumor vessel maturity index (VMI) were higher than baseline values during 3-12 d after treatment. CD31 positive staining rate and VMI had the strongest correlations with K(trans) values (r=0.854 and 0.795), followed by AUC(180) (r=0.750 and 0.808), Ve (r=0.744 and 0.712) and K(ep) values (r=0.729 and 0.758), all P<0.05. R2* value positively correlated with the positive staining rate of HIF-1α and fibronectin (r=0.810 and 0.816), all P<0.05. Conclusion: DCE-MRI and BOLD-MRI are adequate to observe the tumor perfusion and hypoxia during anti-vascular treatment, and the R2* value can predict the tumor metastatic potential during the process of vascular normalization.

M1-like macrophages change tumor blood vessels and microenvironment in murine melanoma.

Tumor-associated macrophages (TAMs) play a significant role in at least two key processes underlying neoplastic progression: angiogenesis and immune surveillance. TAMs phenotypic changes play important role in tumor vessel abnormalization/ normalization. M2-like TAMs stimulate immunosuppression and formation of defective tumor blood vessels leading to tumor progression. In contrast M1-like TAMs trigger immune response and normalize irregular tumor vascular network which should sensitize cancer cells to chemo- and radiotherapy and lead to tumor growth regression. Here, we demonstrated that combination of endoglin-based DNA vaccine with interleukin 12 repolarizes TAMs from tumor growth-promoting M2-like phenotype to tumor growth-inhibiting M1-like phenotype. Combined therapy enhances tumor infiltration by CD4+, CD8+ lymphocytes and NK cells. Depletion of TAMs as well as CD8+ lymphocytes and NK cells, but not CD4+ lymphocytes, reduces the effect of combined therapy. Furthermore, combined therapy improves tumor vessel maturation, perfusion and reduces hypoxia. It caused that suboptimal doses of doxorubicin reduced the growth of tumors in mice treated with combined therapy. To summarize, combination of antiangiogenic drug and immunostimulatory agent repolarizes TAMs phenotype from M2-like (pro-tumor) into M1-like (anti-tumor) which affects the structure of tumor blood vessels, improves the effect of chemotherapy and leads to tumor growth regression.

VEGF121 and VEGF165 differentially promote vessel maturation and tumor growth in mice and humans.

Tumor angiogenesis depends on the vascular endothelial growth factor (VEGF), which exists in multiple splicing isoforms, including the most abundant VEGF165 and VEGF121. We have previously shown that the differential capacity of these two VEGF isoforms to bind Neuropilin-1 accounts for their diverse ability to recruit Nrp1-expressing monocytes (NEMs), resulting in a different arteriogenic potential. Here we measure the expression of VEGF165 and VEGF121 in human cancer and their influence on tumor growth and vascularization. We measured the expression levels of VEGF165 and VEGF121 in human colorectal cancer and found that VEGF121 was more expressed than VEGF165, particularly in patients with extensive lymph node infiltration. Overexpressing either VEGF165 or VEGF121 in a cancer mouse model, we observed that the former decreased, whereas the latter increased tumor growth. In both clinical and experimental tumors, VEGF165 expression resulted in the recruitment of NEMs, paralleled by maturation of the tumor vascular network. Finally, hypoxia induced a shift toward the VEGF165 isoform in the central core of human cancers, as well as in various types of cultured cells. These results demonstrate that the two VEGF splicing isoforms are differentially expressed in colorectal cancers, exerting opposite effects on tumor growth and vessel maturation.

Quantitatively evaluating the evolution of the tumor perfusion in A549 lung adenocarcinoma transplantation model induced by antiangiogenic treatment

To quantitatively evaluate the evolution of the tumor perfusion in A549 lung adenocarcinoma transplantation model induced by antiangiogenic treatment. To establish the preclinical transplantation model of lung adenocarcinoma, 60 BALB/c nu/nu mice was inoculated with A549 cell lines via axilla. Sixty mice were randomly divided into 2 groups. The treatment group was treated with intravenous Bevacizumab (10 mg/kg weight, in a single injection), and the control group received saline only in the same dose. Five times of volume perfusion CT (VPCT) scan was performed before treatment, and on the second, forth, sixth and tenth days of treatment, respectively. The values of blood flow (BF) in the A549 tumors were measured after scanning. The microvessel density (MVD), vessel maturity index (VMI) in the tumors were determined using multiplexed QDs-based immunohistochemical staining. Comparing the values of BF, VMI and MVD between the two groups on the same day before treatment, the values of BF, VMI and MVD of the treatment group were (13.5±1.5) ml·(100 ml)(-1)·min(-1,) 0.14±0.04, (45.7±16.5)/HPF, respectively, and those in the control group were (13.4±1.6) ml·(100 ml)(-1)·min(-1) , 0.14±0.05, (48.0±7.0) /HPF , respectively. There was no significant difference between the two groups (all P>0.05). And on the second, forth, sixth, tenth days of treatment, the values of BF of the treatment group were (17.9±7.3), (32.2±6.9), (18.5±2.4) and (13.8±1.8) ml·(100 ml)(-1)·min(-1,) respectively, and those in the control group were (10.5±0.6), (9.6±0.8), (5.7±1.2) and (1.9±1.0) ml·(100 ml)(-1)·min(-1,) respectively. The values of VMI of the treatment group were 1.17±0.22, 3.25±0.23, 2.94±0.31 and 1.07±0.18, respectively, and those in the control group were 0.12±0.03, 0.13±0.03, 0.15±0.03, and 0.13±0.03, respectively. The values of MVD of the treatment group were (38.0±6.3), (24.3±5.4), (15.2±3.4) and (13.5±4.7)/HPF, respectively, and those in the control group were (44.8±5.9), (48.0±12.8), (41.8±5.7) and (45.7±20.3)/HPF, respectively. In treated mice, BF and VMI were significantly higher than those in the control group (all P<0.01). BF and VMI increased from day2, and reached the peak at day4 (P<0.01), then decreased at day6, however the value of BF at day6 was still higher than that in the baseline (P<0.01) and decreased to the baseline level at day10; while the value of VMI was still higher than that in the baseline at day10. And on the forth, sixth, tenth days of treatment, in treated mice, the values of MVD were significantly lower than those in the control group and the baseline level before treatment (all P<0.01). In control mice, BF decreased (all P<0.01) with the time, while MVD and VMI had no changes. The tumor perfusion and vessel maturity are transiently increased in A549 lung adenocarcinoma transplantation model induced by antiangiogenic treatment. VPCT is helpful to quantify the evolution of the tumor perfusion and then evaluate the functional changes of tumor vessel maturity.

Apelin as a marker for monitoring the tumor vessel normalization window during antiangiogenic therapy.

Antiangiogenic agents transiently normalize tumor vessel structure and improve vessel function, thereby providing a window of opportunity for enhancing the efficacy of chemotherapy or radiotherapy. Currently, there are no reliable predictors or markers reflecting this vessel normalization window during antiangiogenic therapy. Apelin, the expression of which is regulated by hypoxia, and which has well-described roles in tumor progression, is an easily measured secreted protein. Here, we show that apelin can be used as a marker for the vessel normalization window during antiangiogenic therapy. Mice bearing s.c. tumors resulting from inoculation of the colon adenocarcinoma cell line HT29 were treated with a single injection of bevacizumab, a mAb neutralizing vascular endothelial growth factor. Tumor growth, vessel density, pericyte coverage, tumor hypoxia, and small molecule delivery were determined at four different times after treatment with bevacizumab (days 1, 3, 5, and 8). Tumor growth and vessel density were significantly reduced after bevacizumab treatment, which also significantly increased tumor vessel maturity, and improved tumor hypoxia and small molecule delivery between days 3 and 5. These effects abated by day 8, suggesting that a time window for vessel normalization was opened between days 3 and 5 during bevacizumab treatment in this model. Apelin mRNA expression and plasma apelin levels decreased transiently at day 5 post-treatment, coinciding with vessel normalization. Thus, apelin is a potential indicator of the vessel normalization window during antiangiogenic therapy.

Activation of mechanosensitive ion channel TRPV4 normalizes tumor vasculature and improves cancer therapy.

Tumor vessels are characterized by abnormal morphology and hyper-permeability that together cause inefficient delivery of chemotherapeutic agents. Although VEGF has been established as a critical regulator of tumor angiogenesis, the role of mechanical signaling in the regulation of tumor vasculature or tumor endothelial cell (TEC) function is not known. Here, we show that the mechanosensitive ion channel TRPV4 regulates tumor angiogenesis and tumor vessel maturation via modulation of TEC mechanosensitivity. We found that TEC exhibit reduced TRPV4 expression and function, which is correlated with aberrant mechanosensitivity towards ECM stiffness, increased migration and abnormal angiogenesis by TEC. Further, syngeneic tumor experiments revealed that the absence of TRPV4 induced increased vascular density, vessel diameter and reduced pericyte coverage resulting in enhanced tumor growth in TRPV4 KO mice. Importantly, overexpression or pharmacological activation of TRPV4 restored aberrant TEC mechanosensitivity, migration and normalized abnormal angiogenesis in vitro by modulating Rho activity. Finally, a small molecule activator of TRPV4, GSK1016790A, in combination with anti-cancer drug Cisplatin, significantly reduced tumor growth in WT mice by inducing vessel maturation. Our findings demonstrate TRPV4 channels to be critical regulators of tumor angiogenesis and represent a novel target for anti-angiogenic and vascular normalization therapies.

Prostaglandin I2 analog suppresses lung metastasis by recruiting pericytes in tumor angiogenesis.

Prostaglandin I2 (PGI2) agonist has been reported to reduce tumor metastasis by modifying tumor angiogenesis; however, the mechanisms of how PGI2 affects the endothelial cells or pericytes in tumor vessel maturation are still unclear. The purpose of this study was to clarify the effects of PGI2 on tumor metastasis in a mouse lung metastasis model using Lewis lung carcinoma (LLC) cells. The mice were treated continuously with beraprost sodium (BPS), a PGI2 analog, for 3 weeks and then examined for lung metastases. The number and size of lung metastases were decreased significantly by BPS treatment. In addition, scanning electron microscopy and immunohistochemistry revealed that BPS increased the number of tumor‑associated pericytes and improved intratumor hypoxia. Collectively, this study suggests that BPS attenuated vascular functional maturation in metastatic tumors.

Use of genetic variants in pericyte-driven tumor vessel maturation genes to predict treatment efficacy in mCRC patients treated with FOLFIRI/bevacizumab.

3566 Background: Angiogenesis is maintained by the presence of PDGFB (platelet derived growth factor beta) which binds to the receptor, PDGFRB on pericytes, signaling them to stabilize stalk cells adjacent to tip cells. PDGFB signaling by endothelial tip cells promotes proliferation, migration, and stabilization of pericytes which are closely associated with endothelial cells. VEGF driven angiogenesis in response to tumor hypoxia relies on several genes. Previously, studies showed RALBP1, RGS5, and CSPG4 greatly impact the structure of blood vessels during angiogenesis. Recognizing these genes are important in angiogenesis through pericytes, we investigated the correlation between polymorphisms in these genes and clinical outcomes in patients with mCRC treated with FOLFIRI/bevacizumab (BV). Methods: Genomic DNA was extracted from 455 patients’ blood or tissue treated with first-line BV + FOLFIRI and prospectively enrolled in a prospective pharmacogenomic translational study. Median follow up is 24 months and median PFS and OS were 10.5 and 29.9 months, respectively. Eight functionally significant SNPs; RGS5 (rs1056515, rs2661280), PDGFRB (rs2229562, rs2302271), CSPG4 (rs8023621, rs1127648), and RALBP1 (rs10989, rs329007) were analyzed by PCR-based direct sequencing. All candidate SNPs were analyzed for association with tumor response rate (RR), progression free survival (PFS), and overall survival (OS). Results: Four SNPs showed significant results in the univariate analysis of either OS or PFS. PDGFRB rs2302273 remained significant upon multivariate analysis. Univariate analysis of PDGFRB rs2302273 concluded patients with any T (CT/TT) allele were significantly associated with longer PFS compared to those with CC genotypes (median 10.2 vs 11.6 months, HR=0.78 [95%CI: 0.63-.98], p=0.031, log-rank test) which remained significant upon multivariate analysis (HR=0.73 [95% CI: 0.57-0.92], p=0.008, log-rank test). Conclusions: Our results show that the PDGFRB polymorphism rs2302273 may serve as a prognostic marker for the efficacy of FOLFIRI + BV treatment in patients with mCRC. Studies to confirm and update these preliminary findings are ongoing.

Angiopoietin-2 expression is correlated with angiogenesis and overall survival in oral squamous cell carcinoma

This study sought to determine the expression of angiopoietin-2 (Ang-2) and vascular endothelial cell growth factor (VEGF) in oral squamous cell carcinoma (OSCC) and assess their correlations with tumor progression, angiogenesis, vessel maturation, and clinical survival. Tumor tissue from 102 OSCC patients, adjacent noncancerous oral tissue from 79 OSCC patients, and normal oral mucosa from 35 control patients were examined for Ang-2 and VEGF expression using conventional immunohistochemistry. Microvessel density (MVD) and vessel maturation index (VMI) were assessed by double-label immunohistochemistry staining using anti-CD34 and anti-alpha-smooth muscle actin, respectively. Although the proportion of OSCC samples positive for Ang-2 or VEGF expression was significantly higher than that observed in the adjacent noncancerous tissue and normal oral mucosa (P<0.001), neither Ang-2 nor VEGF expression was associated with the clinicopathological parameters analyzed in OSCC patients. However, MVD and VMI were significantly associated with the expression of Ang-2 (P=0.001 and P=0.014, respectively); VEGF expression was associated MVD (P=0.004). The MVD of OSCC tissues expressing both Ang-2 and VEGF was significantly higher than observed in the double-negative samples (P<0.05). Multivariate regression and Kaplan-Meier analyses revealed that Ang-2 was negatively associated with the overall survival of OSCC patients. Expression of Ang-2 was associated with angiogenesis and vessel maturation in OSCC. Further studies will evaluate the prognostic value of determining Ang-2 expression in OSCC.

Abstract 1619: Combination of VEGFR inhibitor lenvatinib (E7080) and Met/EphB4 inhibitor golvatinib (E7050) overcomes VEGFR inhibitor-resistant tumor vascular.

Abstract Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several types of tumors, however, some tumor shows intrinsic resistance to VEGFR inhibitors. In addition, patients initially responding develop acquired resistance to VEGFR inhibitors after varying periods of time. Thus it is urgent to develop strategy to overcome anti-VEGF therapy resistance. Hepatocyte growth factor (HGF)/Met signaling is activated in various types of tumors. Recently, it is reported that HGF/Met signaling contributes to resistance to tyrosine kinase inhibitors such as EGFR, BRAF, ALK inhibitors. This suggests that Met inhibitors may be promising in treating HGF/Met activated anti-VEGF therapy-resistant tumors. We have identified lenvatinib (E7080) as a highly-potent VEGFR inhibitor and phase III trial is ongoing in thyroid cancer. Lenvatinib inhibited the growth of HUVEC induced by VEGF, however, addition of HGF to HUVEC conferred resistance to lenvatinib. This resistance was cancelled by the addition of golvatinib (E7050) which is identified as a potent Met/EphB4 inhibitor and phase I trial of the lenvatinib/golvatinib combination therapy is ongoing. In mouse models bearing various tumor cell lines expressing VEGF and HGF, treatment with lenvatinib alone demonstrated only moderate antitumor activity, however, combination with golvatinib demonstrated synergistic antitumor activity with decrease of tumor vessel density. Furthermore, it is reported that EphB4 has critical roles in tumor vessel development and maturation. Our unique in vitro assay systems, endothelial cells/pericyte 2D co-culture assay and endothelial cells/pericyte 3D co-culture sprouting assay, suggested that EphB4 inhibitory activity of golvatinib disrupted anti-VEGF therapy-resistant tumor vascular in the combination with lenvatinib. Together, these data demonstrated that combination therapy with golvatinib may be promising to overcome anti-VEGF therapy resistance. Citation Format: Satoshi Kawano, Takayuki Nakagawa, Yuya Nakazawa, Yoshiaki Sato, Tomohiro Matsushima, Yasuhiro Funahashi. Combination of VEGFR inhibitor lenvatinib (E7080) and Met/EphB4 inhibitor golvatinib (E7050) overcomes VEGFR inhibitor-resistant tumor vascular. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1619. doi:10.1158/1538-7445.AM2013-1619

Regulatory T cells get nervous in the melanoma microenvironment

Regulatory T cells get nervous in the melanoma microenvironment

Neuropilin-1 Identifies a Subset of Bone Marrow Gr1− Monocytes That Can Induce Tumor Vessel Normalization and Inhibit Tumor Growth

Improving tumor perfusion, thus tempering tumor-associated hypoxia, is known to impair cancer progression. Previous work from our laboratory has shown that VEGF-A165 and semaphorin 3A (Sema3A) promote vessel maturation through the recruitment of a population of circulating monocytes expressing the neuropilin-1 (Nrp1) receptor (Nrp1-expressing monocytes; NEM). Here, we define the characteristics of bone marrow NEMs and assess whether these cells might represent an exploitable tool to induce tumor vessel maturation. Gene expression signature and surface marker analysis have indicated that NEMs represent a specific subset of CD11b+ Nrp1+ Gr1- resident monocytes, distinctively recruited by Sema3A. NEMs were found to produce several factors involved in vessel maturation, including PDGFb, TGF-β, thrombospondin-1, and CXCL10; consistently, they were chemoattractive for vascular smooth muscle cells in vitro. When directly injected into growing tumors, NEMs, isolated either from the bone marrow or from Sema3A-expressing muscles, exerted antitumor activity despite having no direct effects on the proliferation of tumor cells. NEM inoculation specifically promoted mural cell coverage of tumor vessels and decreased vascular leakiness. Tumors treated with NEMs were smaller, better perfused and less hypoxic, and had a reduced level of activation of HIF-1α. We conclude that NEMs represent a novel, unique population of myeloid cells that, once inoculated into a tumor, induce tumor vessel normalization and inhibit tumor growth.

Evaluation of novel combined carbogen USPIO (CUSPIO) imaging biomarkers in assessing the antiangiogenic effects of cediranib (AZD2171) in rat C6 gliomas

The recently described combined carbogen USPIO (CUSPIO) magnetic resonance imaging (MRI) method uses spatial correlations in independent imaging biomarkers to assess specific components of tumor vascular structure and function. Our study aimed to evaluate CUSPIO biomarkers for the assessment of tumor response to antiangiogenic therapy. CUSPIO imaging was performed in subcutaneous rat C6 gliomas before and 2 days after treatment with the potent VEGF-signaling inhibitor cediranib (n = 12), or vehicle (n = 12). Histological validation of Hoechst 33342 uptake (perfusion), smooth muscle actin staining (maturation), pimonidazole adduct formation (hypoxia) and necrosis were sought. Following treatment, there was a significant decrease in fractional blood volume (-43%, p < 0.01) and a significant increase in hemodynamic vascular functionality (treatment altered ΔR(2) *(carbogen) from 1.2 to -0.2 s(-1) , p < 0.05). CUSPIO imaging revealed an overall significant decrease in plasma perfusion (-27%, p < 0.05) following cediranib treatment, that was associated with selective effects on immature blood vessels. The CUSPIO responses were associated with a significant 15% reduction in Hoechst 33342 uptake (p < 0.05), but no significant difference in vascular maturation or necrosis. Additionally, treatment with cediranib resulted in a significant 40% increase in tumor hypoxia (p < 0.05). The CUSPIO imaging method provides novel and more specific biomarkers of tumor vessel maturity and vascular hemodynamics, and their response to VEGF-signaling inhibition, compared to current MR imaging biomarkers utilized in the clinic. Such biomarkers may prove effective in longitudinally monitoring tumor vascular remodeling and/or evasive resistance in response to antiangiogenic therapy.

The Antiangiogenic 16K Prolactin Impairs Functional Tumor Neovascularization by Inhibiting Vessel Maturation

BackgroundAngiogenesis, the formation of new blood vessels from existing vasculature, plays an essential role in tumor growth, invasion, and metastasis. 16K hPRL, the antiangiogenic 16-kDa N-terminal fragment of human prolactin was shown to prevent tumor growth and metastasis by modifying tumor vessel morphology.Methodology/Principal FindingsHere we investigated the effect of 16K hPRL on tumor vessel maturation and on the related signaling pathways. We show that 16K hPRL treatment leads, in a murine B16-F10 tumor model, to a dysfunctional tumor vasculature with reduced pericyte coverage, and disruption of the PDGF-B/PDGFR-B, Ang/Tie2, and Delta/Notch pathways. In an aortic ring assay, 16K hPRL impairs endothelial cell and pericyte outgrowth from the vascular ring. In addition, 16K hPRL prevents pericyte migration to endothelial cells. This event was independent of a direct inhibitory effect of 16K hPRL on pericyte viability, proliferation, or migration. In endothelial cell-pericyte cocultures, we found 16K hPRL to disturb Notch signaling.Conclusions/SignificanceTaken together, our data show that 16K hPRL impairs functional tumor neovascularization by inhibiting vessel maturation and for the first time that an endogenous antiangiogenic agent disturbs Notch signaling. These findings provide new insights into the mechanisms of 16K hPRL action and highlight its potential for use in anticancer therapy.

Tumor Angiogenesis: Pericytes and Maturation Are Not to Be Ignored

Angiogenesis, an essential component of tumor growth and survival, is regulated by complex interactions between several cell types and soluble mediators. Heterogeneous tumor vasculature originates from the collective effect of the nature of carcinoma and the complexity of the angiogenic network. Although the application of angiogenesis inhibitors in some types of cancers has shown clinical benefits, predictive markers to assess treatment effects have yet to be established. In this review, we focus on tumor vessel maturity as a potential marker for evaluating treatment response.

Augmented therapeutic efficacy of irinotecan is associated with enhanced drug accumulation

The goal of this study is to determine whether treatment with methylselenocysteine (MSC) results in differential uptake of irinotecan and its active metabolite (SN-38) between tumors of head and neck squamous cell carcinomas and normal tissue. The in vivo synergy between MSC and irinotecan is influenced by treatment schedule and associated with enhancement of tumor vessel maturation, intra-tumor concentration of SN-38 and apoptotic death of tumor cells. Normal tissue drug concentrations were not impacted by selenium treatment. The finding is of clinical relevance for enabling the delivery of higher doses of irinotecan to reverse tumor resistance, recurrence and ultimately enhancing cure rates.

CEACAM1 creates a pro-angiogenic tumor microenvironment that supports tumor vessel maturation

We have studied the effects of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) on tumor angiogenesis in murine ductal mammary adenocarcinomas. We crossed transgenic mice with whey acidic protein promoter-driven large T-antigen expression (WAP-T mice) with oncogene-induced mammary carcinogenesis with CEACAM1null mice, and with Tie2-Ceacam1 transgenics, in which the Tie2 promoter drives endothelial overexpression of CEACAM1 (WAP-T × CEACAM1(endo+) mice), and analyzed tumor vascularization, angiogenesis and vessel maturation in these mice. Using flat-panel volume computed tomography (fpVCT) and histology, we found that WAP-T × CEACAM1(endo+) mice exhibited enhanced tumoral vascularization owing to CEACAM1(+) vessels in the tumor periphery, and increased intratumoral angiogenesis compared with controls. In contrast, vascularization of CEACAM1null/WAP-T-derived tumors was poor, and tumor vessels were dilated, leaky and showed poor pericyte coverage. Consequently, the tumoral vasculature could not be visualized in CEACAM1null/WAP-T mice by fpVCT, and we observed poor organization of the perivascular extracellular matrix (ECM), accompanied by the accumulation of collagen IV-degrading matrix metalloproteinase 9(+) (MMP9(+)) leukocytes and stromal cells. Vascular instability and alterations in ECM structure were accompanied by a significant increase in pulmonary metastases in CEACAM1null/WAP-T mice, whereas only occasional metastases were observed in CEACAM1(+) hosts. In CEACAM1(+) hosts, intratumoral vessels did not express CEACAM1, but they were intact, extensively covered with pericytes and framed by a well-organized perivascular ECM. MMP9(+) accessory cells were largely absent. Orthotopic transplantation of primary WAP-T- and CEACAM1null/WAP-T tumors into all three mouse lines confirmed that a CEACAM1(+) host environment is a prerequisite for productive angiogenic remodeling of the tumor microenvironment. Hence, CEACAM1 expression in the tumor periphery determines the vascular phenotype in a tumor, whereas systemic absence of CEACAM1 interferes with the formation of an organized tumor matrix and intratumoral vessel maturation.