Changes In Microvessel Density Research Articles

Neutrophil extracellular traps promote angiogenesis in gastric cancer

Although antiangiogenic therapy has been used in gastric cancer, disease progression due to drug resistance remains common. Neutrophils play an important role in the occurrence and progression of cancer via neutrophil extracellular traps (NETs). However, few studies have investigated angiogenic regulation in gastric cancer. We aimed to determine the role of NETs in promoting angiogenesis in gastric cancer. Multiple immunohistochemical staining was used to analyze the spatial distribution of NETs and microvessels in patient tissue samples. A mouse subcutaneous tumor model was established to determine the effect of NETs on tumor growth, and changes in microvessel density were observed via immunohistochemical staining. We screened differentially expressed proteins in HUVECs stimulated by NETs via proteomics. Cell Counting Kit-8, EdU labeling, and tubule formation assays were used to verify the effect of NETs on HUVEC proliferation, migration, and tubule formation. Blocking NETs, which was related to decreased microvessel density, significantly inhibited tumor growth in the murine subcutaneous tumor model. Compared with those of the control group, tumor volume and mass among mice in the inhibition group decreased by 61.3% and 77.9%, respectively. The NET-DNA receptor CCDC25 was expressed in HUVECs, providing a platform for NETs to promote HUVEC proliferation, migration, and tubulation. In an in vitro rat aortic explant model, NETs induced HUVEC proliferation, survival, and chemotaxis, which were not significantly different from those observed in the VEGF stimulation group. Our results confirm that NETs promote angiogenesis in gastric cancer, providing a theoretical basis for identifying new anti-vascular therapeutic targets.Graphical 1Sf8jYXSegxbAWaiHcqZYYVideo

Therapeutic ultrasound combined with microbubbles improves atherosclerotic plaque stability by selectively destroying the intraplaque neovasculature.

Objective: The current antiangiogenic therapy for atherosclerotic plaques was mainly achieved by the use of antiangiogenic drugs, but serious side effects have limited the clinical application. The present study investigated whether therapeutic ultrasound (TUS) treatment with appropriate pressure could selectively deplete the neovasculature in vulnerable plaques to improve its stability with no side effects on the body; the underlying mechanisms were also explored.Methods and Results: A mouse model of advanced atherosclerosis was generated by maintaining apolipoprotein E-deficient (ApoE-/-) mice on a hypercholesterolemic diet (HCD). Plaque, skeletal muscle, mesentery and skin tissue from 114 atheroma-bearing mice were subjected to sham therapy, an ultrasound application combined with microbubbles at four different ultrasound pressures (1.0, 2.0, 3.0, 5.0 MPa), or ultrasound at 5.0 MPa alone. Microvessel density (MVD) was assessed by immunofluorescence and immunohistochemical methods. The plaque necrotic center/fiber cap (NC/FC) ratio and vulnerability index were calculated to evaluate plaque vulnerability. Twenty-four hours after TUS treatment at 3.0 MPa, the MVD in the plaque was substantially decreased by 84% (p < 0.05), while there was almost no change in MVD and neovessel density (NVD) in normal tissues, including skeletal muscle, mesentery and skin. Additionally, a marked reduction in the number of immature vessels was observed in the plaques (reduced by 90%, p < 0.05), whereas the number of mature vessels was not significantly decreased. Furthermore, TUS treatment at 3.0 MPa significantly improved plaque stability, as reflected by the NC/FC ratio and vulnerability index, which may be due to the selective destruction of intraplaque neovascularization by TUS treatment, thereby decreasing the extravasation of erythrocytes and leading to vascular inflammation alleviation and thin-cap fibroatheroma reduction.Conclusions: TUS treatment at 3.0 MPa selectively depleted plaque neovessels and improved the stability of vulnerable plaques through a reduction in erythrocyte extravasation and inflammatory mediator influx, with no significant effect on normal tissue.

Placenta Of Late Onset Preeclampsia Without Fetal Growth Restriction : Is It Different From The Normal?

Background: Preeclampsia (PE) is one of the most common pregnancy complications affecting approximately 5-7% of pregnant women worldwide. Pathogenesis of PE is still mysterious. Literature studies reported differences between early and late onset PE. Also, whether placenta of late-onset PE without fetal growth restriction (FGR) is different from normal one needs further declaration.Objective: This study aims at evaluating the placental microvessel density (MVD), apoptosis and endoglin (CD105) expression in late-onset PE without FGR.Patients and Methods: Placentae of 15 PE and 15 matched control ones were evaluated grossly, microscopically and by immunohistochemistry for caspase-3, CD 34 and CD105.Results: Placentas in PE group showed a statistically significant difference as regard size, syncytial knots, perivillous fibrin deposition, villous infarction increased apoptosis, and endoglin (CD105) expression. However, the percentage of terminal villi and microvessel density (MVD) were comparable in both groups.Conclusion: Placenta from late onset PE without FGR is still different from the normal placenta whatever time onset of PE pathogenesis may be similar. Absence of changes in MVD may explain good fetal outcome.

Phase II clinical trial of pazopanib in patients with acute myeloid leukemia (AML), relapsed or refractory or at initial diagnosis without an intensive treatment option (PazoAML)

We evaluated pazopanib (800mg orally QD) in patients not eligible for intensive treatment with relapsed/refractory AML or at initial diagnosis. Patients receiving pazopanib for > 14days were analyzed for safety, tolerability, and efficacy. Co-primary endpoints were cumulative response rate and reduction of bone marrow microvessel density. Twenty patients (median age 76years, range 52-86) were treated. Fifteen had relapsed/refractory and five had newly diagnosed AML. Median ECOG performance status was 1 (range 1-3). Four patients had adverse, 15 intermediate, and 1 patient favorable cytogenetic/molecular risk (ELN 2010 criteria). The safety profile of pazopanib was as reported. The most common adverse events of any grade were gastrointestinal. Two patients achieved PR (blast reduction > 50%), 14 stable disease (SD), and 4 progressive disease. Median PFS was 65days (95% CI 29-105). After the end of the study, 1 CRi and 1 CRp occurred on demethylating agents, and 1 CR upon alloSCT. In these patients, SD and improved general condition on pazopanib allowed therapy escalation. Median OS for the overall study population was 191days (95% CI 87-435) and 1-year survival was 35%. There was no significant change in microvessel density. Clinical trial information: NCT01361334.

Vitamin D3 promotes cerebral angiogenesis after cerebral infarction in rats by activating Shh signaling pathway.

This study aims at investigating the neuroprotective role of Vitamin D3 (VitD3) in rats with cerebral infarction and its molecular mechanisms. Male Sprague- Dawley (SD) rats were selected and randomly divided into sham operation group, middle cerebral artery occlusion (MCAO) model group, and VitD3 treatment group. The therapeutic effect of VitD3 was evaluated via neurobehavioral scoring and triphenyltetrazolium chloride (TTC) staining. For the evaluation of VitD3 influence on cerebral blood perfusion, Micro-PET imaging technique was applied. The mRNA levels of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) gene were detected via Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Immunofluorescence staining assay was employed to determine the changes in micro-vessel density. Bromodeoxyuridine (Brdu) assay was used to count the number of new vascular endothelial cells. Protein expressions of key genes in the Shh signaling pathway were detected by Western blotting. Our results showed that VitD3 improved the score of neurological function and decreased the size of cerebral infarction in MCAO rats. VitD3 improved cerebral perfusion in the ischemic area after MCAO. VitD3 up-regulated levels of vascular growth-related factors. VitD3 elevated micro-vessel density after cerebral infarction and promoted the proliferation of vascular endothelial cells in the ischemic cortex. The sonic hedgehog (Shh) signaling pathway in the ischemic cortex of MCAO rats was activated after VitD3 treatment. We showed that VitD3 improves cerebral perfusion and reduces neurological impairment in MCAO rats via activating the Shh signaling pathway.

ADAMTS5 acts as a tumor suppressor by inhibiting migration, invasion and angiogenesis in human gastric cancer.

ADAMTS5 has been reported to be involved in the progression of several human tumors. Nevertheless, the role of ADAMTS5 in gastric cancer (GC) remains poorly defined. ADAMTS5 expression levels were analyzed by quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC) in GC cell lines and tissues, and the correlations between ADAMTS5 expression and clinicopathological features and survival were also examined. In vitro assays, including transwell assays, wound healing assays and cell adhesion assays, were employed to further explore the biological functions of ADAMTS5. A MAP kinase pathway microarray was used to identify the underlying mechanisms. The expression of ADAMTS5 and ETS1 and the microvessel density (MVD) were also analyzed using IHC to determine correlations with angiogenesis in GC. ADAMTS5 expression was downregulated in gastric cancer tissues. Low expression of ADAMTS5 was associated with gender, histological type, degree of differentiation, M stage, TNM stage and vascular invasion, and was also an independent indicator of a poor prognosis for patients with GC. ADAMTS5 overexpression markedly inhibited GC cell migration and invasion and enhanced cell adhesion to the extracellular matrix (ECM), whereas knockdown of ADAMTS5 exerted the opposite effects. Furthermore, the ADAMTS5 expression status was negatively correlated with ETS1 expression and MVD. ADAMTS5 is downregulated in GC and suppresses tumor metastasis and angiogenesis by inhibiting ETS1-mediated changes in MVD and potentially acts as a novel prognostic marker and a potential therapeutic target in human GC.

Characterisation of tumour microvessel density during progression of high-grade serous ovarian cancer: clinico-pathological impact (an OCTIPS Consortium study).

BackgroundHigh-grade serous ovarian cancer (HGSOC) intratumoural vasculature evolution remains unknown. The study investigated changes in tumour microvessel density (MVD) in a large cohort of paired primary and recurrent HGSOC tissue samples and its impact on patients’ clinico-pathological outcome.MethodsA total of 222 primary (pOC) and recurrent (rOC) intra-patient paired HGSOC were assessed for immunohistochemical expression of angiogenesis-associated biomarkers (CD31, to evaluate MVD, and VEGF-A). Expression profiles were compared between pOCs and rOCs and correlated with patients' data.ResultsHigh intratumoural MVD and VEGF-A expression were observed in 75.7% (84/111) and 20.7% (23/111) pOCs, respectively. MVDhigh and VEGF(+) samples were detected in 51.4% (57/111) and 20.7% (23/111) rOCs, respectively. MVDhigh/VEGF(+) co-expression was found in 19.8% (22/111) and 8.1% (9/111) of pOCs and rOCs, respectively (p = 0.02). Pairwise analysis showed no significant change in MVD (p = 0.935) and VEGF-A (p = 0.121) levels from pOCs to rOCs. MVDhigh pOCs were associated with higher CD3(+) (p = 0.029) and CD8(+) (p = 0.013) intratumoural effector TILs, while VEGF(+) samples were most frequently encountered among BRCA-mutated tumours (p = 0.019). Multivariate analysis showed VEGF and MVD were not independent prognostic factors for OS.ConclusionsHGSOC intratumoural vasculature did not undergo significant changes during disease progression. High concentration of CD31(+) vessels seems to promote recruitment of effector TILs. The study also provides preliminary evidence of the correlation between VEGF-positivity and BRCA status.

Erythropoietin attenuates hyperoxia-induced lung injury by upregulating epidermal growth factor-like domain 7 in newborn rats.

The aim of the present study was to observe the effects of recombinant human erythropoietin (rhEPO) on the expression of epidermal growth factor-like domain 7 (EGFL7) and cell apoptosis in lung tissue following hyperoxic lung injury in newborn rats. The 96 Sprague-Dawley newborn rats were randomly divided into 4 groups (n=24) as follows: Room air-exposed control group, room air-exposed rhEPO-treated group, hyperoxia-exposed group and the hyperoxia-exposed rhEPO-treated group. Pups (n=8) from each group were sacrificed on postnatal days 3, 7 and 14. The pulmonary morphometric and microvessel density changes were observed. In addition, the mRNA and protein expression levels of EGFL7, B-cell lymphoma 2 (Bcl-2) and Bcl-2-like protein 4 (Bax) in lung tissue samples were measured. The rats in the hyperoxia-exposed group exhibited alveolar and pulmonary vascular dysplasia, as well as low mRNA and protein expression levels of EGFL7 and Bcl-2, in addition to high level of Bax in the lung tissue samples when compared with the room air-exposed control group (P<0.05). However, in the hyperoxia-exposed rhEPO-treated group the lung histopathology was improved, and the protein and mRNA expression levels of EGFL7 and Bcl-2 were increased compared with the hyperoxia-exposed group (P<0.05). Furthermore, the expression level of Bax was lower than that of the hyperoxia-exposed group (P<0.05). The present study demonstrated that rhEPO promotes alveolar development and increases pulmonary vascular density by upregulating the expression level of EGFL7 in hyperoxia-induced lung injury of newborn rats.

Abstract LB-347: Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival

Abstract OBJECTIVE: We aimed to enhance the efficacy of anti-VEGF therapy in glioblastoma (GBM) through additional inhibition of Angiopoietin-2 (Ang-2), a potential mediator of resistance to antiangiogenic therapy using VEGF inhibition. INTRODUCTION: Glioblastoma (GBM) is a uniformly lethal primary brain tumor affecting more than 12.000 patients every year in the US alone. The standard therapy regimen for this highly angiogenic tumor entity comprises maximal safe resection and chemoradiation with temozolomide. The addition of antiangiogenic (anti-VEGF) therapy to the standard of care regimen improved progression-free survival, but failed to improve overall survival of GBM patients. Preclinical and clinical data suggest that resistance to anti-VEGF therapy in GBM is mediated by Ang-2, making this pathway a potential target. EXPERIMENTAL DESIGN: We tested the effect of dual Ang-2/VEGF blockade with A2V on mouse survival using a syngeneic (Gl261) model and a human xenograft (MGG8) model, compared to anti-VEGF antibody therapy (B20). In addition, we used blood-based Gaussian Luciferase (GLUC) assays, immunohistochemistry and flow cytometry to measure changes in tumor growth, microvessel density (MVD), and immune microenvironment, respectively. RESULTS: Gl261 tumors have a highly abnormal tumor vasculature. In this model, treatment with A2V reduced MVD compared to B20. The decrease in MVD was due to a reduction in pericyte-low tumor vessels, while pericyte-high vessels were unaffected. These vascular changes were accompanied by reduced tumor burden and enhanced survival. Interestingly, in the MGG8 tumors, which have a vasculature similar to the normal brain, we detected no change in MVD after A2V treatment. Nevertheless, we found a reduced tumor burden and prolonged animal survival in the MGG8 model. Since vascular normalization may impact immune cell infiltration and function in tumors, we next evaluated these cell populations. We found that A2V therapy reduced pro-tumor M2 polarization of macrophages and microglia and reprogrammed these cells toward the M1 phenotype in both the Gl261 and MGG8 models. Collectively, our data indicate that therapy-induced anti-tumor immunity is mediated by M1-type macrophages but not by T-cell infiltration or function. CONCLUSION: Dual Ang-2/VEGF therapy with A2V reprogrammed macrophages and microglia from pro-tumor M2 toward the anti-tumor M1 phenotype in two GBM models, in addition to normalizing vasculature in tumors with abnormal vessels. These data indicate that dual anti-angiogenic therapy has the potential to overcome resistance to anti-VEGF therapy and confer clinical benefits in GBM patients through vascular and immuno-modulatory effects. Citation Format: Jonas Kloepper, Lars Riedemann, Zohreh Amoozgar, Giorgio Seano, Katharina H. Susek, Veronica Yu, Nisha Dalvie, Robin L. Amelung, Meenal Datta, Jonathan W. Song, Vasileios Askoxylakis, Jennie W. Taylor, Christine Lu-Emerson, Ana Batista, Nathaniel D. Kirkpatrick, Keehoon Jung, Matija Snuderl, Alona Muzikansky, Kay G. Stubenrauch, Oliver Krieter, Hiroaki Wakimoto, Lei Xu, Lance L. Munn, Dan G. Duda, Dai Fukumura, Tracy T. Batchelor, Rakesh K. Jain. Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-347.

Abstract 3267: Eltd1 as an anti-angiogenic therapy against glioma tumors

Abstract Gliomas consist of up to 80% of malignant brain tumors that are invasive and typically resistant to radiotherapy and chemotherapy. Finding biomarkers to high-grade gliomas can enable better diagnosis and therapeutic intervention for this disease. We have identified ELTD1 as a biomarker for high-grade human gliomas. Here, we report our findings in vivo using anti-ELTD1 (at two different concentrations), Bevacizumab, and IgG antibodies on human G55 xenograft glioma models. Using MRI, we investigate tumor growth, perfusion, tumor blood flow, and microvessel density changes in mice. In addition, survival rates were measured. Mice were implanted with human G55 xenograft glioma cells and were left untreated, administered anti-ELTD1, (1 mg/kg or 2 mg/kg every 2-3 days) Bevacizumab (2 mg/kg every 2-3 days) or IgG (1 mg/kg every 2-3 days). MRI experiments were performed to assess tumor growth and calculate tumor volumes. In order to assess angiogenesis, representative histology slides were obtained and stained for blood vessels using CD34 antibody and microvessel density (MVD) was then calculated. Finally, cerebral blood flow rates from MR perfusion imaging was obtained as well as MR angiography to determine tumor blood volume from mice in each group. Our results show a significant decrease in tumor volumes and increase in percent survival for mice treated with 1 mg/kg and 2 mg/kg of ELTD1 antibody compared to untreated mice and IgG treated mice. Mice also had an increase in perfusion, decrease in tumor blood volume and decrease in MVD. Anti-ELTD1 antibody therapy reduced tumor volumes, prolonged life, and overall decreased angiogenesis in our mouse model. Anti-ELTD1 therapy may be an ideal anti-angiogenic treatment for high-grade gliomas. Citation Format: Jadith Ziegler, Nataliya Smith, Debra Saunders, Blake Evans, James D. Battiste, Michael Sughrue, Paul Tompkins, Jake Sutton, Megan Lerner, Patricia Coutinho de Souza, Kar-Ming Fung, Jonathan Wren, Rheal Towner. Eltd1 as an anti-angiogenic therapy against glioma tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3267.

High-Accuracy Ultrasound Contrast Agent Detection Method for Diagnostic Ultrasound Imaging Systems

An accurate method for detecting contrast agents using diagnostic ultrasound imaging systems is proposed. Contrast agents, such as microbubbles, passing through a blood vessel during ultrasound imaging are detected as blinking signals in the temporal axis, because their intensity value is constantly in motion. Ultrasound contrast agents are detected by evaluating the intensity variation of a pixel in the temporal axis. Conventional methods are based on simple subtraction of ultrasound images to detect ultrasound contrast agents. Even if the subject moves only slightly, a conventional detection method will introduce significant error. In contrast, the proposed technique employs spatiotemporal analysis of the pixel intensity variation over several frames. Experiments visualizing blood vessels in the mouse tail illustrated that the proposed method performs efficiently compared with conventional approaches. We also report that the new technique is useful for observing temporal changes in microvessel density in subiliac lymph nodes containing tumors. The results are compared with those of contrast-enhanced computed tomography.

Intestinal tumor suppression in ApcMin/+ mice by prostaglandin D2 receptor PTGDR.

Our earlier work showed that knockout of hematopoietic prostaglandin D synthase (HPGDS, an enzyme that produces prostaglandin D2) caused more adenomas in ApcMin/+ mice. Conversely, highly expressed transgenic HPGDS allowed fewer tumors. Prostaglandin D2 (PGD2) binds to the prostaglandin D2 receptor known as PTGDR (or DP1). PGD2 metabolites bind to peroxisome proliferator-activated receptor γ (PPARG). We hypothesized that Ptgdr or Pparg knockouts may raise numbers of tumors, if these receptors take part in tumor suppression by PGD2. To assess, we produced ApcMin/+ mice with and without Ptgdr knockouts (147 mice). In separate experiments, we produced ApcMin/+ mice expressing transgenic lipocalin-type prostaglandin D synthase (PTGDS), with and without heterozygous Pparg knockouts (104 mice). Homozygous Ptgdr knockouts raised total numbers of tumors by 30–40% at 6 and 14 weeks. Colon tumors were not affected. Heterozygous Pparg knockouts alone did not affect tumor numbers in ApcMin/+ mice. As mentioned above, our Pparg knockout assessment also included mice with highly expressed PTGDS transgenes. ApcMin/+ mice with transgenic PTGDS had fewer large adenomas (63% of control) and lower levels of v-myc avian myelocytomatosis viral oncogene homolog (MYC) mRNA in the colon. Heterozygous Pparg knockouts appeared to blunt the tumor-suppressing effect of transgenic PTGDS. However, tumor suppression by PGD2 was more clearly mediated by receptor PTGDR in our experiments. The suppression mechanism did not appear to involve changes in microvessel density or slower proliferation of tumor cells. The data support a role for PGD2 signals acting through PTGDR in suppression of intestinal tumors.

Dynamic Hypoxia in Scar Tissue During Human Hypertrophic Scar Progression

The skin color of human hypertrophic scar changes dynamically during scar progression. To investigate whether hypoxia is dynamic during scar progression. Thirty-five patients with early, proliferative, regressive, and mature scars were involved in this study. Tissue oxygen tension was measured before scar surgery. After surgery, the scar stage was further defined using hematoxylin and eosin staining, and microvessel density and hypoxia inducible factor-1 (HIF-1) expression were detected using immunohistochemistry to determine a correlation with oxygen level. Mild hypoxia is present in early scars, moderate hypoxia in proliferative scars, and severe hypoxia in regressive scars. Oxygen levels then return to normal in mature scars, which was consistent with the dynamic change in microvessel density. Meanwhile, HIF-1 expression also changed dynamically along with alteration in oxygen levels. Hypoxia is dynamic in scar tissue and is possibly correlated with scar formation and regression.

Experimental study on residual tumor angiogenesis after cryoablation.

To explore the mechanism and significance of tumor angiogenesis by observing changes of micro-vessel density (MVD) and expression of vascular endothelial growth factor (VEGF) in residual tumor tissues after cryoablation. A total of 18 nude mice xenograft models with transplanted lung adenocarcinoma cell line A549 were established and randomly divided into 3 groups when the maximum diameter of tumor reached 1 cm: control, cisplatin (DDP) and cryoablation. The nude mice were sacrificed after 21-d cryoablation to obtain the tumor tissues. Then immunohistochemistry was applied to determine MVD and the expression of VEGF in tumor tissues. The tumor volumes of control group, DDP group and cryoablation group were 1.48 ± 0.14 cm3, 1.03 ± 0.12 cm3 and 0.99 ± 0.06 cm3 respectively and the differences were significant (P < 0.01), whereas MVD values were 21.1 ± 0.86, 24.7 ± 0.72 and 29.2 ± 0.96 (P < 0.01) and the positive expression rates of VEGF were 36.2 ± 1.72%, 39.0 ± 1.79% and 50.8 ± 2.14% (P < 0.01), respectively, showing that MVD was proportional to the positive expression of VEGF (r = 0.928, P < 0.01). Cryoablation can effectively inhibit tumor growth, but tumor angiogenesis significantly increases in residual tumors, with high expression of VEGF playing an important role in the residual tumor angiogenesis.

Artemisinin derivative artesunate induces radiosensitivity in cervical cancer cells in vitro and in vivo

ObjectiveCervical cancer is the third most common type of cancer in women worldwide and radiotherapy remains its predominant therapeutic treatment. Artesunate (ART), a derivative of artemisinin, has shown radiosensitization effect in previous studies. However, such effects of ART have not yet been revealed for cervical cancer cells.MethodsThe effect of ART on radiosensitivity of human cervical cancer cell lines HeLa and SiHa was assessed using the clonogenic assay. Cell cycle progression and apoptosis alterations were analyzed by flow cytometry. For in vivo study, HeLa or SiHa cells were inoculated into nude mice to establish tumors. Tissues from xenografts were obtained to detect the changes of microvessel density, apoptosis and cell cycle distribution. Microarray was used to analyze differentially expressed genes.ResultsART increased the radiosensitivity of HeLa cells (SER = 1.43, P < 0.001) but not of SiHa cells. Apoptosis and the G2-M phase transition induced by X-ray irradiation (IR) were enhanced by ART via increased Cyclin B1 expression in HeLa cells. Tumor growth of xenografts from HeLa but not SiHa cells was significantly inhibited by irradiation combined with ART (tumor volume reduction of 72.34% in IR + ART group vs. 41.22% in IR group in HeLa cells and 48.79% in IR + ART group vs. 44.03% in IR alone group in SiHa cells). Compared with the irradiated group, cell apoptosis was increased and the G2/M cell cycle arrest was enhanced in the group receiving irradiation combined with ART. Furthermore, compared with radiation alone, X-ray irradiation plus ART affected the expression of 203 genes that function in multiple pathways including RNA transport, the spliceosome, RNA degradation and p53 signaling.ConclusionART potently abrogates the G2 checkpoint control in HeLa cells. ART can induce radiosensitivity of HeLa cells in vitro and in vivo.

Tumor angiogenesis in esophageal cancer

High invasive potential of esophageal carcinoma is often associated with angiogenesis,accompanied by the changes of microvessel density,vascular endothelial growth factor,angiogenic factors and their receptors.Studies show that there are significant correlations between increased serum levels of angiogenic factors and insufficient treatment response and poor prognosis in patients with esophageal cancer.Such angiogenesis-related molecules may represent novel targets in treating esophageal cancer,which deserve further exploration. Key words: Esophageal neoplasms; Neovascularization, pathologic; Vascular endothelial growth factors ; Targeted therapy

Abstract P4-01-14: Two-week change in optical tomography predicts residual cancer burden score in women treated with neoadjuvant chemotherapy

Abstract Background: A low Residual Cancer Burden (RCB) score after neoadjuvant chemotherapy (NACT) predicts an improved survival in patients (pts) with breast cancer (BC). While an early predictor of tumor response to NACT would allow for treatment optimization, no ideal method exists. Diffuse optical tomography (DOT) is a novel, fast, and low-cost imaging approach that assesses the distribution of water (H2O), oxyhemoglobin (HbO), and deoxyhemoglobin (Hb) concentrations, as a measure of tissue vascularity, without the use of ionizing radiation or breast compression, allowing for safe repeated measurements. We hypothesize that changes in DOT parameters will predict the RCB score following NACT. Methods: Women with stage II-IIIc invasive BC scheduled to undergo NACT with 12 cycles of a weekly taxane followed by 4 cycles of doxorubicin with cyclophosphamide were enrolled. Treatment with biologic therapies was allowed. DOT assessments were made before starting NACT, 2 weeks into treatment, and before surgery. DOT data were reconstructed into 3D images of the tumor region, from which HbO, Hb, and H2O concentrations were extracted. Final pathology specimens were scored for the RCB index (continuous measure), RCB class (0, 1, 2, 3), and a dichotomized RCB score (RCB class 0 or 1: responders to NACT; RCB class 2 or 3: non-responders). Ki-67 was measured on baseline tumor biopsies and surgical specimens. Correlation analysis, ANOVA testing, and two sample t-tests were used to evaluate the relationship between the 2-week changes in DOT parameters and the RCB score and Ki-67 level. Results: Since July 2011, we have recruited 24 pts of a total planned accrual of 40. 19 pts have undergone surgery and complete data is available for 13 at this time. Of the 13 pts, 4 had a pCR (RCB 0), 2 had RCB 1, 6 had RCB 2, and 1 had RCB 3. The Pearson correlations between the 2-week change in HbO, Hb, and H2O with the continuous RCB index were 0.76 (p = 0.0022), 0.87 (p = 0.0001), and 0.74 (p = 0.0038), respectively. There was a significant difference in the 2-week Hb change for pts with RCB 0 compared to pts with RCB 1, 2, or 3. There were significant differences in the 2-week change in H2O and HbO for pts with RCB 0 compared to pts with RCB 2. There were also significant differences between DOT parameters by the dichotomized RCB score (table 1). Ki-67 change was correlated with 2-week H2O change (Pearson r = 0.61 p = 0.045). 2-week DOT% change by RCB class and dichotomized RCB score HbOHbOH2ORCB 0-38%-28.5%-6.7%RCB 1-3.9%-2.9%-0.2%RCB 2+2.3%-0.6%+0.7%RCB 3+1.0%+10.9%-0.4% Responders (RCB 0/1)-26.6%-20%-4.5%Non-Responders (RCB 2/3)+2.1%+1%+0.6%P value0.010.00690.014 Conclusions: Two-week DOT change is an early predictor of response to NACT as measured by the RCB score. We found significant associations between the RCB index and Ki-67 with 2-week changes in HbO, Hb, and H2O. Significantly different changes in DOT parameters were associated with the other RCB classifications. We are analyzing DOT data on the remaining pts and will conduct biomarker assessments of microvessel density changes. Additional pts are being recruited to evaluate DOT's predictive ability by tumor subtype. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-01-14.

Experimental Study of Efficacy of Multi-target Inhibitor Combined with Cytotoxic Treatment on Malignant Glioma

Objective To investigate the influence of multi-target inhibitor vandetanib combined cytotoxic drugs carmustine(BCNU) on the proliferation and invasion of rat glioma cell C6.Methods The MTT assay was used to test the impact of drugs on cell viability and changes of cell morphology were observed by HE staining.Rat glioma cell C6 model was established by situ injection,and six days later they were divided into the vandetanib group [50 mg/(kg·d)],BCNU group [25 mg/(kg·d)],the combination group(vandetanib+ BCNU) and the control group.Dose the rats 7 times every other day for 2 weeks,then take the rats' brain after the decapitation,section the tumor tissues and calculate the volume of the tumor.Immunohistochemical SP assay was used to mark tumor vascular endothelial cells,and detect the microvessel density(MVD) in transplanted tumors and the expression of MMP1 protein related to tumor invasiveness.Apoptotic index was tested by TUNEL technique.Results The combination group was much remarkable to promote the apoptosis than that of the BCNU group(P0.01).Compared with the control group,tumor volumes of the BCNU group decreased by(P0.05) The expression of MMP1 in xenografts was weak positive(+ +),and there was no significant change in MVD,but the apoptosis rate of transplanted tumor cells was 62.6%(P0.05).Tumor volume of the combination group was decreased about 56% and the expression of MMP1 was negative(+),and MVD decreased by 53%(P0.05).Conclusion The combination therapy can not only significantly inhibit the growth of glioma cell C6,but also decrease the expression of MMP1 related to neoplasm invasiveness.Therefore,it provides experimental basis for the joint multi-target inhibitors and cytotoxic drugs in clinical treatment of malignant glioma.

Expression and localization of the vascular endothelial growth factor and changes of microvessel density during hair follicle development of Liaoning cashmere goats

Vascular endothelial growth factors (VEGFs) play important roles in neovascularization, tissue development, and angiogenesis. In this study, changes in VEGF expression patterns and microvessel density (MVD), and their correlations, were investigated during hair follicle development in epidermal appendages of Liaoning cashmere goats. Polyclonal antibodies to VEGF and microvessels were used for monthly immunohistochemical examinations of normal skin specimens from adult female goats for one year. VEGF was expressed in the hair bulb of primary and secondary hair follicles, the outer and inner root sheaths, sebaceous glands (ductal and secretory portions), eccrine sweat glands (ductal and secretory portions), and the epidermis. Abundant expression of VEGF was observed in the follicular basement membrane zone surrounding the bulb matrix and in ductal and secretory portions of eccrine sweat glands. The change in VEGFs in primary hair follicles showed a bimodal pattern, with the first peak observed from March to May, and the second in August. Maximal expression in secondary hair follicles occurred in May and August. Therefore, VEGF expression in primary and secondary hair follicles is synchronized throughout the year, and is correlated to hair development. In the later telogen and anagen phases, VEGF expression was higher in the secondary, compared to the primary, hair follicle. Changes in MVD also showed a bimodal pattern with peaks in May and August. VEGF expression and MVD showed moderate and strongly positive correlation in the primary and secondary hair follicles, respectively. Therefore, MVD and VEGF are closely related to the processes involved in hair cycle regulation.

Effects of electroacupuncture on the microvasculature of the cerebral cortex after focal cerebral ischemia and reperfusion in rats

Objective To study the effects of electroacupuncture (EA) on micro-vessel density (MVD) in the cerebral cortex after focal cerebral ischemia and reperfusion.Methods Forty Sprague-Dawley rats were randomly divided into a normal group ( n =4), a sham-operated group (n =4) , a model group (n = 16) and an EA group (n = 16). The middle cerebral artery was sutured to establish a model of middle cerebral artery occlusion (MCAO). The Baihui (DU20), Shuigou (DU26) and Zusanli (ST36) acupoints were stimulated with slow-fast wave current for 30 min daily. Streptavidin-biotin-peroxidase complex was used to detect the expression of CD34 and MVD changes in the rats' right cerebral cortex on the 1st, 2nd, 4th and 8th day.Results Compared with the normal group and the sham-operated group, MVD in the ischemic cortex of all subgroups of the model group had increased. It peaked on the 4th day and then gradually declined through the 8th day. MVD in all the subgroups of the EA group increased significantly, and more than in the model group at every time point, but the difference between the 2 groups was significant only on the 4th day.Conclusions Regulating angiogenesis in ischemic brain tissue is one of the ways EA can protect the brain. Key words: Cerebral ischemia; Reperfusion; Electroacupuncture; Angiogenesis