Cell Microvessel Research Articles

Space radiation damage rescued by inhibition of key spaceflight associated miRNAs

Our previous research revealed a key microRNA signature that is associated with spaceflight that can be used as a biomarker and to develop countermeasure treatments to mitigate the damage caused by space radiation. Here, we expand on this work to determine the biological factors rescued by the countermeasure treatment. We performed RNA-sequencing and transcriptomic analysis on 3D microvessel cell cultures exposed to simulated deep space radiation (0.5 Gy of Galactic Cosmic Radiation) with and without the antagonists to three microRNAs: miR-16-5p, miR-125b-5p, and let-7a-5p (i.e., antagomirs). Significant reduction of inflammation and DNA double strand breaks (DSBs) activity and rescue of mitochondria functions are observed after antagomir treatment. Using data from astronaut participants in the NASA Twin Study, Inspiration4, and JAXA missions, we reveal the genes and pathways implicated in the action of these antagomirs are altered in humans. Our findings indicate a countermeasure strategy that can potentially be utilized by astronauts in spaceflight missions to mitigate space radiation damage.

Surrogate vascular input function measurements from the superior sagittal sinus are repeatable and provide tissue-validated kinetic parameters in brain DCE-MRI

Accurate vascular input function (VIF) derivation is essential in brain dynamic contrast-enhanced (DCE) MRI. The optimum site for VIF estimation is, however, debated. This study sought to compare VIFs extracted from the internal carotid artery (ICA) and its branches with an arrival-corrected vascular output function (VOF) derived from the superior sagittal sinus (VOFSSS). DCE-MRI datasets from sixty-six patients with different brain tumours were retrospectively analysed and plasma gadolinium-based contrast agent (GBCA) concentration-time curves used to extract VOF/VIFs from the SSS, the ICA, and the middle cerebral artery. Semi-quantitative parameters across each first-pass VOF/VIF were compared and the relationship between these parameters and GBCA dose was evaluated. Through a test–retest study in 12 patients, the repeatability of each semiquantitative VOF/VIF parameter was evaluated; and through comparison with histopathological data the accuracy of kinetic parameter estimates derived using each VOF/VIF and the extended Tofts model was also assessed. VOFSSS provided a superior surrogate global input function compared to arteries, with greater contrast-to-noise (p < 0.001), higher peak (p < 0.001, repeated-measures ANOVA), and a greater sensitivity to interindividual plasma GBCA concentration. The repeatability of VOFSSS derived semi-quantitative parameters was good to excellent (ICC = 0.717–0.888) outperforming arterial based approaches. In contrast to arterial VIFs, kinetic parameters obtained using a SSS derived VOF permitted detection of intertumoural differences in both microvessel surface area and cell density within resected tissue specimens. These results support the usage of an arrival-corrected VOFSSS as a surrogate vascular input function for kinetic parameter mapping in brain DCE-MRI.

Stem cell–secreted factor therapy regenerates the ovarian niche and rescues follicles

Ovarian senescence is a normal age-associated phenomenon, but increasingly younger women are affected by diminished ovarian reserves or premature ovarian insufficiency. There is an urgent need for developing therapies to improve ovarian function in these patients. In this context, previous studies suggest that stem cell-secreted factors could have regenerative properties in the ovaries. This study aimed to test the ability of various human plasma sources, enriched in stem cell-secreted factors, and the mechanisms behind their regenerative properties, to repair ovarian damage and to promote follicular development. In the first phase, the effects of human plasma enriched in bone marrow stem cell soluble factors by granulocyte colony-stimulating factor mobilization, umbilical cord blood plasma, and their activated forms on ovarian niche, follicle development, and breeding performance were assessed in mouse models of chemotherapy-induced ovarian damage (n=7 per group). In addition, the proteomic profile of each plasma was analyzed to find putative proteins and mechanism involved in their regenerative properties in ovarian tissue. In the second phase, the most effective plasma treatment was validated in human ovarian cortex xenografted in immunodeficient mice (n=4 per group). Infusion of human plasma enriched bone marrow stem cell soluble factors by granulocyte colony-stimulating factor mobilization or of umbilical cord blood plasma-induced varying degrees of microvessel formation and cell proliferation and reduced apoptosis in ovarian tissue to rescue follicular development and fertility in mouse models of ovarian damage. Plasma activation enhanced these effects. Activated granulocyte colony-stimulating factor plasma was the most potent inducing ovarian rescue in both mice and human ovaries, and proteomic analysis indicated that its effects may be mediated by soluble factors related to cell cycle and apoptosis, gene expression, signal transduction, cell communication, response to stress, and DNA repair of double-strand breaks, the most common form of age-induced damage in oocytes. Our findings suggested that stem cell-secreted factors present in both granulocyte colony-stimulating factor-mobilized and umbilical cord blood plasma could be an effective treatment for increasing the reproductive outcomes in women with impaired ovarian function owing to several causes. The activated granulocyte colony-stimulating factor plasma, which is already enriched in both stem cell-secreted factors and platelet-enclosed growth factors, seems to be the most promising treatment because of its most potent restorative effects on the ovary together with the autologous source.

Predicting pathological complete response (pCR) after stereotactic ablative radiation therapy (SABR) of lung cancer using quantitative dynamic [18F]FDG PET and CT perfusion: a prospective exploratory clinical study

BackgroundStereotactic ablative radiation therapy (SABR) is effective in treating inoperable stage I non-small cell lung cancer (NSCLC), but imaging assessment of response after SABR is difficult. This prospective study aimed to develop a predictive model for true pathologic complete response (pCR) to SABR using imaging-based biomarkers from dynamic [18F]FDG-PET and CT Perfusion (CTP).MethodsTwenty-six patients with early-stage NSCLC treated with SABR followed by surgical resection were included, as a pre-specified secondary analysis of a larger study. Dynamic [18F]FDG-PET and CTP were performed pre-SABR and 8-week post. Dynamic [18F]FDG-PET provided maximum and mean standardized uptake value (SUV) and kinetic parameters estimated using a previously developed flow-modified two-tissue compartment model while CTP measured blood flow, blood volume and vessel permeability surface product. Recursive partitioning analysis (RPA) was used to establish a predictive model with the measured PET and CTP imaging biomarkers for predicting pCR. The model was compared to current RECIST (Response Evaluation Criteria in Solid Tumours version 1.1) and PERCIST (PET Response Criteria in Solid Tumours version 1.0) criteria.ResultsRPA identified three response groups based on tumour blood volume before SABR (BVpre-SABR) and change in SUVmax (ΔSUVmax), the thresholds being BVpre-SABR = 9.3 mL/100 g and ΔSUVmax = − 48.9%. The highest true pCR rate of 92% was observed in the group with BVpre-SABR < 9.3 mL/100 g and ΔSUVmax < − 48.9% after SABR while the worst was observed in the group with BVpre-SABR ≥ 9.3 mL/100 g (0%). RPA model achieved excellent pCR prediction (Concordance: 0.92; P = 0.03). RECIST and PERCIST showed poor pCR prediction (Concordance: 0.54 and 0.58, respectively).ConclusionsIn this study, we developed a predictive model based on dynamic [18F]FDG-PET and CT Perfusion imaging that was significantly better than RECIST and PERCIST criteria to predict pCR of NSCLC to SABR. The model used BVpre-SABR and ΔSUVmax which correlates to tumour microvessel density and cell proliferation, respectively and warrants validation with larger sample size studies.Trial registrationMISSILE-NSCLC, NCT02136355 (ClinicalTrials.gov). Registered May 8, 2014, https://clinicaltrials.gov/ct2/show/NCT02136355

Effect of Treatment Sequencing on the Tumor Response to Combined Treatment With Ultrasound-Stimulated Microbubbles and Radiotherapy.

To investigate whether timing and sequencing of ultrasound-stimulated microbubbles (USMBs) and external beam radiotherapy (XRT) affect the treatment response in a preclinical prostate cancer model. Prostate cancer xenografts were treated with ultrasound-stimulated lipid microspheres before and after 8-Gy XRT. Treatments were separated by 0, 3, 6, 12, and 24 hours, with 5 tumors per group. Tumor effects were evaluated by microvessel density (measured by CD31 staining), cell death (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labeling and hematoxylin-eosin staining), and hypoxia (carbonic anhydrase 9 staining). Administering USMBs 6 hours before XRT showed the maximum treatment effect using all 3 assays. At this time, the mean cell death index ± SD was 36% ± 10%, compared with 19% ± 4% for no separation between USMB treatment and XRT; the microvessel density was 9 ± 3 counts per field (19 ± 5 without separation); and the percentage of hypoxic cells was 10% ± 5% (21% ± 4%). The observed treatment effect was greater with USMBs before XRT than when administering XRT first, but these differences were not statistically significant. The maximum tumor effect was observed with USMBs delivered 6 hours before XRT. The sequencing of treatment did not have a significant effect on the tumor response.

Intravital assessment of angioarchitecture in rat hepatocellular nodules using in vivo fluorescent microscopy.

To prospectively evaluate the stepwise changes that occur in intra-nodular microvessels and microcirculation during the carcinogenesis process of hepatocellular nodules by using in vivo fluorescent microscopy, and to compare these with pathological changes. Forty-five 10-week-old male Wistar rats received drinking water containing N-nitrosomorpholine at 10 mg/100 mL for 18weeks to develop multiple hepatocellular carcinomas (HCC) and dysplastic nodules (DN) in the liver; meanwhile, the non-lesion liver tissues become fibrotic. The microvascular morphological change and hemodynamic change of two lesion areas (HCC or DN) and one non-lesion area in each rat were observed with in vivo fluorescent microscope. After in vivo microscopy, 90 nodules and 45 non-lesion liver tissues that were observed were removed for pathological study. The microvessel density (MVD), branch density (BD), and cell density (CD) of these lesions were compared with the Kruskal-Wallis test and Mann-Whitney test, with an overall statistical significance of 0.05. The intra-nodular microvessels appeared tortuous, with irregular branching and abrupt diameter changes to form irregular convoluted networks in the HCC. This was distinctly different from the appearance of DN and non-lesion liver parenchyma. The MVD and BD of HCC were less than that of the DN and non-lesion liver parenchyma (P<0.01), and the BD of DN was also less than that of the non-lesion liver parenchyma (P<0.05). However, the MVD of the DN was similar to that of the non-lesion liver parenchyma (P>0.05). The CD of HCC was more than that of the DN and non-lesion liver parenchyma (P<0.05), and the CD of DN was also more than that of the non-lesion liver parenchyma (P<0.05). Concurrent with the carcinogenesis process of the hepatocellular nodule, both the intra-nodular microvascular morphology and hemodynamics were stepwise changed, and the number of the intravascular lumen of intranodular microvessels decreased due to the infiltration and compression of intra-nodular parenchymal cells.

An Inhibitory Ligand of Neuropilin 2 Blocks Pancreatic Cancer Progression and Impedes Tumor Angiogenesis

The proliferation and branching pattern of blood vessels and nerves is mediated during development, in part, by the interaction between the Neuropilin‐2 (NRP2) receptor and its endogenous secreted ligand, Semaphorin‐3F (SEMA3F). This interaction induces a repulsive signal, which participates in the guidance of both neuronal axons and blood vessels (veins and capillaries). Furthermore, upon binding vascular endothelial growth factor‐A (VEGFA), NRP2 forms a complex with the VEGF Receptor‐2 (VEGFR2), which mediates a potent pro‐angiogenic signal. Although NRP2 expression is dormant in adult endothelial tissue, its expression is upregulated in a variety of physiological and pathological conditions. Interestingly, pancreatic ductal adenocarcinoma (PDAC) induces the over‐expression of NRP2 in endothelial cells and in the pancreatic cancer cells themselves. Consequently, this project is aimed at studying the anti‐angiogenic and repulsive effects of SEMA3F in order to inhibit the growth of the primary tumor and the incidence of liver metastases, respectively, in PDAC. Our preclinical trials utilized an orthotopic, syngeneic luciferase‐labelled mouse pancreatic cancer xenograft model in C57BL/6J mice. The delivery of SEMA3F was performed through a systemic intravenous injection of SEMA3F‐encoding adenovirus. Next, we performed a series of three independent preclinical trials, in which we compared the disease progression of PDAC in SEMA3F‐virus treated mice (n=5) compared to control‐virus treated mice (n=5). In the Prevention Trial, the mice received the virus injection three days prior to receiving the PDAC implantation. The Experimental Prevention Trial followed the same timeline, but the PDAC cells were injected into the spleen in order to accelerate the incidence of liver metastases. Lastly, in the Intervention Trial the SEMA3F‐encoding virus was injected after PDAC tumors were established in order to simulate a more realistic clinical setting. Tumorigenicity was monitored during the trials via bioluminescence imaging following systemic luciferin injection. Mice were euthanized and necropsied 20 days after tumor injection, and livers and pancreata were weighed to asses primary tumor growth as well as metastatic burden. Furthermore, the tumors were stained with antibodies directed against CD31 and KI67, in order to quantify tissue microvessel density and cell proliferation respectively. Qualitative analysis of the livers consisted in staining them with antibodies directed against PDX1 and NRP2, in order to confirm that the metastases are of pancreatic origin and that they retain their NRP2 expression after migrating to the liver. Our results indicate a potent anti‐angiogenic effect of SEMA3F compared to control across all experimental groups. Furthermore, a reduced incidence of metastases to the liver supports the chemorepulsive effects of SEMA3F. Overall, SEMA3F shows great potential as a novel therapy in PDAC.Support or Funding InformationNIH CA155728This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Treatment Sequencing on Tumor Response to Combined Treatment With Ultrasound-Driven Microbubbles and Radiation Therapy

Ultrasound-driven microbubbles (USMB) have synergistic effects with radiotherapy (RT). Sequence and timing of RT and vascular-disrupting agents have a large effect on cytotoxicity. We investigated outcomes based on USMB and RT sequencing in a preclinical prostate cancer model. PC-3 prostate cancer xenografts were treated with ultrasound-driven lipid microspheres and 8 Gy of external-beam RT. Sequence 1 (S1) consisted of USMB treatment preceding RT by 3, 6, 12, and 24 hours; Sequence 2 (S2) delivered USMB after RT at the same times. 5 tumors were treated at baseline (no treatment) and at each time point. Tumor effect was assessed via CD31 staining, TUNEL and H&E staining, and Carbonic Anhydrase 9 (CA9) staining to measure microvessel density, cell death and hypoxia, respectively. For the tumors treated using S1, microvessel density (MVD) was 18.8±4.8 counts per field (cpf) at baseline and 9.8±2.3, 9.1±2.5, 11.0±3.9 and 12.8±3.3 cpf when USMB preceded RT by 3, 6, 12 and 24 hours, respectively. Using S2, MVD was 18.0±1.8 cpf at baseline and 12.2±0.7, 10.7±1.1, 12.1±1.9 and 14.2±1.8 cpf, respectively, when RT preceded USMB by the same times. Each MVD measurement was significantly lower than baseline (p<0.05) except for S2 separated by 24 hours (S2-24). The MVD using S1 was larger than S2 at all time points, but none of these differences were statistically significant. Percent cell death (CD) using S1 was 19.2%±4.4% at baseline and 28.0%±6.4%, 35.6%±10.4%, 30.8%±9.0% and 26.4%±3.4% when USMB preceded RT by 3, 6, 12 and 24 hours, respectively. Using S2, CD was 14.8%±4.4% at baseline and 24.2%±7.0%, 27.8%±5.2%, 24.8%±6.1% and 22.4%±5.2%, respectively, at the same times. Only the S1-6 measurement differed significantly from baseline (p<0.01). There was more cell death with S1 than S2 at all time points but these differences were not statistically significant (p>0.05). By both MVD and CD, maximum effect occurred using S1-6. Among S2 measurements, maximum effect was using S2-6. However, despite the clear trend of increasing effect with longer separation time up to 6 hours followed by a decrease with longer separation, the measurements at 3, 6, 12 and 24 hours were not significantly different from each other using either S1 or S2 (p>0.05). The only hypoxia measurement significantly different from baseline was S1-6 (p<0.01). The lowest S2 measurement was S2-6, but it did not differ significantly from baseline (p>0.05). Less hypoxia was measured using S1 than S2 at all time points but these differences were not statistically significant (p>0.05). A trend was seen toward S1 yielding greater tumor effect than S2 and toward maximum tumor effect with treatment separation of 6 hours. However, our sample was not large enough to establish statistical significance. Testing of a larger sample should confirm this finding after which further testing is warranted to develop clinical protocols for combining USMB and RT.

Hypoxic and Reoxygenated Microenvironment: Stemness and Differentiation State in Glioblastoma.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Hypoxia is a distinct feature in GBM and plays a significant role in tumor progression, resistance to treatment, and poor outcome. However, there is lack of studies relating type of cell death, status of Akt phosphorylation on Ser473, mitochondrial membrane potential, and morphological changes of tumor cells after hypoxia and reoxygenation. The rat glioma C6 cell line was exposed to oxygen deprivation (OD) in 5% fetal bovine serum (FBS) or serum-free media followed by reoxygenation (RO). OD induced apoptosis on both 5% FBS and serum-free groups. Overall, cells on serum-free media showed more profound morphological changes than cells on 5% FBS. Moreover, our results suggest that OD combined with absence of serum provided a favorable environment for glioblastoma dedifferentiation to cancer stem cells, since nestin, and CD133 levels increased. Reoxygenation is present in hypoxic tumors through microvessel formation and cell migration to oxygenated areas. However, few studies approach these phenomena when analyzing hypoxia. We show that RO caused morphological alterations characteristic of cells undergoing a differentiation process due to increased GFAP. In the present study, we characterized an in vitro hypoxic microenvironment associated with GBM tumors, therefore contributing with new insights for the development of therapeutics for resistant glioblastoma.

Therapeutic effect of IGF-1R-targeting inhibitor (TAE226) on malignant pleural effusion in nude mice

To study the therapeutic effect of IGF-1R inhibitor TAE226 on malignant pleural effusion (MPE) in nude mice. Human lung carcinoma A549 cells were injected into the pleural cavity of nude mice to establish MPE model. The mice were randomly divided into model group and treatment group, and were orally administered with distilled water and TAE226 (20 mg/kg) in the same volume, respectively. The volume of pleural effusion and tumor weight of the two groups were observed. HE staining was used to reveal the histological changes and enzyme-linked immunosorbent assay (ELISA) was used to detect the IGF-1R protein expression. IGF-1R mRNA level in the tumor tissue was determined by RT-PCR. Microvessel density (MVD) and cell proliferation index (PI) were assessed by immunohistochemical analysis. The protein expression levels of IGF-1R, p-IGF-1R, PI3K and p-PI3K in the tumor tissue were determined by Western blotting. The volumes of pleural effusion were (241.4±89.7) μl and (121.7±78.8) μl in the model and treatment groups, respectively (P<0.05). The tumor weight of treatment group was (316.7±186.3) mg, significantly lower than that of the model group (671.4±281.4) mg (P<0.05). RT-PCR analysis showed that IGF-1R mRNA level was 0.914±0.029 in the treatment group, significantly lower than that of the model group (1.152±0.037, P<0.01). The ELISA data revealed that IGF-1R protein expression level of the model group was significantly higher than that of the treatment group [(41.0±4.7) μg/L vs. (24.0±3.1) μg/L, P<0.01]. Immunohistochemical analysis showed that there were significant differences between MVD and PI in the model and treatment groups [MVD, 34.75±3.49 vs. 22.25±3.63; PI, (75.25±7.15)% vs. (45.75±5.12)%; P<0.01 for both). Western blot data showed that IGF-1R and PI3K protein expression levels were not significantly different between the model and treatment groups (1.03±0.33 vs. 0.98±0.37 and 1.05±0.28 vs. 0.98±0.19), respectively (P>0.05), but p-IGF-1R and p-PI3K protein expression levels had significant differences between the two groups (1.08±0.10 vs. 0.51±0.08 and 1.12±0.09 vs. 0.86±0.09), respectively (P<0.01 for both). The IGF-1R inhibitor can effectively inhibit the formation of malignant pleural effusion. Its mechanism may be related to the suppression of tumor cell proliferation, invasion and angiogenesis through inhibition of PI3K signaling. TAE226 treatment may be a potential therapeutic regimen of treating malignant pleural effusion.

Abstract P6-04-01: Melatonin on angiogenesis in breast cancer

Abstract The rapid tumor growth results in hypoxia on tumor microenvironment, leading to a cascade of events that induce angiogenesis and subsequent cancer progression. Thus, the identification of therapeutic agents that can inhibit angiogenesis is essential for the control of tumor progression. Exogenous administration of melatonin, a hormone secreted by the pineal gland, has been shown several oncostatics effects on different types of cancers. The aim of this study was to evaluate the effectiveness of melatonin treatment on angiogenesis in breast cancer, in the in vitro and in vivo studies. In the in vitro study, breast cancer cell lines (MCF-7 and MDA-MB-231) were treated with melatonin under cobalt chloride (CoCl2)-induced hypoxic conditions. Cell viability was measured by MTT assay, the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF-A) was assessed by real-time PCR and immunocytochemistry. Additionally, other proteins involved in angiogenesis were evaluated by the Protein Array. In the in vivo study, the MDA-MB-231 cells were implanted in athymic nude mice, which were treated with melatonin (40 mg/kg) for 21 days. The tumor was measured weekly and evaluation of angiogenesis was performed by single-photon emission computed tomography (SPECT) with Tc-99m-HYNIC-VEGF-c, which is specific for VEGF receptors (VEGFR2/VEGF3). Moreover, VEGFR2, VEGFR3, von Willebrand factor (vWF) and cell proliferation marker (Ki-67) were evaluated in tumor tissue by immunohistochemistry, and other angiogenic proteins by Protein Array. Results from the in vitro study showed that 1 mM of melatonin under hypoxic conditions (200 µM CoCl2) led to decreased cell viability, protein levels of HIF-1α and gene and protein expression of VEGF-A in both cell lines (p &amp;lt; 0.05). Among other proteins evaluated, melatonin treatment under hypoxia resulted in a decrease of VEGF-C, VEGFR2, VEGFR3, matrix metalloproteinase 9 (MMP-9) and angiogenin in MCF-7 cell line (p &amp;lt; 0.05). For MDA-MB-231, a significant reduction was observed for VEGFR2 protein, epidermal growth receptor (EGFR), and angiogenin (p &amp;lt;0.05). In vivo study, mice treated with melatonin showed reduced tumor growth compared to control animals (144.90 ± 38.38 mm3 vs 282.00 ± 88.53 mm3, p &amp;lt; 0.05). Furthermore, one animal showed tumor regression during melatonin treatment (Day 7 = 27.38 mm3, 8.79 mm3 Day = 14, mm3 Day 21 = 4.8). SPECT detect less radioactivity of Tc-99m-HYNIC-VEGF-c and consequent reduced expression of VEGFR2/3 in tumors treated with melatonin (150,46 ± 17,06 % vs 183,55 ± 20,92 %) but statistical significance was not achieved (p &amp;gt; 0.05). The reduction of VEGFR2 in tumors treated with melatonin was confirmed by immunohistochemistry (p &amp;lt;0.05), as well as the reduction of micro-vessel density (vWF) and cell proliferation (ki-67) (p &amp;lt; 0.05). There was no change in the expression of the other proteins evaluated, however a significant increase in EGFR and Insulin-like growth factor (IGF-I) was observed in tumors treated with melatonin (p&amp;lt; 0.05). Taken together, our results showed that melatonin has an important anti-angiogenic effect, suggesting its potential therapeutic action in breast cancer. Support: FAPESP. Citation Format: Debora C Zuccari, Bruna V Jardim-Perassi, Mateus R Lourenço, Gabriel M Doho, Gabriela B Gelaleti, Lívia C Ferreira, Thaiz F Borin, Marina G Moschetta. Melatonin on angiogenesis in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-04-01.

Abstract A02: Effect of curcumin on the tumor growth and angiogenesis of breast cancer

Abstract Background: Angiogenesis plays an important role in the pathogenesis of breast cancer. For growth, tumor requires the formation of new blood vessels, which are stimulated by angiogenic factors to initiate the proliferation of endothelial cells. The major contributors to angiogenesis are the vascular endothelial growth factor (VEGF) and its receptors, which promote cell proliferation, migration, permeability and survival. Controlling tumor-associated angiogenesis and metastasis are promising tactics in limiting cancer progression. Curcumin, a polyphenolic compound derived from the spice plant Curcuma longa, displays multiple actions on solid tumours including anti-angiogenic effects. In this study we evaluated the benefits of curcumin treatment on tumor growth and angiogenesis in breast cancer. Methods: MDA-MB-231 breast cancer cell line was cultured and cell viability was measured by MTT assay after incubation with different concentrations of curcumin (25 uM, 50 uM, 100 uM). We performed an in vivo study implanting cells in athymic nude mice. Mice were treated with 300 mg/kg of curcumin (n = 5) or vehicle (n = 8) daily, starting in the same day as tumor implantation and continued for 21 days. Tumors were measured with a digital caliper on day 7, 14 and 21 and the animals underwent SPECT scanning with Tc-99m tagged VEGF-c to detect in vivo angiogenesis (expression of VEGFR2/3). In addition, the markers of angiogenesis (VEGF-A, VEGF-C, VEGFR2, VEGFR3 and von Willebrand Factor (vWF)) and cell proliferation (Ki-67) were evaluated by immunohistochemistry in mammary tumor tissues. Results: Curcumin treatment in vitro was able to significantly decrease cell viability, after 4 hours of exposure, maintaining its efficacy at higher doses after 24 hours exposure (p&amp;lt;0.05). After 21 days of treatment, curcumin retards the breast tumor growth, showed by the slower tumoral development of the treated group compared to the control group (p&amp;lt;0.05). The mean tumor volume of control and treated animals were 282.00 ± 88.53 mm3 232.5± 53.2 mm3, respectively. Animals treated with curcumin showed lower activity of Tc-99-VEGF-C in the tumors compared to that of the animals that were treated with vehicle. The intensity of radioactivity in the control animals was 183.55 ± 20.92%, while the intensity of radioactivity in animals treated with curcumin was 134.9 ± 10.3%. Although there was difference in the radioactivity in the tumors between the two group, statistically significant difference was not achieved (p&amp;gt;0.05). However, decrease of VEGF-A, VEGF-C and VEGFR2 in curcumin treated mice was confirmed by immunohistochemistry (p&amp;lt;0.05). In addition, there was a decrease of micro-vessel density (vWF) and cell proliferation (Ki-67) in curcumin treated tumors (p&amp;lt;0.05). Conclusion: In summary, our results demonstrate that curcumin affect the viability of the breast cancer cells in vitro. In addition, in vivo study, curcumin is able to retards the breast tumor growth when compared to the control group. Both SPECT and histochemical analysis indicate decrease in angiogenesis following treatment with curcumin. Citation Format: Lívia Carvalho Ferreira, Ali Syed Arbab, Bruna Victorasso Jardim-Perassi, Thaiz Ferraz Borin, Naiane Nascimento Gonçalves, Ravi Sankara Varma Nadimpalli, Debora Aparecida Pires de Campos Zuccari. Effect of curcumin on the tumor growth and angiogenesis of breast cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A02. doi:10.1158/1538-7445.CHTME14-A02

Critical role of histone demethylase RBP2 in human gastric cancer angiogenesis

BackgroundThe molecular mechanisms responsible for angiogenesis and abnormal expression of angiogenic factors in gastric cancer, including vascular endothelial growth factor (VEGF), remain unclear. The histone demethylase retinoblastoma binding protein 2 (RBP2) is involved in gastric tumorgenesis by inhibiting the expression of cyclin-dependent kinase inhibitors (CDKIs).MethodsThe expression of RBP2, VEGF, CD31, CD34 and Ki67 was assessed in 30 human gastric cancer samples and normal control samples. We used quantitative RT-PCR, western blot analysis, ELISA, tube-formation assay and colony-formation assay to characterize the change in VEGF expression and associated biological activities induced by RBP2 silencing or overexpression. Luciferase assay and ChIP were used to explore the direct regulation of RBP2 on the promoter activity of VEGF. Nude mice and RBP2-targeted mutant mice were used to detect the role of RBP2 in VEGF expression and angiogenesis in vivo.ResultsRBP2 and VEGF were both overexpressed in human gastric cancer tissue, with greater microvessel density (MVD) and cell proliferation as compared with normal tissue. In gastric epithelial cell lines, RBP2 overexpression significantly promoted the expression of VEGF and the growth and angiogenesis of the cells, while RBP2 knockdown had the reverse effect. RBP2 directly bound to the promoter of VEGF to regulate its expression by histone H3K4 demethylation. The subcutis of nude mice transfected with BGC-823 cells with RBP2 knockdown showed reduced VEGF expression and MVD, with reduced carcinogenesis and cell proliferation. In addition, the gastric epithelia of RBP2 mutant mice with increased H3K4 trimethylation showed reduced VEGF expression and MVD.ConclusionsThe promotion of gastric tumorigenesis by RBP2 was significantly associated with transactivation of VEGF expression and elevated angiogenesis. Overexpression of RBP2 and activation of VEGF might play important roles in human gastric cancer development and progression.

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.

Conformational remodeling of the fibronectin matrix selectively regulates VEGF signaling.

The fibronectin matrix plays a crucial role in the regulation of angiogenesis during development, tissue repair and pathogenesis. Previous work has identified a fibronectin-derived homophilic binding peptide, anastellin, as an effective inhibitor of angiogenesis; however, its mechanism of action is not well understood. In the present study, we demonstrate that anastellin selectively inhibits microvessel cell signaling in response to the VEGF165 isoform, but not VEGF121, by preventing the assembly of the complex containing the VEGF receptor and neuropilin-1. Anastellin treatment resulted in the inactivation of α5β1 integrins but was not accompanied by a change in either adhesion complexes or adhesion-based signaling. Integrin inactivation was associated with a masking of the fibronectin synergy site within the extracellular matrix (ECM), indicating that α5β1 inactivation resulted from a decrease in available ligand. These data demonstrate that anastellin influences the microvessel cell response to growth factors by controlling the repertoire of ligated integrins and point to anastellin as an effective regulator of fibronectin matrix organization. These studies further suggest that homophilic fibronectin binding peptides might have novel applications in the field of tissue regeneration as tools to regulate neovascularization.

Microvessel and mast cell densities in malignant laryngeal neoplasm

Laryngeal neoplasm contributes to 30-40% of carcinomas of the head and neck. Mast cells are normal connective tissue residents, well represented in the respiratory tract. Experimental evidence suggests that the growth of a tumor beyond a certain size requires angiogenesis, which may also permit metastasis. The aim of this study was to evaluate the correlation between mast cell density, microvascular density, histopathological type and histological grade. Our study included 38 laryngeal carcinomas as follows: adenoid cystic carcinoma (2 cases), malignant papilloma (2 cases) and squamous cell carcinoma (34 cases). The combined technique of CD 34-alcian blue safranin (ABS) was used to identify microvessel and mast cell density, which was quantified by the hot spot method. A significant correlation was found between both mast cell and microvascular density, and G1/G2 histological grade (p=0.002 and p=0.004, respectively). Squamous cell carcinoma was significantly correlated with mast cell density (p=0.003), but not with microvascular density (p=0.454).

Abstract A009: Effect of melatonin on the tumor growth and angiogenesis of breast cancer

Abstract Background: Breast cancer has a high mortality rate, mainly due to tumor progression and metastatic spread. Once tumor lesion exceeds a few millimeters in diameter, hypoxia triggers a cascade of events to allow neovascularization. The major contributors to angiogenesis are the vascular endothelial growth factor (VEGF) and its receptors, which promote cell proliferation, migration, permeability and survival. Thus, controlling tumor-associated angiogenesis is a promising tactic in limiting cancer development. The melatonin administration, a hormone naturally produced and secreted in the pineal gland, appears to play an important oncostatic role in cancer and have been currently proposed different mechanisms of action. In this study we evaluated the benefits of melatonin treatment on tumor growth and angiogenesis in breast cancer. Methods: MDA-MB-231 breast cancer cell line was cultured and cell viability was measured by MTT assay after incubation with different concentrations of melatonin (5 mM, 10 mM, 20 mM). We performed an in vivo study implanting cells in athymic nude mice. Mice were treated with 1mg of melatonin (n = 5) or vehicle (n = 8) daily, starting in the same day as tumor implantation and continued for 21 days. Tumors were measured with a digital caliper on day 7, 14 and 21 and the animals underwent SPECT scanning with Tc-99m tagged VEGF-c to detect in vivo angiogenesis (expression of VEGFR2/3). In addition, the markers of angiogenesis (VEGFR2, VEGFR3 and von Willebrand Factor (vWF)) and cell proliferation (Ki-67) were evaluated by immunohistochemistry in mammary tumor tissues. Results: Melatonin treatment in vitro was able to significantly decrease cell viability, after 4 hours of exposure, maintaining its efficacy at higher doses after 24 hours exposure (p&amp;lt;0.05). The animals treated with melatonin showed reduced tumor size compared to control animals after 21 days of treatment (p&amp;lt;0.05). The mean tumor volume of control and treated animals were 282.00 ± 88.53 mm3 and 144.90 ± 38.38 mm3, respectively. The mean of tumor volume in control animals increased significantly from day 14 (118.90 ± 40.17 mm3) to day 21 (282.00 ± 88.53 mm3) (p&amp;lt;0.05), which was not observed in the treated group. Furthermore, there was tumor regression in an animal treated with melatonin (Day 7 = 27.38 mm3; Day 14 = 8.79 mm3; Day 21 = 4.8 mm3), similar pattern was not seen in any of the control. Animals treated with melatonin showed lower activity of Tc-99-VEGF-C in the tumors compared to that of the animals that were treated with vehicle. The intensity of radioactivity in the control animals was 183.55 ± 20.92%, while the intensity of radioactivity in animals treated with melatonin was 150.46 ± 17.06%. Although there was difference in the radioactivity in the tumors between the two group, statistically significant difference was not achieved (p&amp;gt;0.05). However, decrease of VEGFR2 in melatonin treated mice was confirmed by immunohistochemistry (p&amp;lt;0.05). In addition, there was a decrease of micro-vessel density (vWF) and cell proliferation (Ki-67) in melatonin treated tumors (p&amp;lt;0.05). Conclusion: Taken together, our results showed that melatonin inhibits tumor growth, cell proliferation and blocks tumor angiogenesis in breast cancer, directing the development of a clinical study to determine its therapeutic potential for breast cancer. Citation Format: Bruna Victorasso Jardim-Perassi, Ali S. Arbab, Lívia Carvalho Ferreira, Thaiz Ferraz Borin, Nadimpalli Ravi S. Varma, Adarsh Shankar, Asm Iskander, Meser M. Ali, Debora Aparecida Pires de Campos Zuccari. Effect of melatonin on the tumor growth and angiogenesis of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A009.

Combination Therapy of VEGF-Trap and Gemcitabine Results in Improved Anti-Tumor Efficacy in a Mouse Lung Cancer Model

BackgroundAngiogenesis is essential for the growth and metastasis of cancer. Although anti-angiogenic agents, particularly vascular endothelial growth factor (VEGF) inhibitors, have exhibited single-agent activity, there is considerable interest in combining these novel drugs with conventional chemotherapy reagents to achieve an optimal clinical efficacy. The objective of this study was to evaluate the benefits of the combination therapy of vascular endothelial growth factor trap (VEGF-Trap) with gemcitabine in a lung tumor model.MethodsA luciferase-expressing Lewis lung carcinoma (LLC) model was established in C57BL/6J mice and tumor-bearing mice were randomized into control, VEGF-Trap, gemcitabine and VEGF-Trap/gemcitabine combination groups. Tumor growth and animal survival were monitored. Tumor microvessel density and cell proliferation were evaluated by CD31 and Ki-67 immunohistochemical analysis. TUNEL assay was performed to detect apoptotic cells. The protein levels of Cyclin D1, Pro-Caspase-3, Bcl-2, MMP2 and MMP9 in tumor extracts were examined by western blot.ResultsVEGF-Trap in combination with gemcitabine showed significantly enhanced inhibition of tumor growth and prolonged mouse survival compared to the VEGF-Trap or gemcitabine monotherapy. The VEGF-Trap/gemcitabine combination therapy not only potently inhibited tumor angiogenesis and cell proliferation, but also increased cellular apoptosis within tumor tissues. In addition, the combination treatment markedly down-regulated the expression of proliferation, anti-apoptosis and invasion related proteins.ConclusionCombination therapy using VEGF-Trap and gemcitabine resulted in improved anti-tumor efficacy in a lung cancer model and VEGF-Trap/gemcitabine combination might represent a promising strategy in the treatment of human lung cancer.

Combination of the ERK inhibitor AZD6244 and low-dose sorafenib in a xenograft model of human renal cell carcinoma

Sorafenib, a multikinase inhibitor, is currently used as monotherapy for advanced renal cell carcinoma (RCC). However, adverse effects associated with its use have been experienced by some patients. In this study, we examined the antitumor and antiangiogenic activities of low-dose sorafenib in combination with the MEK inhibitor AZD6244 (sorafenib/AZD6244) in a preclinical model of RCC. Primary RCC 08-0910 and RCC786-0 cells as well as patient-derived RCC models were used to study the antitumor and antiangiogenic activities of sorafenib/AZD6244. Changes of biomarkers relevant to angiogenesis and cell cycle were determined by western immunoblotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry. Treatment of RCC 786-0 cells with sorafenib/AZD6244 resulted in G1 cell cycle arrest and blockade of serum-induced cell migration. Sorafenib/AZD6244 induced apoptosis in primary RCC 08-0910 cells at low concentrations. Invivo addition of AZD6244 to sorafenib significantly augmented the antitumor activity of sorafenib and allowed dose reduction of sorafenib without compromising its antitumor activity. Sorafenib/AZD6244 potently inhibited angiogenesis and phosphorylation of VEGFR-2, PDGFR-β and ERK, p90RSK, p70S6K, cdk-2 and retinoblastoma. Sorafenib/AZD6244 also caused upregulation of p27, Bad and Bim but downregulation of survivin and cyclin B1. These resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. Our findings showed that AZD6244 and sorafenib complement each other to inhibit tumor growth. This study provides sound evidence for the clinical investigation of low-dose sorafenib in combination with AZD6244 in patients with advanced RCC.

Abstract 2314: Dovitinib demonstrates antitumor and anti-metastatic activities in xenograft models of hepatocellular carcinoma

Abstract Background and aims: Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. Although sorafenib has been shown to improve survival of patients with advanced HCC, this improvement is modest and patients eventually have refractory disease. This study aims to investigate the antitumor, antiangiogenesis and antimetastatic activities of dovitinib in preclinical models of HCC. Methods: 21-0208 and SK-HEP1 cells as well as patient-derived HCC models were employed to study of the antitumor effect of dovitinib. Changes of biomarkers relevant to FGFR/VEGFR/PDGFR pathways were determined by Western blotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry. Results: Treatment of SK-HEP1 cells with dovitinib resulted in G2/M cell cycle arrest, inhibition of colony formation in soft agar and blockade of bFGF-induced cell migration. Dovitinib inhibited basal expression and FGF-induced phosphorylation of FGFR-1, FRS2-α and ERK1/2. In vivo, dovitinib potently inhibited tumor growth of six HCC lines. Inhibition of angiogenesis correlated with inactivation of FGFR/PDGFR-α/VEGFR-2 signaling pathways. Dovitinib also caused dephosphorylation of retinoblastoma, upregulation of p-histone H2A-X and p27, and downregulation of p-cdk-2 and cyclin B1, which resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. In an orthotopic model, dovitinib potently inhibited primary tumor growth and lung metastasis and significantly prolonged mouse survival. Conclusion: Dovitinib demonstrated significant antitumor and antimetastatic activities in HCC xenograft models. This study provides a compelling rationale for clinical investigation in patients with advanced HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2314. doi:1538-7445.AM2012-2314