Extracellular matrix stiffness controls VEGF165 secretion and neuroblastoma angiogenesis via the YAP/RUNX2/SRSF1 axis.

Aberrant variations in angiogenesis have been observed in tumor tissues with abnormal stiffness of extracellular matrix (ECM). However, it remains largely unclear how ECM stiffness influences tumor angiogenesis. Numerous studies have reported that vascular endothelial growth factor-A (VEGF-A) released from tumor cells plays crucial roles in angiogenesis. Hence, we demonstrated the role of ECM stiffness in VEGF-A release from neuroblastoma (NB) cells and the underlying mechanisms. Based on 17 NB clinical samples, a negative correlation was observed between the length of blood vessels and stiffness of NB tissues. In vitro, an ECM stiffness of 30 kPa repressed the secretion of VEGF165 from NB cells which subsequently inhibited the tube formation of human umbilical vein endothelial cells (HUVECs). Knocked down VEGF165 in NB cells or blocked VEGF165 with neutralizing antibodies both repressed the tube formation of HUVECs. Specifically, 30 kPa ECM stiffness repressed the expression and nuclear accumulation of Yes-associated protein (YAP) to regulate the expression of Serine/Arginine Splicing Factor 1 (SRSF1) via Runt-related transcription factor 2 (RUNX2), which may then subsequently induce the expression and secretion of VEGF165 in NB tumor cells. Through implantation of 3D col-Tgels with different stiffness into nude mice, the inhibitory effect of 30 kPa on NB angiogenesis was confirmed in vivo. Furthermore, we found that the inhibitory effect of 30 kPa stiffness on NB angiogenesis was reversed by YAP overexpression, suggesting the important role of YAP in NB angiogenesis regulated by ECM stiffness. Overall, our work not only showed a regulatory effect of ECM stiffness on NB angiogenesis, but also revealed a new signaling axis, YAP-RUNX2-SRSF1, that mediates angiogenesis by regulating the expression and secretion of VEGF165 from NB cells. ECM stiffness and the potential molecules revealed in the present study may be new therapeutic targets for NB angiogenesis.

Perfusion-guided sonopermeation of neuroblastoma: a novel strategy for monitoring and predicting liposomal doxorubicin uptake in vivo.

Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, and imposes significant morbidity and mortality in this population. The aggressive chemoradiotherapy required to treat high-risk NB results in survival of less than 50%, yet is associated with significant long-term adverse effects in survivors. Boosting efficacy and reducing morbidity are therefore key goals of treatment for affected children. We hypothesize that these may be achieved by developing strategies that both focus and limit toxic therapies to the region of the tumor. One such strategy is the use of targeted image-guided drug delivery (IGDD), which is growing in popularity in personalized therapy to simultaneously improve on-target drug deposition and assess drug pharmacodynamics in individual patients. IGDD strategies can utilize a variety of imaging modalities and methods of actively targeting pharmaceutical drugs, however in vivo imaging in combination with focused ultrasound is one of the most promising approaches already being deployed for clinical applications. Over the last two decades, IGDD using focused ultrasound with "microbubble" ultrasound contrast agents (UCAs) has been increasingly explored as a method of targeting a wide variety of diseases, including cancer. This technique, known as sonopermeation, mechanically augments vascular permeability, enabling increased penetration of drugs into target tissue. However, to date, methods of monitoring the vascular bioeffects of sonopermeation in vivo are lacking. UCAs are excellent vascular probes in contrast-enhanced ultrasound (CEUS) imaging, and are thus uniquely suited for monitoring the effects of sonopermeation in tumors. Methods: To monitor the therapeutic efficacy of sonopermeation in vivo, we developed a novel system using 2D and 3D quantitative contrast-enhanced ultrasound imaging (qCEUS). 3D tumor volume and contrast enhancement was used to evaluate changes in blood volume during sonopermeation. 2D qCEUS-derived time-intensity curves (TICs) were used to assess reperfusion rates following sonopermeation therapy. Intratumoral doxorubicin (and liposome) uptake in NB was evalauted ex vivo along with associated vascular changes. Results: In this study, we demonstrate that combining focused ultrasound therapy with UCAs can significantly enhance chemotherapeutic payload to NB in an orthotopic xenograft model, by improving delivery and tumoral uptake of long-circulating liposomal doxorubicin (L-DOX) nanoparticles. qCEUS imaging suggests that changes in flow rates are highly sensitive to sonopermeation and could be used to monitor the efficacy of treatment in vivo. Additionally, initial tumor perfusion may be a good predictor of drug uptake during sonopermeation. Following sonopermeation treatment, vascular biomarkers show increased permeability due to reduced pericyte coverage and rapid onset of doxorubicin-induced apoptosis of NB cells but without damage to blood vessels. Conclusion: Our results suggest that significant L-DOX uptake can occur by increasing tumor vascular permeability with microbubble sonopermeation without otherwise damaging the vasculature, as confirmed by in vivo qCEUS imaging and ex vivo analysis. The use of qCEUS imaging to monitor sonopermeation efficiency and predict drug uptake could potentially provide real-time feedback to clinicians for determining treatment efficacy in tumors, leading to better and more efficient personalized therapies. Finally, we demonstrate how the IGDD strategy outlined in this study could be implemented in human patients using a single case study.

Runt-related transcription factor 1 promotes apoptosis and inhibits neuroblastoma progression in vitro and in vivo.

BACKGROUND: Runt-related transcription factor 1 (RUNX1) is a heterodimeric transcription factor that binds to the core element of many enhancers and promoters and can accelerate apoptosis in various tumors. However, the regulatory mechanisms underlying RUNX1 expression in neuroblastoma (NB), a highly malignant tumor in childhood, remain largely unclear. In this study, we aimed to assess the role of RUNX1 in NB and to reveal the underlying mechanisms that may contribute to finding a potential therapeutics strategy against NB. METHODS: Growth, invasion, metastasis and angiogenesis were assessed using Cell Counting Kit-8 (CCK-8) immunocytochemistry, and studies involving soft agar, cell invasion, tube formation and whole animals. The levels of expression were measured using real-time quantitative PCR for RNA, Western blot and immunostaining analyses for proteins. Luciferase reporter and chromatin immunoprecipitation assays indicated that RUNX1 directly binds within the BIRC5, CSF2RB and NFKBIA promoter regions to facilitate transcription. The level of apoptosis was assessed by determining mitochondrial membrane potential and flow cytometry. RESULTS: RUNX1 was highly expressed in ganglioneuroma (GN) and well-differentiated (WD) tissues relative to the poorly differentiated (PD) and undifferentiated (UD) ones. Moreover, RUNX1 effectively reduced cell viability, invasion, metastasis, angiogenesis, and promoted apoptosis in vitro and in vivo. RUNX1 reduced BIRC5 transcription and increased CSF2RB and NFKBIA transcription by directly binding BIRC5, CSF2RB and NFKBIA promoters. In addition, cytotoxic drugs, especially cisplatin, significantly increased RUNX1 expression in NB cells and promoted apoptosis. CONCLUSIONS: These data show that RUNX1 is an independent surrogate marker for the progression of NB and it can be used for monitoring NB prognosis during therapy.

Repotrectinib (TPX-0005), effectively reduces growth of ALK driven neuroblastoma cells.

Neuroblastoma is the most commonly diagnosed extracranial tumor in the first year of life. Approximately 9% of neuroblastoma patients present germline or somatic aberrations in the gene encoding for anaplastic lymphoma kinase (ALK). This increases in high-risk neuroblastomas, which have a 14% frequency of ALK aberrations at the time of diagnosis and show increasing numbers at relapse. Abrogating ALK activity with kinase inhibitors is employed as clinical therapy in malignancies such as non-small cell lung cancer and has shown good results in pediatric inflammatory myofibroblastic tumors and anaplastic large cell lymphomas. A phase I clinical trial of the first generation ALK inhibitor, crizotinib, in neuroblastoma patients showed modest results and suggested that further investigation was needed. Continuous development of ALK inhibitors has resulted in the third generation inhibitor repotrectinib (TPX-0005), which targets the active kinase conformations of ALK, ROS1 and TRK receptors. In the present study we investigated the effects of repotrectinib in a neuroblastoma setting in vitro and in vivo. Neuroblastoma cell lines were treated with repotrectinib to investigate inhibition of ALK and to determine its effect on proliferation. PC12 cells transfected with different ALK mutant variants were used to study the efficacy of repotrectinib to block ALK activation/signaling. The in vivo effect of repotrectinib was also analyzed in a neuroblastoma xenograft model. Our results show that repotrectinib is capable of inhibiting signaling activity of a range of ALK mutant variants found in neuroblastoma patients and importantly it exhibits strong antitumor effects in a xenograft model of neuroblastoma.

MRI Imaging of the Hemodynamic Vasculature of Neuroblastoma Predicts Response to Antiangiogenic Treatment.

Childhood neuroblastoma is a hypervascular tumor of neural origin, for which antiangiogenic drugs are currently being evaluated; however, predictive biomarkers of treatment response, crucial for successful delivery of precision therapeutics, are lacking. We describe an MRI-pathologic cross-correlative approach using intrinsic susceptibility (IS) and susceptibility contrast (SC) MRI to noninvasively map the vascular phenotype in neuroblastoma Th-MYCN transgenic mice treated with the vascular endothelial growth factor receptor inhibitor cediranib. We showed that the transverse MRI relaxation rate R 2* (second-1) and fractional blood volume (fBV, %) were sensitive imaging biomarkers of hemorrhage and vascular density, respectively, and were also predictive biomarkers of response to cediranib. Comparison with MRI and pathology from patients with MYCN-amplified neuroblastoma confirmed the high degree to which the Th-MYCN model vascular phenotype recapitulated that of the clinical phenotype, thereby supporting further evaluation of IS- and SC-MRI in the clinic. This study reinforces the potential role of functional MRI in delivering precision medicine to children with neuroblastoma. SIGNIFICANCE: This study shows that functional MRI predicts response to vascular-targeted therapy in a genetically engineered murine model of neuroblastoma.

Discrimination of histopathologic types of childhood peripheral neuroblastic tumors based on clinical and biological factors.

The aim of this study was to discriminate the children malignant peripheral neuroblastic tumors (PNTs) from those with benign histotype ganglioneuroma (GN) based on clinical and biological characteristics in all PNTs. Four hundred and seventy-six patients were included in this study, containing 345 patients for model development and 131 patients for external validation. Multivariate logistic regression analysis was conducted to select potentially useful characteristics for discrimination of histopathology. External validation was performed for model evaluation. Compared with the main characteristics of GN (85/345, 24.6%), those of malignant PNTs (260/345, 75.4%) showed significant differences. Multivariate analysis was performed to further find the characteristics linked to histopathology. The results indicated that for the patients younger than 49 months, the primary site of adrenal and thoracic, the level of serum neuron-specific enolase (NSE) > 33 ng/mL, and tumor encasing blood vessels were the extremely important discrimination factors of malignant PNTs. The area under the receiver-operating-characteristic of the discrimination model was 0.96. The accuracy rate, sensitivity and specificity were 93.4%, 96.3% and 83.8%, respectively. Meanwhile, the accuracy rate of the external validation from the 131 patients was 97.0%. Overall, histopathologic type of childhood malignant PNTs can be discriminated based on age, primary site, NSE level and the relationship between primary tumor and blood vessels.

Predictive Modeling of Neuroblastoma Growth Dynamics in Xenograft Model After Bevacizumab Anti-VEGF Therapy.

Neuroblastoma is the leading cause of cancer death in young children. Although treatment for neuroblastoma has improved, the 5-year survival rate of patients still remains less than half. Recent studies have indicated that bevacizumab, an anti-VEGF drug used in treatment of several other cancer types, may be effective for treating neuroblastoma as well. However, its effect on neuroblastoma has not been well characterized. While traditional experiments are costly and time-consuming, mathematical models are capable of simulating complex systems quickly and inexpensively. In this study, we present a model of vascular tumor growth of neuroblastoma IMR-32 that is complex enough to replicate experimental data across a range of tumor cell properties measured in a suite of in vitro and in vivo experiments. The model provides quantitative insight into tumor vasculature, predicting a linear relationship between vasculature and tumor volume. The tumor growth model was coupled with known pharmacokinetics and pharmacodynamics of the VEGF blocker bevacizumab to study its effect on neuroblastoma growth dynamics. The results of our model suggest that total administered bevacizumab concentration per week, as opposed to dosage regimen, is the major determining factor in tumor suppression. Our model also establishes an exponentially decreasing relationship between administered bevacizumab concentration and tumor growth rate.

Hypoxia and hypoxia-inducible factors in neuroblastoma.

Hypoxia (i.e., low oxygen levels) is a known feature of aggressive tumors. Cells, including tumor cells, respond to conditions of insufficient oxygen by activating a transcriptional program mainly driven by hypoxia-inducible factors (HIF)-1 and HIF-2. Both HIF-1α and HIF-2α expression levels have been shown to correlate to patient outcome in various tumor forms and in neuroblastoma, a solid childhood tumor of the sympathetic nervous system, in particular, HIF-2α marks a subpopulation of immature neural crest-like perivascularly located cells and associates with aggressive disease and distant metastasis. It has for long been recognized that the HIF-α subunits are oxygen-dependently regulated at the post-translational level, via ubiquitination and proteasomal degradation. Evidence of oxygen-independent mechanisms of regulation, transcriptional control of EPAS1/HIF2A and possible cytoplasmic activities of HIF-2α has also emerged during recent years. In this review, we discuss these non-conventional actions of HIF-2α, its putative role as a therapeutic target and the constraints it carries, as well as the importance of HIF-2 activity in a vascularized setting, the so-called pseudo-hypoxic state.

Vascularized Tissue-Engineered Model for Studying Drug Resistance in Neuroblastoma.

Neuroblastoma is a vascularized pediatric tumor derived from neural crest stem cells that displays vasculogenic mimicry and can express a number of stemness markers, such as SOX2 and NANOG. Tumor relapse is the major cause of succumbing to this disease, and properties attributed to cancer stem-like cells (CSLC), such as drug-resistance and cell plasticity, seem to be the key mechanisms. However, the lack of controllable models that recapitulate the features of human neuroblastoma limits our understanding of the process and impedes the development of new therapies. In response to these limitations, we engineered a perfusable, vascularized in vitro model of three-dimensional human neuroblastoma to study the effects of retinoid therapy on tumor vasculature and drug-resistance. METHODS: The in vitro model of neuroblastoma was generated using cell-sheet engineering and cultured in a perfusion bioreactor. Firstly, we stacked three cell sheets containing SKNBE(2) neuroblastoma cells and HUVEC. Then, a vascular bed made of fibrin, collagen I and HUVEC cells was placed onto a collagen-gel base with 8 microchannels. After gelling, the stacked cell sheets were placed on the vascular bed and cultured in the perfusion bioreactor (perfusion rate: 0.5 mL/min) for 4 days. Neuroblastoma models were treated with 10µM isotretionin in single daily doses for 5 days. RESULTS: The bioengineered model recapitulated vasculogenic mimicry (vessel-like structure formation and tumor-derived endothelial cells-TECs), and contained CSLC expressing SOX2 and NANOG. Treatment with Isotretinoin destabilized vascular networks but failed to target vasculogenic mimicry and augmented populations of CSLCs expressing high levels of SOX2. Our results suggest that CSLCs can transdifferentiate into drug resistant CD31+-TECs, and reveal the presence of an intermediate state STEC (stem tumor-derived endothelial cell) expressing both SOX2 and CD31. CONCLUSION: Our results reveal some roles of SOX2 in drug resistance and tumor relapse, and suggest that SOX2 could be a therapeutic target in neuroblastoma.

Pharmacokinetics and Pharmacodynamics-Based Mathematical Modeling Identifies an Optimal Protocol for Metronomic Chemotherapy.

Metronomic chemotherapy is usually associated with better tolerance than conventional chemotherapy, and encouraging response rates have been reported in various settings. However, clinical development of metronomic chemotherapy has been hampered by a number of limitations, including the vagueness of its definition and the resulting empiricism in protocol design. In this study, we developed a pharmacokinetic/pharmacodynamic mathematical model that identifies in silico the most effective administration schedule for gemcitabine monotherapy. This model is based upon four biological assumptions regarding the mechanisms of action of metronomic chemotherapy, resulting in a set of 6 minimally parameterized differential equations. Simulations identified daily 0.5-1 mg/kg gemcitabine as an optimal protocol to maximize antitumor efficacy. Both metronomic protocols (0.5 and 1 mg/kg/day for 28 days) were evaluated in chemoresistant neuroblastoma-bearing mice and compared with the standard MTD protocol (100 mg/kg once a week for 4 weeks). Systemic exposure to gemcitabine was 14 times lower in the metronomic groups compared with the standard group. Despite this, metronomic gemcitabine significantly inhibited tumor angiogenesis and reduced tumor perfusion and inflammation in vivo, while standard gemcitabine did not. Furthermore, metronomic gemcitabine yielded a 40%-50% decrease in tumor mass at the end of treatment as compared with control mice (P = 0.002; ANOVA on ranks with Dunn test), while standard gemcitabine failed to significantly reduce tumor growth. Stable disease was maintained in the metronomic groups for up to 2 months after treatment completion (67%-72% reduction in tumor growth at study conclusion, P < 0.001; ANOVA on ranks with Dunn test). Collectively, our results confirmed the superiority of metronomic protocols in chemoresistant tumors in vivoCancer Res; 77(17); 4723-33. ©2017 AACR.

Nuclear FOXO3 predicts adverse clinical outcome and promotes tumor angiogenesis in neuroblastoma.

Neuroblastoma is the most frequent, extracranial solid tumor in children with still poor prognosis in stage IV disease. In this study, we analyzed FOXO3-phosphorylation and cellular localization in tumor biopsies and determined the function of this homeostasis regulator in vitro and in vivo. FOXO3-phosphorylation at threonine-32 (T32) and nuclear localization in biopsies significantly correlated with stage IV disease. DNA-damaging drugs induced nuclear accumulation of FOXO3, which was associated with elevated T32-phosphorylation in stage IV-derived neuroblastoma cells, thereby reflecting the in situ results. In contrast, hypoxic conditions repressed PKB-activity and caused dephosphorylation of FOXO3 in both, stroma-like SH-EP and high-stage-derived STA-NB15 cells. The activation of an ectopically-expressed FOXO3 in these cells reduced viability at normoxia, but promoted growth at hypoxic conditions and elevated VEGF-C-expression. In chorioallantoic membrane (CAM) assays STA-NB15 tumors with ectopic FOXO3 showed increased micro-vessel formation and, when xenografted into nude mice, a gene-dosage-dependent effect of FOXO3 in high-stage STA-NB15 cells became evident: low-level activation increased tumor-vascularization, whereas hyper-activation repressed tumor growth.The combined data suggest that, depending on the mode and intensity of activation, cellular FOXO3 acts as a homeostasis regulator promoting tumor growth at hypoxic conditions and tumor angiogenesis in high-stage neuroblastoma.

Smad4 suppresses the tumorigenesis and aggressiveness of neuroblastoma through repressing the expression of heparanase.

Heparanase (HPSE) is the only endo-ß-D-glucuronidase that is correlated with the progression of neuroblastoma (NB), the most common extracranial malignancy in childhood. However, the mechanisms underlying HPSE expression in NB still remain largely unknown. Herein, through analyzing cis-regulatory elements and mining public microarray datasets, we identified SMAD family member 4 (Smad4) as a crucial transcription regulator of HPSE in NB. We demonstrated that Smad4 repressed the HPSE expression at the transcriptional levels in NB cells. Mechanistically, Smad4 suppressed the HPSE expression through directly binding to its promoter and repressing the lymphoid enhancer binding factor 1 (LEF1)-facilitated transcription of HPSE via physical interaction. Gain- and loss-of-function studies demonstrated that Smad4 inhibited the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo. Restoration of HPSE expression prevented the NB cells from changes in these biological features induced by Smad4. In clinical NB specimens, Smad4 was under-expressed and inversely correlated with HPSE levels, while LEF1 was highly expressed and positively correlated with HPSE expression. Patients with high Smad4 expression, low LEF1 or HPSE levels had greater survival probability. These results demonstrate that Smad4 suppresses the tumorigenesis and aggressiveness of NB through repressing the HPSE expression.

The novel kinase inhibitor ponatinib is an effective anti-angiogenic agent against neuroblastoma.

Background High-risk neuroblastoma has poor outcomes with high rates of relapse despite aggressive treatment, and novel therapies are needed to improve these outcomes. Ponatinib is a multi-tyrosine kinase inhibitor that targets many pathways implicated in neuroblastoma pathogenesis. We hypothesized that ponatinib would be effective against neuroblastoma in preclinical models. Methods We evaluated the effects of ponatinib on survival and migration of human neuroblastoma cells in vitro. Using orthotopic xenograft mouse models of human neuroblastoma, we analyzed tumors treated with ponatinib for growth, gross and histologic appearance, and vascularity. Results Ponatinib treatment of neuroblastoma cells resulted in decreased cell viability and migration in vitro. In mice with orthotopic xenograft neuroblastoma tumors, treatment with ponatinib resulted in decreased growth and vascularity. Conclusions Ponatinib reduces neuroblastoma cell viability in vitro and reduces tumor growth and vascularity in vivo. The antitumor effects of ponatinib suggest its potential as a novel therapeutic agent for neuroblastoma, and further preclinical testing is warranted.

SPARC overexpression combined with radiation retards angiogenesis by suppressing VEGF-A via miR­410 in human neuroblastoma cells.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children and despite aggressive therapy survival rates remain low. One of the contributing factors for low survival rates is aggressive tumor angiogenesis, which is known to increase due to radiation, one of the standard therapies for neuroblastoma. Therefore, targeting tumor angiogenesis can be a viable add-on therapy for the treatment of neuroblastomas. In the present study, we demonstrate that overexpression of secreted protein acidic and rich in cysteine (SPARC) suppresses radiation induced angiogenesis in SK-N­BE(2) and NB1691 neuroblastoma cells. We observed that overexpression of SPARC in SK-N-BE(2) and NB1691 cells reduced radiation induced angiogenesis in an in vivo mouse dorsal skin model and an ex vivo chicken CAM (chorioallantoic-membrane) model and also reduced tumor size in subcutaneous mouse tumor models of NB. We also observed that SPARC overexpression reduces VEGF-A expression, in SK-N-BE(2) and NB1691 NB cells via miR-410, a VEGF-A targeting microRNA. SPARC overexpression alone or in combination with miR-410 and radiation was shown to be effective at reducing angiogenesis. Moreover, addition of miR-410 inhibitors reversed SPARC mediated inhibition of VEGF-A in NB1691 cells but not in SK-N-BE(2) NB cells. In conclusion, the present study demonstrates that the overexpression of SPARC in combination with radiation reduced tumor angiogenesis by downregulating VEGF-A via miR-410.

Detection of Phenotypic Alterations Using High-Content Analysis of Whole-Slide Images.

Tumors exhibit spatial heterogeneity, as manifested in immunohistochemistry (IHC) staining patterns. Current IHC quantification methods lose information by reducing this heterogeneity in each whole-slide image (WSI) or in selective fields of view to a single staining index. The aim of this study was to investigate the sensitivity of an IHC quantification method that uses this heterogeneity to reliably compare IHC staining patterns. We virtually partitioned WSIs by a grid of square tiles, and computed the staining index distributions to quantify heterogeneities. We used samples from these distributions as inputs to non-parametric statistical comparisons. We applied our grid method to fixed tumor samples from 26 tumors obtained from a double-blind preclinical study of a patient-derived orthotopic xenograft model of pediatric neuroblastoma in CD1 nude mice. We compared the results of our grid method to the results based on whole-slide indices, the current practice. We show that our grid method reliably detects phenotypic alterations that other tests based on whole-slide indices fail to detect. Based on robustness and increased sensitivity of statistical inference, we conclude that our method of whole-slide grid quantification is superior to existing whole-slide quantification techniques.

Vascular patterns provide therapeutic targets in aggressive neuroblastic tumors.

Angiogenesis is essential for tumor growth and metastasis, nevertheless, in NB, results between different studies on angiogenesis have yielded contradictory results. An image analysis tool was developed to characterize the density, size and shape of total blood vessels and vascular segments in 458 primary neuroblastic tumors contained in tissue microarrays. The results were correlated with clinical and biological features of known prognostic value and with risk of progression to establish histological vascular patterns associated with different degrees of malignancy. Total blood vessels were larger, more abundant and more irregularly-shaped in tumors of patients with associated poor prognostic factors than in the favorable cohort. Tumor capillaries were less abundant and sinusoids more abundant in the patient cohort with unfavorable prognostic factors. Additionally, size of post-capillaries & metarterioles as well as higher sinusoid density can be included as predictive factors for survival. These patterns may therefore help to provide more accurate pre-treatment risk stratification, and could provide candidate targets for novel therapies.

High-Dose, Single-Fraction Irradiation Rapidly Reduces Tumor Vasculature and Perfusion in a Xenograft Model of Neuroblastoma.

PURPOSE: To characterize the effects of high-dose radiation therapy (HDRT) on neuroblastoma tumor vasculature, including the endothelial cell (EC)-pericyte interaction as a potential target for combined treatment with antiangiogenic agents. METHODS AND MATERIALS: The vascular effects of radiation therapy were examined in a xenograft model of high-risk neuroblastoma. In vivo 3-dimensional contrast-enhanced ultrasonography (3D-CEUS) imaging and immunohistochemistry (IHC) were performed. RESULTS: HDRT significantly reduced tumor blood volume 6 hours after irradiation compared with the lower doses used in conventionally fractionated radiation. There was a 63% decrease in tumor blood volume after 12-Gy radiation compared with a 24% decrease after 2 Gy. Analysis of tumor vasculature by lectin angiography showed a significant loss of small vessel ends at 6 hours. IHC revealed a significant loss of ECs at 6 and 72 hours after HDRT, with an accompanying loss of immature and mature pericytes at 72 hours. CONCLUSIONS: HDRT affects tumor vasculature in a manner not observed at lower doses. The main observation was an early reduction in tumor perfusion resulting from a reduction of small vessel ends with a corresponding loss of endothelial cells and pericytes.

The long noncoding RNA MALAT1 promotes tumor-driven angiogenesis by up-regulating pro-angiogenic gene expression.

Neuroblastoma is the most common solid tumor during early childhood. One of the key features of neuroblastoma is extensive tumor-driven angiogenesis due to hypoxia. However, the mechanism through which neuroblastoma cells drive angiogenesis is poorly understood. Here we show that the long noncoding RNA MALAT1 was upregulated in human neuroblastoma cell lines under hypoxic conditions. Conditioned media from neuroblastoma cells transfected with small interfering RNAs (siRNA) targeting MALAT1, compared with conditioned media from neuroblastoma cells transfected with control siRNAs, induced significantly less endothelial cell migration, invasion and vasculature formation. Microarray-based differential gene expression analysis showed that one of the genes most significantly down-regulated following MALAT1 suppression in human neuroblastoma cells under hypoxic conditions was fibroblast growth factor 2 (FGF2). RT-PCR and immunoblot analyses confirmed that MALAT1 suppression reduced FGF2 expression, and Enzyme-Linked Immunosorbent Assays revealed that transfection with MALAT1 siRNAs reduced FGF2 protein secretion from neuroblastoma cells. Importantly, addition of recombinant FGF2 protein to the cell culture media reversed the effects of MALAT1 siRNA on vasculature formation. Taken together, our data suggest that up-regulation of MALAT1 expression in human neuroblastoma cells under hypoxic conditions increases FGF2 expression and promotes vasculature formation, and therefore plays an important role in tumor-driven angiogenesis.

PI3K-mTORC2 but not PI3K-mTORC1 regulates transcription of HIF2A/EPAS1 and vascularization in neuroblastoma.

Hypoxia-inducible factor (HIF) is a master regulator of cellular responses to oxygen deprival with a critical role in mediating the angiogenic switch in solid tumors. Differential expression of the HIF subunits HIF1α and HIF2α occurs in many human tumor types, suggesting selective implications to biologic context. For example, high expression of HIF2α that occurs in neuroblastoma is associated with stem cell-like features, disseminated disease, and poor clinical outcomes, suggesting pivotal significance for HIF2 control in neuroblastoma biology. In this study, we provide novel insights into how HIF2α expression is transcriptionally controlled by hypoxia and how this control is abrogated by inhibition of insulin-like growth factor-1R/INSR-driven phosphoinositide 3-kinase (PI3K) signaling. Reducing PI3K activity was sufficient to decrease HIF2α mRNA and protein expression in a manner with smaller and less vascularized tumors in vivo. PI3K-regulated HIF2A mRNA expression was independent of Akt or mTORC1 signaling but relied upon mTORC2 signaling. HIF2A mRNA was induced by hypoxia in neuroblastoma cells isolated from metastatic patient-derived tumor xenografts, where HIF2A levels could be reduced by treatment with PI3K and mTORC2 inhibitors. Our results suggest that targeting PI3K and mTORC2 in aggressive neuroblastomas with an immature phenotype may improve therapeutic efficacy.