Hypoxia Metagene Research Articles

Repurposing Atovaquone as a Tumor Hypoxia Modifier: A Window of Opportunity Study in Patients with Resectable Non-small Cell Lung Cancer

Tumor hypoxia confers profound resistance to radiation. An emerging strategy to tackle hypoxia is by reprogramming tumor metabolism. The antimalarial drug atovaquone inhibits oxidative phosphorylation (OXPHOS) and preclinically has been shown to reduce tumor cell oxygen consumption and alleviate tumor hypoxia resulting in radiosensitization. We conducted a translational clinical trial to determine whether atovaquone reduces tumor hypoxia in patients with non-small cell lung cancer (NSCLC). Patients with NSCLC scheduled for surgical resection were recruited sequentially into two cohorts. Cohort 1 received oral atovaquone 750mg bd until the day before surgery. Cohort 2 received no treatment. Tumor hypoxia imaging with 18F-FMISO or 18F-FAZA PET-CT was undertaken at baseline and repeated just prior to resection. Tumor hypoxic volume (HV) was defined using tumor-to-blood tracer uptake ratio ≥1.4, with ≥10% reduction in HV classed a response to treatment. Patients with <1.5 mL HV at baseline were deemed not evaluable for HV analysis and were replaced. Perfusion CT (pCT) and shMOLLI T1 map MRI were undertaken at the same timepoints to assess for changes in tumor perfusion and biochemistry, respectively. An inter-cohort comparison of hypoxia metagene expression signatures obtained by RNA sequencing was conducted from multiple regions per resected tumor. Adverse event monitoring (CTCAE v4.0) was performed until surgery. Forty-six patients were recruited with 30 (15 per cohort) evaluable for paired HV analysis. Patient and tumor characteristics were similar between cohorts. Median duration of atovaquone treatment between imaging timepoints was 12 days. The median change in HV in atovaquone-treated patients was -28% (95% CI: -58.2, -4.4), with 11 (73.3%) patients classed as responders. In contrast, the median change in untreated patients was +15.5% (95% CI: -6.5, +35.5). Using linear regression with pre-surgery HV as outcome adjusting for cohort and baseline HV and tumor volume (log-transformed), the expected geometric mean HV was 60% (95% CI: 30%, 70%) lower for treated patients (p = 0.004). Transcriptomic analysis revealed significantly decreased expression of multiple hypoxia metagene signatures in treated tumors. Altered expression of OXPHOS related pathways was also observed suggesting wider metabolic reprogramming. pCT and MRI results did not show altered tumor perfusion or suggest change in cellularity, respectively, in keeping with an OXPHOS-dependent mechanism of action. No adverse events related to atovaquone were reported. This the first clinical study to investigate whether an OXPHOS inhibitor reduces tumor hypoxia. The data presented supports our pre-clinical finding that atovaquone rapidly reduces tumor hypoxia through decreased oxygen consumption in NSCLC. We will shortly commence phase I clinical testing combining atovaquone with radical chemoradiotherapy in NSCLC.

Functional Genomics Uncover the Biology behind the Responsiveness of Head and Neck Squamous Cell Cancer Patients to Cetuximab.

To identify the tumor portrait of the minority of head and neck squamous cell carcinoma (HNSCC) patients with recurrent-metastatic (RM) disease who upon treatment with platinum-based chemotherapy plus cetuximab present a long-lasting response. The gene expression of pretreatment samples from 40 HNSCC-RM patients, divided in two groups [14 long-progression-free survival (PFS) and 26 short-PFS (median = 19 and 3 months, respectively)], was associated with PFS and was challenged against a dataset from metastatic colon cancer patients treated with cetuximab. For biologic analysis, we performed functional and subtype association using gene set enrichment analysis, associated biology across all currently available HNSCC signatures, and inferred drug sensitivity using data from the Cancer Genomic Project. The identified genomic profile exhibited a significant predictive value that was essentially confirmed in the single publicly available dataset of cetuximab-treated patients. The main divergence between long- and short-PFS groups was based on developmental/differentiation status. The long-PFS patients are characterized by basal subtype traits such as strong EGFR signaling phenotype and hypoxic differentiation, further validated by the significantly higher association with the hypoxia metagene. The short-PFS patients presented a strong activation of RAS signaling confirmed in an in vitro model of two isogenic HNSCC cell lines sensitive or resistant to cetuximab. The predicted drug sensitivity for all four EGFR inhibitors was higher in long- versus short-PFS patients (P range: <0.0022-1e-07). Our data uncover the biology behind response to platinum-based chemotherapy plus cetuximab in RM-HNSCC cancer and may have translational implications improving treatment selection. Clin Cancer Res; 22(15); 3961-70. ©2016 AACRSee related commentary by Chau and Hammerman, p. 3710.

Estrogen receptor-α directly regulates the hypoxia-inducible factor 1 pathway associated with antiestrogen response in breast cancer

A majority of breast cancers are driven by estrogen via estrogen receptor-α (ERα). Our previous studies indicate that hypoxia-inducible factor 1α (HIF-1α) cooperates with ERα in breast cancer cells. However, whether ERα is implicated in the direct regulation of HIF-1α and the role of HIF-1α in endocrine therapy response are unknown. In this study we found that a subpopulation of HIF-1α targets, many of them bearing both hypoxia response elements and estrogen response elements, are regulated by ERα in normoxia and hypoxia. Interestingly, the HIF-1α gene itself also bears an estrogen response element, and its expression is directly regulated by ERα. Clinical data revealed that expression of the HIF-1α gene or a hypoxia metagene signature is associated with a poor outcome to endocrine treatment in ERα(+) breast cancer. HIF-1α was able to confer endocrine therapy resistance to ERα(+) breast cancer cells. Our findings define, for the first time to our knowledge, a direct regulatory pathway between ERα and HIF-1α, which might modulate hormone response in treatment.

Gene expression profiling of sequential metastatic biopsies for biomarker discovery in breast cancer

The feasibility of longitudinal metastatic biopsies for gene expression profiling in breast cancer is unexplored. Dynamic changes in gene expression can potentially predict efficacy of targeted cancer drugs.Patients enrolled in a phase III trial of metastatic breast cancer with docetaxel monotherapy versus combination of docetaxel + sunitinib were offered to participate in a translational substudy comprising longitudinal fine needle aspiration biopsies and Positron Emission Tomography imaging before (T1) and two weeks after start of treatment (T2). Aspirated tumor material was used for microarray analysis, and treatment-induced changes (T2 versus T1) in gene expression and standardized uptake values (SUV) were investigated and correlated to clinical outcome measures.Gene expression profiling yielded high-quality data at both time points in 14/18 patients. Unsupervised clustering revealed specific patterns of changes caused by monotherapy vs. combination therapy (p = 0.021, Fisher's exact test). A therapy-induced reduction of known proliferation and hypoxia metagene scores was prominent in the combination arm. Changes in a previously reported hypoxia metagene score were strongly correlated to the objective responses seen by conventional radiology assessments after 6 weeks in the combination arm, Spearman's ρ = 1 (p = 0.017) but not in monotherapy, ρ = −0.029 (p = 1). Similarly, the Predictor Analysis of Microarrays 50 (PAM50) proliferation metagene correlated to tumor changes merely in the combination arm at 6 and 12 weeks (ρ = 0.900, p = 0.083 and ρ = 1, p = 0.017 respectively). Reductions in mean SUV were a reliable early predictor of objective response in monotherapy, ρ = 0.833 (p = 0.008), but not in the combination arm ρ = −0.029 (p = 1).Gene expression profiling of longitudinal metastatic aspiration biopsies was feasible, demonstrated biological validity and provided predictive information.

Comprehensive gene expression meta-analysis of head and neck squamous cell carcinoma microarray data defines a robust survival predictor

Head and neck squamous cell carcinoma refers to a heterogeneous disease frequently aggressive in its biologic behavior. Despite the improvements in the therapeutic modalities, the long-term survival rate remained unchanged over the past decade and patients with this type of cancer are at a high risk of developing recurrence. For this reason, there is a great need to find better ways to foresee outcome, to improve treatment choices, and to enable a more personalized approach. Nine microarray gene expression datasets, reporting survival data of a total of 841 samples, were retrieved from publicly repositories. Three datasets, profiled on the same version of microarray chips, were selected and merged following a meta-analysis approach to build a training set. The remaining six studies were used as independent validation sets. The training set led us to identify a 172-gene signature able to stratify patients in low or high risk of relapse [log-rank, P = 2.44e-05; hazard ratio (HR) = 2.44, 95% confidence interval (CI) 1.58-3.76]. The model based on the 172 genes was validated on the six independent datasets. The performance of the model was challenged against other proposed prognostic signatures (radiosensitivity index, 13-gene oral squamous cell carcinoma signature, hypoxia metagene, 42-gene high-risk signature) and was compared with a human papillomavirus (HPV) signature: our model resulted independent and even better in prediction. We have identified and validated a prognostic model based on the expression of 172 genes, independent from HPV status and able to improve assessment of patient's risk of relapse compared with other molecular signatures. In order to transpose our model into a useful clinical grade assay, additional work is needed following the framework established by the Institute of Medicine and REMARK guidelines.

An in vivo hypoxia metagene identifies the novel hypoxia inducible factor target gene SLCO1B3

A hypoxia-associated gene signature (metagene) was previously derived via in vivo data-mining. In this study, we aimed to investigate whether this approach could identify novel hypoxia regulated genes. From an initial list of nine genes, three were selected for further study (BCAR1, IGF2BP2 and SLCO1B3). Ten cell lines were exposed to hypoxia and interrogated for the expression of the three genes. All three genes were hypoxia inducible in at least one of the 10 cell lines with SLCO1B3 induced in seven. SLCO1B3 was studied further using chromatin immunoprecipitation and luciferase assays to investigate hypoxia inducible factor (HIF) dependent transcription. Two functional HIF response elements were identified within intron 1 of the gene. The functional importance of SLCO1B3 was studied by gene knockdown experiments followed by cell growth assays, flow cytometry and Western blotting. SLCO1B3 knockdown reduced cell size and 3-dimensional spheroid volume, which was associated with decreased activation of the mammalian target of rapamycin (mTOR) pathway. Finally, Oncomine analysis revealed that head and neck and colorectal tumours had higher levels of SLCO1B3 compared to normal tissue. Thus, the knowledge based approach for deriving gene signatures can identify novel biologically relevant genes.

Radiogenomic Analysis of Breast Cancer Using MRI: A Preliminary Study to Define the Landscape

Molecular profiling studies have defined the increasing importance of gene expression phenotyping in breast cancer. However, the relationship between global transcriptomic profiles and the information provided by breast MRI remains to be examined. In this pilot study, our aim was to provide a preliminary radiogenomic association map linking MR image phenotypes to underlying global gene expression patterns in breast cancer. From a multiinstitutional study, a total of 353 patients with a diagnosis of breast cancer were examined for gene expression analysis. Radiogenomic analysis was then performed on a subset of these patients (n = 10) who also underwent breast MRI. Two radiologists evaluated each MRI study across 26 predefined imaging phenotypes. Analyses were performed to correlate the expression and imaging data and to define associations between specific MR image phenotypes and gene sets of interest. High-level analysis revealed 21 imaging traits that were globally correlated (p < 0.05), with 71% of the total genes measured in patients with breast cancer. A significant correlation was identified between heterogeneous enhancement patterns and a previously described interferon breast cancer subtype (p < 0.01). We also identified 12 imaging traits that significantly correlated (false discovery rate < 0.25) with gene sets related to breast cancer and 11 traits correlated (false discovery rate < 0.25) to prognostic gene sets (van't Veer, wound response, and hypoxia metagene signatures) using gene enrichment analysis. Radiogenomic analysis of breast cancer with MRI is a novel approach to understanding the underlying molecular biology of breast cancers.

Correction: Close And Stable Relationship Between Proliferation And A Hypoxia Metagene In Aromatase Inhibitor–Treated ER-Positive Breast Cancer

Correction: Close And Stable Relationship Between Proliferation And A Hypoxia Metagene In Aromatase Inhibitor–Treated ER-Positive Breast Cancer

Close and Stable Relationship between Proliferation and a Hypoxia Metagene in Aromatase Inhibitor–Treated ER-Positive Breast Cancer

The majority of breast cancer patients who have estrogen receptor positive (ER(+)) tumors whose proliferation is reduced after estrogen deprivation by aromatase inhibitors (AI). This study investigates any link between proliferation and hypoxia, a major determinant of tumor biology, and defines the effect of estrogen deprivation on hypoxia-associated genes. Genome-wide expression profiles were obtained from tumor biopsies from 81 ER(+) postmenopausal patients, before and after 2 weeks' anastrozole treatment. A hypoxia metagene was developed by identifying genes clustered with classical hypoxia-regulated genes, excluding those associated with proliferation. Proliferation was measured by Ki67 and a proliferation metagene derived from two published breast cancer data sets. Hypoxia and proliferation metagenes were associated at baseline (Pearson correlation coefficient, r = 0.67, P < 10(-4)) and after 2 weeks (r = 0.71, P < 10(-4)). Hypoxia metagene at baseline was associated with 2-week Ki67 (r = 0.43, P = 0.0002) and more weakly with poor 2-week Ki67 change consistent with a weak association with AI resistance. Hypoxia metagene was significantly downregulated with AI. This downregulation was significantly associated with change in the proliferation metagene and with Ki67 but, importantly, not with the substantial change in expression of classical estrogen-dependent genes. Hypoxia metagene is closely associated with proliferation before and after AI treatment. The downregulation of hypoxia metagene after AI therapy is most likely the result of changes in proliferation. There may be a weak effect of hypoxia metagene on de novo resistance to AIs. These findings are important to consider in coapplication of antiproliferative agents with antiangiogenic or antihypoxia agents.

Erratum: Large meta-analysis of multiple cancers reveals a common, compact and highly prognostic hypoxia metagene

Erratum: Large meta-analysis of multiple cancers reveals a common, compact and highly prognostic hypoxia metagene

Erratum: Large meta-analysis of multiple cancers reveals a common, compact and highly prognostic hypoxia metagene

Correction to: British Journal of Cancer (2010) 102, 428–435. doi:10.1038/sj.bjc.6605450 Owing to an error during the web publication of this paper, an element of the Supplementary Material, vital to the understanding of the paper, was omitted. This document, ‘Suppl_online_material.pdf’, has now been uploaded to accompany the paper and appears online (please see ‘Correction to’ link above).

Hsa‐miR‐210 is a marker of tumor hypoxia and a prognostic factor in head and neck cancer

Hypoxia is an important mechanism of treatment resistance in head and neck squamous cell carcinoma (HNSCC). MicroRNAs are short noncoding RNAs that regulate multiple mRNAs and are frequently dysregulated in cancer. The authors have investigated the role of 3 microRNAs, including the hypoxia-induced hsa-miR-210, as potential markers of hypoxia or prognosis. Three hypoxia-related microRNAs, hsa-miR-210, hsa-miR-21, and hsa-miR-10b, were measured in 46 samples from patients with HNSCC. Expression levels were correlated with clinicopathological variables and other markers of hypoxia: a published 99-gene hypoxia metagene, individual hypoxia-related genes such as TWIST1, and immunohistochemical expression of hypoxia-inducible factor 1 and its target gene carbonic anhydrase 9. We then performed survival analyses to investigate the prognostic significance of these microRNAs. Only the level of hsa-miR-210 was significantly correlated with other markers of hypoxia, including the 99-gene hypoxia metagene (rho = 0.67, P < .001). We found no association between hsa-miR-210, hsa-miR-21, or hsa-miR-10b and clinicopathological variables such as tumor size, differentiation, and stage. However, high levels of hsa-miR-210 were associated with locoregional disease recurrence (P = .001) and short overall survival (P = .008). hsa-miR-21 and hsa-miR-10b had no prognostic significance. Expression of hsa-miR-210 in head and neck cancer correlates with other approaches for assessing hypoxia and is associated with prognosis. This warrants further study as a classification marker of patients for therapies involving modulation of hypoxia.

Abstract SY34-03: Hypoxia-metabolic targets for drug development: Identification of targets and their validation

Abstract The low oxygen status of solid tumours relative to normal tissues has been known for over 50 years. However, methods targeting or exploiting the presence of hypoxia in tumours, for therapeutic benefit, have generally met with little success. Initially, these methods included the use of hypoxic cell radiosensitizers and bioreductive drugs. In the light of current knowledge the reason for “failure” was not necessarily a flaw in the hypotheses being tested, but rather an inability to identify those patients most likely to respond to the modifying treatment. It is now known that tumours vary in their level and extent of hypoxia, with those tumours showing the least hypoxia (highest oxygen tension) being the most responsiveness to therapy. The discovery of the hypoxia-dependent transcription factor, HIF-1, and identification of its downstream gene targets revealed new avenues for targeting tumours. Expression of HIF-1, CAIX, and GLUT-1 have been used as molecular markers of hypoxia and there has been some success in their use as prognostic markers. Further, there has been recent development of a hypoxia metagene signature (Cancer Res. 2007, 67, 3441-9) that has shown prognostic significance in multiple cancers. Despite this progress in identifying molecular markers of hypoxia there has only been one well defined example of using this information for developing novel therapeutic targets; this is the identification and validation of lysyl oxidase (Nature 2006, 440, 1222-6). Associated with hypoxia is an abnormal metabolic phenotype often characterized by high levels of lactate in human tumours. Monocarboxylate transporter-4 (mct-4) functions to transport lactate out of the cell preventing intra-cellular accumulation of lactate and therefore enabling sustained high glycolytic rates and maintenance of intra-cellular pH. We have demonstrated that mct-4 is a hypoxia/HIF-dependent gene and we evaluated its potential as a novel therapeutic target. Firstly, we evaluated whether expression of mct-4 protein in a panel of 155 oropharangeal tumours correlated with outcome of treatment with radiotherapy. Using a univariate analysis to assess high mct-4 expression (top 25% of scores) vs low mct-4 expression (lower 75%) we showed that mct-4 is a significant adverse prognostic factor in this series of biopsies. High mct-4 expression correlated with poor loco-regional control (p = 0.017), reduced cancer-specific survival (p = 0.02) and reduced overall survival (p = 0.055). In a multivariate analysis high mct-4 expression retained prognostic significance for poor loco-regional control (p = 0.007). We have therefore identified mct-4 as a potential target in head and neck cancer and we have gone on to validate its therapeutic significance by carrying out various gene knock-down studies in vitro and in vivo. Using siRNA to mct-4 we have shown that cells lacking mct-4 are substantially more radioresponsive under hypoxic conditions. Hence, not only is mct-4 expression an indicator of adverse outcome but it is also a target for the development of potential therapeutics (particularly when combined with radiotherapy). Citation Format: Ian J. Stratford. Hypoxia-metabolic targets for drug development: Identification of targets and their validation [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY34-03

Large meta-analysis of multiple cancers reveals a common, compact and highly prognostic hypoxia metagene

Background:There is a need to develop robust and clinically applicable gene expression signatures. Hypoxia is a key factor promoting solid tumour progression and resistance to therapy; a hypoxia signature has the potential to be not only prognostic but also to predict benefit from particular interventions.Methods:An approach for deriving signatures that combine knowledge of gene function and analysis of in vivo co-expression patterns was used to define a common hypoxia signature from three head and neck and five breast cancer studies. Previously validated hypoxia-regulated genes (seeds) were used to generate hypoxia co-expression cancer networks.Results:A common hypoxia signature, or metagene, was derived by selecting genes that were consistently co-expressed with the hypoxia seeds in multiple cancers. This was highly enriched for hypoxia-regulated pathways, and prognostic in multivariate analyses. Genes with the highest connectivity were also the most prognostic, and a reduced metagene consisting of a small number of top-ranked genes, including VEGFA, SLC2A1 and PGAM1, outperformed both a larger signature and reported signatures in independent data sets of head and neck, breast and lung cancers.Conclusion:Combined knowledge of multiple genes' function from in vitro experiments together with meta-analysis of multiple cancers can deliver compact and robust signatures suitable for clinical application.

Aromatase Inhibitors Reduce the Expression of a Hypoxia Metagene in Oestrogen Receptor Positive Breast Cancer in Postmenopausal Women.

Abstract Aims: To (1) define the effect of oestrogen deprivation on genes related to hypoxia in oestrogen receptor positive (ER+) breast cancer and (2) identify any link between hypoxia and proliferation.Background: The majority of breast cancer patients are postmenopausal women with ER+ tumours and at some point receive an aromatase inhibitor (AI) as part of their treatment. Hypoxia and proliferation are important factors in the progression of ER+ tumours. Proliferation is profoundly reduced in most ER+ cancers after treatment with AIs[1], however little is known on the effects of AIs on hypoxia.Materials and methods: 81 pre- and 2-week post-treatment core-cut tumor biopsies were obtained from postmenopausal women with ER+ breast cancer who received single agent neoadjuvant anastrozole (AI)[2], and from 20 of these patients after 16 weeks of AI treatment. RNA was extracted and analysed on Illumina 48K microarrays. A hypoxia metagene (MG) was developed by identification of genes whose expression clustered with the expression of classical hypoxia-regulated genes[3]. Genes associated with proliferation were removed from the MG. A proliferation MG was derived by selecting the intersection of proliferation clusters from three public breast cancer datasets.Results: Spearman correlations revealed a strong relationship between the hypoxia and proliferation MGs prior to AI treatment (r =0.61, p&amp;lt;10-3), and persisted after 2 weeks (r =0.77, p&amp;lt;10-3) and after 16 weeks of treatment (r=0.72, p =0.002). Baseline expression of the hypoxia MG was (1) positively correlated with 2-week Ki67 (Spearman r =0.37, p =0.002), (2) showed a trend for a positive correlation with poor 2-week Ki67 change (Spearman r=0.22, p =0.06) and with poor reduction in the mean expression of four classical oestrogen dependant genes (TFF1/pS2, GREB1, PDZK1 and PGR) known as AVERG[4] (Spearman r =0.22, p=0.06). Expression of the hypoxia MG was significantly down-regulated after 2 weeks of oestrogen deprivation using AI treatment (p&amp;lt;10-3). The 2-week change in hypoxia showed a positive correlation with the 2-week change in proliferation (Spearman r=0.58, p&amp;lt;10-3), and with the 2-week change in Ki67 (Spearman r=0.35, p=0.005).Conclusions: The expression of a hypoxia MG decreases after oestrogen deprivation. The hypoxia MG is strongly associated with proliferation prior to and after AI treatment. The data are consistent with hypoxia being a secondary effect of proliferation in ER+ breast cancer and could contribute in understanding the need to combine anti-proliferative drugs with anti-angiogenic agents for these patients. There may be a weak effect of hypoxia on de-novo resistance to AIs.1. Dowsett M, et al., Clin Cancer Res 2006; 12(3): p.1024-302. Smith, I.E, et al., J Clin Oncol 2007; 25(25): p.3816-223. Winter, S.C, et al., Cancer Res 2007; 67(7): p.3441-94. Dunbier, A.K, et al., Cancer Res 2009; 69(Suppl.), 78sSupported by The Mary-Jean Mitchell Green Foundation and Breakthrough Breast Cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 408.

Relation of a Hypoxia Metagene Derived from Head and Neck Cancer to Prognosis of Multiple Cancers

Affymetrix U133plus2 GeneChips were used to profile 59 head and neck squamous cell cancers. A hypoxia metagene was obtained by analysis of genes whose in vivo expression clustered with the expression of 10 well-known hypoxia-regulated genes (e.g., CA9, GLUT1, and VEGF). To minimize random aggregation, strongly correlated up-regulated genes appearing in >50% of clusters defined a signature comprising 99 genes, of which 27% were previously known to be hypoxia associated. The median RNA expression of the 99 genes in the signature was an independent prognostic factor for recurrence-free survival in a publicly available head and neck cancer data set, outdoing the original intrinsic classifier. In a published breast cancer series, the hypoxia signature was a significant prognostic factor for overall survival independent of clinicopathologic risk factors and a trained profile. The work highlights the validity and potential of using data from analysis of in vitro stress pathways for deriving a biological metagene/gene signature in vivo.

Vascular endothelial growth factor blockade rapidly elicits alternative proangiogenic pathways in neuroblastoma

Most children with neuroblastoma presenting after infancy have metastatic, chemoresistant disease. Amplification of the MYCN proto-oncogene is a significant marker of these poor-prognosis neuroblastoma tumors. Recent studies suggest that MYCN may function in part by promoting angiogenesis via vascular endothelial growth factor (VEGF). VEGF blockade has been validated as a therapeutic strategy in adult cancers. In these studies, we asked whether inhibition of VEGF signaling via VEGFR2 blockade in established MYCN-amplified neuroblastoma xenografts would: 1) restrict tumor growth; 2) induce hypoxia; and 3) alter tumor vasculature. The MYCN-amplified neuroblastoma human cell line NGP was implanted intrarenally in athymic female mice. After 5 weeks, mice with established tumors were selected, a cohort euthanized to provide day 0 controls, and the rest assigned to receive biweekly injections of DC101 (anti-murine VEGFR2 antibody) or vehicle. DC101 treatment did not inhibit progressive tumor growth in established NGP xenografts. Although tumor vasculature was not significantly disrupted, a modest increase in tumor hypoxia was demonstrated by pimonidazole staining, and expression of a previously described hypoxia metagene was increased by gene set enrichment analysis (GSEA) in DC101-treated tumors. DC101 treatment elicited increased: 1) expression of VEGFR1 and its ligand placental growth factor; and 2) increased Notch activation in tumor vasculature concurrent with expression of the Notch ligand Jagged1. This result suggests that established MYCN-amplified neuroblastoma tumors are relatively VEGF-independent, and display the ability to rapidly up-regulate hypoxia-responsive alternative proangiogenic mechanisms that may stabilize vasculature when VEGF is deficient.