- Open Access
Chemoresistance acquisition induces a global shift of expression of aniogenesis-associated genes and increased pro-angogenic activity in neuroblastoma cells
© Michaelis et al; licensee BioMed Central Ltd. 2009
Received: 3 July 2009
Accepted: 29 September 2009
Published: 29 September 2009
Chemoresistance acquisition may influence cancer cell biology. Here, bioinformatics analysis of gene expression data was used to identify chemoresistance-associated changes in neuroblastoma biology.
Bioinformatics analysis of gene expression data revealed that expression of angiogenesis-associated genes significantly differs between chemosensitive and chemoresistant neuroblastoma cells. A subsequent systematic analysis of a panel of 14 chemosensitive and chemoresistant neuroblastoma cell lines in vitro and in animal experiments indicated a consistent shift to a more pro-angiogenic phenotype in chemoresistant neuroblastoma cells. The molecular mechanims underlying increased pro-angiogenic activity of neuroblastoma cells are individual and differ between the investigated chemoresistant cell lines. Treatment of animals carrying doxorubicin-resistant neuroblastoma xenografts with doxorubicin, a cytotoxic drug known to exert anti-angiogenic activity, resulted in decreased tumour vessel formation and growth indicating chemoresistance-associated enhanced pro-angiogenic activity to be relevant for tumour progression and to represent a potential therapeutic target.
A bioinformatics approach allowed to identify a relevant chemoresistance-associated shift in neuroblastoma cell biology. The chemoresistance-associated enhanced pro-angiogenic activity observed in neuroblastoma cells is relevant for tumour progression and represents a potential therapeutic target.
Neuroblastoma is the most frequent extracranial solid tumour of childhood. About half of all neuroblastoma patients are diagnosed with high-risk disease with overall survival rates below 40% despite intensive multimodal treatment . Therapy failure is basically caused by acquired chemoresistance. Primary tumours usually respond to initial chemotherapy. However, a significant fraction of tumours reappear as chemoresistant recidives .
Acquisition of chemoresistance under therapy may affect the biology of neuroblastoma and other tumour cells [3–9]. Mostly a shift towards a more malignant phenotype is observed indicating cancer progression [3, 4, 6–9]. Molecular changes in different signalling pathways including apoptosis signalling or cell cycle regulation may be involved in this coincidence of cancer cell chemoresistance and increased malignancy [3, 10, 11]. Neuroblastoma cells adapted to different cytotoxic drugs showed increased malignant properties as indicated by enhanced invasive potential in vitro [7, 8] and increased malignancy in nude mice .
Here, differences in angiogenesis signalling were identified by bioinformatics pathway analysis of gene expression data from chemosensitive and chemoresistant neuroblastoma cells. Subsequently, cell culture and animal experiments using 14 human neuroblastoma cell lines indicated a consistently higher pro-angiogenic activity of chemoresistant neuroblastoma cells than of chemosensitive cells. The molecular mechanisms underlying the chemoresistance-associated increased pro-angiogenic potential were individual and differed between individual cell lines. Doxorubicin treatment of doxorubicin-resistant neuroblastoma xenografts resulted in impairment of tumour angiogenesis and growth suggesting the chemoresistance-associated pro-angiogenic phenotype to contribute to tumour progression.
Gene expression analysis
Gene expression analysis using AB1700 Human Genome Survey Microarray V2.0 chips (Applied Biosystems, Darmstadt, Germany) was performed by IMGM laboratories (Martinsried, Germany). Gene expression analysis using GeneChip HGU133 Plus 2.0 (Affymetrix, Santa Clara, CA, USA) was performed by Fraunhofer Institut für Zelltherapie und Immunologie (Leipzig, Germany). mRNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Triplicates of UKF-NB-3 RNA were compared to triplicates of UKF-NB-3rVCR10 RNA, UKF-NB-3rDOX20 RNA, or UKF-NB-3rCDDP1000 RNA.
AB1700 expression data were processed using the R/bioconductor package 'ABarray' (http://www.r-project.org/; http://www.bioconductor.org/) with default parameter (SN threshold > = 3, % Detect Samples: 0.5). This included quality control, quantile normalisation  and filtering of unspecific hybridisation. HGU133 Plus 2.0 expression data were processed using the R/bioconductor packages 'gcrma' and 'limma' (http://www.r-project.org/; http://www.bioconductor.org/).
For every microarray experiment, the expression pattern of 50 randomly chosen genes was verified by quantitative real-time PCR resulting in confirmation of expression of >80% of investigated genes (data not shown).
Signal transduction pathway bioinformatics
Statistical analysis to identify significant expression changes was focusing on a pathway analysis using the PANTHER database  http://www.pantherdb.org, which identifies global patterns in expression. For each expert-curated pathway in the database, potential differential expression was determined by a binomial test , using the PANTHER human gene reference list matching our microarrays (Human AB1700 genes) and lists of differentially expressed genes that passed a false discovery rate threshold  of 0.05 based on a t-test.
A total of 25,909 genes were annotated in the dataset, 3,125 of them included pathway information, and 223 of these (corresponding to 280 AB1700 ProbeIDs or 537 HGU133 Plus 2.0 ProbeIDs) were annotated as related to angiogenesis. For this list of angiogenesis-associated ProbeIDs and angiogenesis-associated genes signal intensities of UKF-NB-3, UKF-NB-3rVCR10, UKF-NB-3rCDDP1000, or UKF-NB-3rDOX20 cells were visualised as heatmaps using R http://www.r-project.org.
Influence of supernatants from different neuroblastoma cell lines on endothelial cell growth and endothelial cell survival
Endothelial cell growth (%)3
Endothelial cell survival (%)4
100 ± 8
100 ± 9
5 ± 7
12 ± 6
15 ± 6
22 ± 8
78 ± 12*
83 ± 12*
89 ± 9*
85 ± 7*
66 ± 13*
70 ± 6*
17 ± 11
20 ± 5
48 ± 13*
55 ± 8*
38 ± 7*
48 ± 10*
35 ± 9*
42 ± 9*
8 ± 6
17 ± 5
63 ± 15*
67 ± 8*
49 ± 7*
58 ± 6*
37 ± 8*
44 ± 14*
92 ± 9#
78 ± 15#
94 ± 16#
90 ± 7#
All cell lines were grown in Iscove's modified Dulbecco's medum (IMDM) supplemented with 10% foetal calf serum (FCS), 100 IU/ml penicillin, and 100 mg/ml streptomycin at 37°C.
Human umbilical vein endothelial cells (HUVECs) were cultivated as described before  using IMDM supplemented with 15% FCS and 5% pooled human serum.
HUVEC viability was investigated using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Mannheim, Germany) following the manufacturer's instructions.
Caspase 3/7 activation was measured using the Caspase-Glo 3/7 Assay (Promega, Mannheim, Germany) following the manufacturer's instructions.
Tube formation assay
Endothelial cellular tube formation was investigated using HUVECs seeded on extracellular matrix (Matrigel, BD Biosciences, Heidelberg, Germany) as described before .
Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Proteins were detected using specific antibodies against β-actin (Sigma, Taufkirchen, Germany), ERK 1/2, the phosphorylated forms of ERK 1/2 (each from New England Biolabs, Frankfurt am Main, Germany), Akt, or the phosphorylated forms of Akt (all Millipore (Upstate), Schwalbach, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).
Electrophoretic mobility shift assay (EMSA)
Electrophoretic mobility shift assay (EMSA) was performed as described .
Experiments using the chick chorioallantoic membrane (CAM) were performed using described methods . 106 cells were placed onto the CAM at day 8. Vessel formation was examined at day 12.
Mouse experiments were performed using female NMRI:nu/nu mice as described before . 107 cells were injected subcutaneously together with Matrigel in a total volume of 100 μl. For doxorubicin treatment, the day when xenograft tumours became palpable was defined to be day 1. Tumour sections were stained for apoptotic cells by TUNEL staining and for cell proliferation by ki67 staining using established methods [24, 25].
All animal experiments were performed in accordance with all relevant declarations on the use of laboratory animals and with the German Animal Protection Law.
Expression of angiogenesis-associated genes
A pathway analysis was performed in order to detect the most strongly influenced signalling pathways between UKF-NB-3 and its chemoresistant sub-lines UKF-NB-3rVCR10 and UKF-NB-3rCDDP1000. Of the 153 pathways mapped at PANTHER, angiogenesis was found to be the fourth most significantly affected signalling pathway (p = 1.87 × 10-4) [see Additional file 1].
Subsequently to these analyses, we compared angiogenesis signalling between UKF-NB-3 and UKF-NB-3rDOX20 cells. Since Applied Biosystems had stopped manufacturing of AB1700 arrays, HGU133 Plus 2.0 arrays (Affymetrix) were used. Results were similar to those obtained from the comparison of UKF-NB-3 with UKF-NB-3rVCR10 and UKF-NB-3rCDDP1000 cells. PANTHER pathway analysis indicated angiogenesis to be the fourth most significantly differentially regulated signalling pathway [see Additional file 4]. Hierarchical cluster analysis of angiogenesis-associated genes separated UKF-NB-3 from UKF-NB-3rDOX20 cells [see Additional file 5]. 65 angiogenesis-associated genes were found significantly differentially regulated between UKF-NB-3 and UKF-NB-3rDOX20 cells. 38 genes were up-regulated in UKF-NB-3rDOX20 cells and 27 genes were down-regulated in UKF-NB-3rDOX20 relative to UKF-NB-3 cells [see Additional file 6]. The relatively high number of significantly differentially regulated genes compared to the comparisons of UKF-NB-3 vers. UKF-NB-3rVCR10 or UKF-NB-3rCDDP1000 cells most likely results from the different statistical procedures used to analyse HGU133 Plus 2.0 or AB1700 data.
To further investigate the influence of chemoresistance acquisition on the pro-angiogenic potential of cancer cells, a panel of chemsensitive and chemoresistant neuroblastoma cell lines was systematically investigated for their angiogenic phenotypes.
Influence of neuroblastoma cell line supernatants on endothelial cell growth and survival
Next, the influence of neuroblastoma cell culture supernatants was examined on HUVEC survival. Confluent HUVEC monolayers were washed and incubated for 48 h with supernatants of UKF-NB-3, UKF-NB-3rVCR10, UKF-NB-3rCDDP1000, or UKF-NB-3rDOX20 cells and HUVEC viability was determined. Results revealed increased HUVEC viability in cultures incubated with supernatants of chemoresistant cells (Table 1). Lack of growth factors or nutrients induces apoptosis in endothelial cells [26–28]. Therefore, we investigated caspase 3/7 activation as indicator of apoptosis in confluent HUVEC monolayers incubated for 48 h with supernatants of UKF-NB-3, UKF-NB-3rVCR10, UKF-NB-3rCDDP1000 cells or UKF-NB-3rDOX20 cells. Results indicated decreased caspase activation in HUVECs incubated with supernatants from chemoresistant cells (Figure 2B).
Influence of neuroblastoma cell line supernatants on endothelial cell tube formation
Influence of neuroblastoma cell line supernatants on activation of pro-angiogenic signalling events in endothelial cells
The phosphoinositide-3-kinase (PI3K) - Akt (also known as protein kinase B, PKB) signalling pathway, "classical" mitogen-activated protein kinase (MAPK) signalling via Ras-Raf-MEK-ERK, and activation of nuclear factor κB (NFκB) are involved in angiogenesis signalling in endothelial cells [29–31]. The influence of supernatants of chemoresistant cells on Akt phosphorylation or ERK 1/2 phosphorylation in HUVECs is shown in Figure 3C. Densitometric analysis of Western blot data is given in Additional file 8. Akt may be activated through phosphorylation at Ser473 and/or at Thr308. The supernatants of UKF-NB-3rVCR10 or UKF-NB-3rCDDP1000 cells induced enhanced Akt phosphorylation at Thr308 and ERK 1/2 phosphorylation in comparison to UKF-NB-3 supernatants. All supernatants of chemoresistant cells caused enhanced NFκB activation relative to supernatants of chemosensitive UKF-NB-3 cells (Figure 3D).
Chemoresistant cancer cells induce increased vessel formation in animal models
Vessel formation was further investigated in xenografts formed of UKF-NB-3, UKF-NB-3rVCR10, or UKF-NB-3rDOX20 cells in female NMRI:nu/nu mice. Tumour take in mice injected with UKF-NB-3rVCR10 cells was 100%, in mice injected with UKF-NB-3rDOX20 cells it was 90% while only 10% of UKF-NB-3 cell-injected mice formed tumours. UKF-NB-3rVCR10 cells and UKF-NB-3rDOX20 cells also formed considerably bigger and stronger vascularised xenograft tumours than UKF-NB-3 cells (Figure 4B-D).
Increased pro-angiogenic activity of chemoresistant neuroblastoma cells is mediated by individual molecular mechanisms
VEGF is a pro-angiogenic factor that has frequently been associated with neuroblastoma angiogenesis [32, 33]. However, increased VEGF levels were not consistently found in supernatants of chemoresistant cells [see Additional file 9]. Acute cisplatin treament has been described to induce tumour progression through VEGF expression in paediatric tumour cells including the neuroblastoma cell line SK-N-BE2 . In cisplatin-resistant neuroblastoma cells, VEGF expression has not been investigated, yet. Increased VEGF levels were detected in UKF-NB-3rCDDP1000 cells versus UKF-NB-3 cells and in IMR-32rCDDP1000 cells versus IMR-32 cells but not in UKF-NB-2rCDDP10 cells versus UKF-NB-2 cells [see Additional file 9]. Moreover, the pro-angiogenic factors interleukin-8 (IL-8), angiogenin, basic fibroblast growth factor, or tumour necrosis factor α (TNF-α) were not generally found to be increased in supernatants of chemoresistant cells [see Additional file 9]. Two angiogenesis-associated genes (ARHGAP8, FGFR2) were found commonly up-regulated in UKF-NB-3rCDDP1000, UKF-NB-3rVCR10, or UKF-NB-3rDOX20 cells versus UKF-NB-3 cells [see Additional files 2, 3, 6]. However, these genes were not consistently found up-regulated in chemoresistant neuroblastoma cells (data not shown).
Expression of a number of further pro- and anti-angiogenic factors has been suggested to be relevant for neuroblastoma angiogenesis including platelet-derived growth factor α (PDGFα), matrix metalloproteinase 2 (MMP-2), MMP-9, erythropoietin (EPO), EPO receptor, activin A, interleukin-6 (IL-6), leukemia inhibitory factor (LIF), tissue inhibitor of metalloproteinase 2 (TIMP2), pigment epithelial-derived growth factor (PEDGF), secreted protein acidic and rich in cysteine (SPARC), thrombospondin-1, and thrombospondin-2 . However, analysis of gene microarray data from neuroblastoma cell lines did not reveal specific expression of these or other angiogenesis-related genes that would suggest a single common molecular event underlying increased neuroblastoma tumour angiogenesis in all chemoresistant cells (data not shown).
N-myc amplification has also been reported to result in increased neuroblastoma tumour angiogenesis through different mechanisms [33, 35–37]. However, UKF-NB-3rDOX20 cells showed enhanced pro-angiogenic potential compared to UKF-NB-3 cells although both cell lines do neither differ in N-myc amplification nor in N-myc expression [8, 18]. This indicates that the N-myc status may not generally be critical for increased pro-angiogenic potential of chemoresistant cells. Furthermore, the loss of functional p53 during tumourigenesis has been correlated to a more pro-angiogenic tumour phenotype . However, in our experiments pro-angiogenic activity was enhanced in both p53-mutated and p53-wild-type chemoresistant neuroblastoma cells (Table 1). Taken together, the more pro-angiogenic phenotype observed in chemoresistant neuroblastoma cells appears to result from different individual shifts in the expression of angiogenesis-associated genes.
Doxorubicin inhibits tumour angiogenesis and growth of doxorubicin-resistant neuroblastoma xenografts
Data had indicated individual changes in the expression of angiogenesis-related genes to be responsible for the proangiogenic phenotype of chemoresistant neuroblastoma cells (see above). To investigate if the increased pro-angiogenic activity of chemoresistant neuroblastoma cells may be relevant for enhanced growth of chemoresistant neuroblastoma xenografts, doxorubicin-resistant UKF-NB-3rDOX20 neuroblastoma cells were treated with doxorubicin that is known to interfere with angiogenesis by direct influence on endothelial cells [39, 40].
Here, we used a bioinformatics-based approach based on transcriptomics data to identify signalling pathways associated with increased malignant behaviour of chemoresistant neuroblastoma cells. Angiogenesis signalling belonged to the top 5 pathways most strongly differentially regulated between chemosensitive and chemoresistant neuroblastoma cells. Systematic evaluation of a panel of neuroblastoma cell lines in cell culture and animal models showed consitently increased pro-angiogenic acivity exerted by chemoresistant cells. These findings are in accordance with previous reports showing that human melanoma and breast cancer cells selected for resistance to chemotherapeutic agents produced higher levels of multiple angiogenic factors [44, 45]. Moreover, an increased microvessel density (MVD) was detected in chemotherapy resistant xenograft tumours [44, 45].
Selection of cancer stem cells has been suggested to play a role in the enhanced pro-angiogenic activity seen in chemoresistant cancer cells. In lung cancer cells, treatment with cisplatin, doxorubicin, or etoposide resulted in the selection of cancer stem cells as indicated by cell biology and analysis of expression of stemness genes . These chemotherapy-selected cancer stem cells were responsible for the observed increased pro-angiogenic properties of lung cancer cells. In the absence of cytotoxic drugs, lung cancer cell lines returned to their initial phenotype and re-acquired drug sensitivity . In contrast, UKF-NB-3rVCR10 and UKF-NB-3rCDDP1000 cells remained chemoresistant and did not loose their pro-angiogenic phenotype even when they were cultivated for up to six months in the absence of drugs (data not shown). This suggests that chemoresistance and pro-angiogenic activity in these cell lines are not consequence of a simple chemotherapy-induced selection of cancer stem cells that are already present in the parental UKF-NB-3 cell line. Moreover, acute cisplatin treatment increased VEGF expression together with expression of the stemness genes Nanog, Bmi-1, and Oct 4 in osteosarcoma (HOS), rhabdomyosarcoma (RH-4) and neuroblastoma (SK-N-BE2) cell lines . However, none of these stemness genes was found up-regulated in UKF-NB-3rVCR10 or UKF-NB-3rCDDP1000 cells relative to UKF-NB-3 cells [see Additional file 10].
The finding that cell culture supernatants from chemoresistant cells exerted stronger pro-angiogenic effects than those from chemosensitive cells suggests that soluble factors contribute to the enhanced pro-angiogenic activity exerted by chemoresistant neuroblastoma cells. Statistical analysis of the expression of angiogenesis-related genes indicated clear differences between chemosensitive UKF-NB-3 cells and the chemoresistant sub-lines UKF-NB-3rVCR10, UKF-NB-3rCDDP1000, or UKF-NB-3rDOX20 (Figure 1) [see Additional files 1, 4, 5]. Obviously, chemoresistance development resulted in a global change of expression of angiogenesis-associated genes towards a more pro-angiogenic phenotype. The resistance-related changes in expression patterns appear to differ between individual chemoresistant neuroblastoma cell lines. This suggests that the enhanced pro-angiogenic phenotype observed in all chemoresistant neuroblastoma cell lines in comparison to the chemosensitive cell lines is caused by different (individual) changes in the expression patterns of angiogenesis-associated genes. Notably, hierarchical clustering of expression of angiogenesis-associated genes also clearly discriminated UKF-NB-2 cells from UKF-NB-2rVCR10 and UKF-NB-2rCDDP1000 cells [see Additional file 11], as well as IMR-32 cells from IMR-32rVCR10 cells [see Additional file 12].
The view that individual chemoresistant neuroblastoma cell lines exert pro-angiogenic effects by individual mechanisms is supported by the results derived from the examination of pro-angiogenic signalling in endothelial cells incubated with supernatants from different neuroblastoma cell lines. Supernatants of chemoresistant UKF-NB-3rDOX20, UKF-NB-3rVCR10, and UKF-NB-3rCDDP1000 cells enhanced NFκB activation compared to supernatants of chemosensitive UKF-NB-3 cells However, only supernatants of UKF-NB-3rVCR10 and UKF-NB-3rCDDP1000 cells but not UKF-NB-3rDOX20 cells elevated Akt and ERK 1/2 phosphorylation in endothelial cells. Based on these differences in the activation of pro-angiogenic signalling events in endothelial cells, it appears plausible that endothelial cell activation might be caused by different chemoresistant neuroblastoma cell lines by different molecular mechanisms resulting in up- or down-regulation of varying pro- or anti-angiogenic factors.
Possibly, there is an overlap between gene products involved in angiogenesis and gene products relevant in chemoresistance. Indeed, among between the aniogenesis-associated genes that were differentially expressed there are those that are also considered to contribute to chemoresistance. Three arbitrarily chosen examples are BIRC5, MAPK3, and AKT1. BIRC5 (increased expression in UKF-NB-3rVCR10 vs. UKF-NB-3, [see Additional file 2], and in UKF-NB-3rDOX20 vers. UKF-NB-3, [see Additional file 6]) encodes for a protein that is also named survivin and plays a prominent role in apoptosis inhibition and cancer cell chemoresistance . Moreover, BIRC5 expression in cancer cells has been linked to tumour angiogenesis  and inhibition of BIRC5 expression in tumour cells decreased tumour angiogenesis [49, 50]. MAPK3 (increased expression in UKF-NB-3rVCR10 vs. UKF-NB-3, [see Additional file 2]) encodes for a protein that is also called extracellular signal-regulated kinase 1 (ERK1) and is a constituent of the "classical" MAP kinase pathway Ras/Raf/MEK/ERK. ERK1 phosphorylation protects cancer cells from different entities against chemotherapy-induced apoptosis [51–53]. Moreover, MAPK3 activation/phosphorylation induces production of pro-angiogenic factors in renal carcinoma cells . AKT1 (increased expression in UKF-NB-3rDOX20 vers. UKF-NB-3, [see additional file 6]) encodes for a protein also called protein kinase B (PKB) that is a central mediator of survival signals transduced by the phosphatidylinositol 3-kinase and is involved in chemoresistance [55–57] as well as in cancer cell expression of pro-angiogenic factors [58–60]. Remarkably, an angiogenesis-associated gene expression signature had been described before to predict the sensitivity of cancer cells to artemisinins, an anti-cancer active group of anti-malaria drugs .
The complexicity of pro-angiogenic mechanisms observed in chemoresistant neuroblastoma cells is in accordance with other reports demonstrating that pro-angiogenic activity of cancer cells is commonly caused by complex changes in angiogenesis signalling and that inhibition of one pro-angiogenic event may not be enough to interfere with tumour vessel formation . N-myc-amplified neuroblastoma cells that exert pro-angiogenic activity mainly through VEGF have very recently been shown to rapidly develop alternative pro-angiogenic mechanisms when VEGF signalling is inhibited . In addition, up-regulation of multiple pro-angiogenic factors enabled carcinoma cells to escape from angiogenesis inhibition by the three endogenous anti-angiogenic molecules thrombospondin-1, endostatin, and tumstatin . Notably, combination therapy of metastatic breast cancer with paclitaxel and the anti-VEGF-A antibody bevacizumab resulted in prolonged progression-free survival but did not influence overall survival relative to paclitaxel in a phase III trial . In the light of the findings presented here, one may speculate that anti-angiogenic therapy may prolong progression-free survival but that resistance development (to chemotherapy and/or anti-angiogenic therapy) may result in a more aggressive cancer cell phenotype, which might be the reason for the decreased time period observed between tumour re-onset and patients' deaths.
High tumour angiogenesis and high-level expression of pro-angiogenic factors at diagnosis have previously been suggested to be correlated with advanced disease stages in neuroblastoma [32, 66, 67]. However, the prognostic value of angiogenesis in neuroblastoma at diagnosis is still a matter of debate [68, 69]. Notably, analysis of two different data sets reporting on gene expression profiles in tumours from poor outcome or bad outcome N-myc amplified  or non-N-myc amplified  neuroblastoma patients indicated statistically significant differences in angiogenesis signalling between these groups [see Additional files 13, 14]. To investigate if the increased pro-angiogenic phenotype observed in chemoresistant cells may contribute to tumour progression, xenografts grown from doxorubicin-resistant (UKF-NB-3rDOX20) cells were treated with doxorubicin, an anti-cancer drug that exerts anti-angiogenic activity by direct effect on endothelial cells [39, 40]. Tumour vessel formation and growth were strongly reduced by doxorubicin in doxorubicin-resistant xenografts. Although it cannot be concluded without a doubt that the entire effect on xenograft growth can be attributed to inhibition of angiogenesis, microvessel density was statistically reduced supporting the view that inhibition of angiogenesis has definitely contributed. Therefore, these data suggest that increased pro-angiogenic activity of doxorubicin-resistant cells contributes to their more malignant phenotype and that anti-angiogenic strategies that target endothelial cells might represent a therapeutic option for neuroblastoma treatment.
Bioinformatics pathway analysis indicated differences in the expression of angiogenesis-associated genes between chemosensitive and chemoresistant neuroblastoma cell lines. Cell culture and animal data showed that acquired resistance to different anti-cancer drugs resulted in increased pro-angiogenic activity of neurobastoma cells. The changes in angiogenesis signalling observed in chemoresistant neuroblastoma cells are very complex and differ between individual cell lines. Therefore, individual molecular mechanisms appear to be responsible for the enhanced pro-angiogenic activity that was consistently observed in all investigated chemoresistant neuroblastoma cell lines relative to chemosensitive cells. Doxorubicin treatment of doxorubicin-resistant neuroblastoma xenografts resulted in decreased vessel formation and tumour growth suggesting that the more pro-angiogenic phenotype of chemoresistant cells may contribute to increased malignancy of chemoresistant neuroblastoma cells and that endothelial cell targeting may represent a possibility for therapeutic intervention. The complex nature of the chemoresistance-associated changes responsible for the more pro-angiogenic phenotype strongly stresses the need for an improved understanding of biological processes like angiogenesis on a systems biology level.
The authors thank Mrs. Eva Bechtold and Mrs. Janette Spitznagel technical assistance.
The work was in part supported by the friendly society "Hilfe für krebskranke Kinder Frankfurt e.V.", its foundation "Frankfurter Stiftung für krebskranke Kinder" and the European Commission (project acronym: SYNLET, contract no. 043312).
- Maris JM, Hogarty MD, Bagatell R, Cohn SL: Neuroblastoma. Lancet. 2007, 369: 2106-2120. 10.1016/S0140-6736(07)60983-0View ArticlePubMedGoogle Scholar
- Maris JM, Matthay KK: Molecular biology of neuroblastoma. J Clin Oncol. 1999, 17: 2264-2279.PubMedGoogle Scholar
- Goldsmith KC, Hogarty MD: Targeting programmed cell death pathways with experimental therapeutics: opportunities in high-risk neuroblastoma. Cancer Lett. 2005, 228: 133-141. 10.1016/j.canlet.2005.01.048View ArticlePubMedGoogle Scholar
- Shah AN, Gallick GE: Src, chemoresistance and epithelial to mesenchymal transition: are they related?. Anticancer Drugs. 2007, 18: 371-375. 10.1097/CAD.0b013e32801265d7View ArticlePubMedGoogle Scholar
- Ogbomo H, Michaelis M, Klassert D, Doerr HW, Cinatl J: Resistance to cytarabine induces the up-regulation of NKG2D ligands and enhances natural killer cell lysis of leukemic cells. Neoplasia. 2008, 10: 1402-1410.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu F, Chen Z, Wang J, Shao X, Cui Z, Yang C, Zhu Z, Xiong D: Overexpression of cell surface cytokeratin 8 in multidrug-resistant MCF-7/MX cells enhances cell adhesion to the extracellular matrix. Neoplasia. 2008, 10: 1275-1284.PubMed CentralView ArticlePubMedGoogle Scholar
- Blaheta RA, Daher FH, Michaelis M, Hasenberg C, Weich EM, Jonas D, Kotchetkov R, Doerr HW, Cinatl J: Chemoresistance induces enhanced adhesion and transendothelial penetration of neuroblastoma cells by down-regulating NCAM surface expression. BMC Cancer. 2006, 6: 294- 10.1186/1471-2407-6-294PubMed CentralView ArticlePubMedGoogle Scholar
- Blaheta RA, Michaelis M, Natsheh I, Hasenberg C, Weich E, Relja B, Jonas D, Doerr HW, Cinatl J: Valproic acid inhibits adhesion of vincristine- and cisplatin-resistant neuroblastoma tumour cells to endothelium. Br J Cancer. 2007, 96: 1699-1706. 10.1038/sj.bjc.6603777PubMed CentralView ArticlePubMedGoogle Scholar
- Michaelis M, Fichtner I, Behrens D, Haider W, Rothweiler F, Mack A, Cinatl J, Doerr HW, Cinatl J: Anti-cancer effects of bortezomib against chemoresistant neuroblastoma cell lines in vitro and in vivo. Int J Oncol. 2006, 28: 439-446.PubMedGoogle Scholar
- Müller M, Schleithoff ES, Stremmel W, Melino G, Krammer PH, Schilling T: One, two, three--p53, p63, p73 and chemosensitivity. Drug Resist Updat. 2006, 9: 288-306. 10.1016/j.drup.2007.01.001View ArticlePubMedGoogle Scholar
- Fernandez-Luna JL: Regulation of pro-apoptotic BH3-only proteins and its contribution to cancer progression and chemoresistance. Cell Signal. 2008, 20: 1921-1926. 10.1016/j.cellsig.2008.04.015View ArticlePubMedGoogle Scholar
- Bolstad BM, Irizarry RA, Astrand M, Speed TP: A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003, 19: 185-193. 10.1093/bioinformatics/19.2.185View ArticlePubMedGoogle Scholar
- Mi H, Guo N, Kejariwal A, Thomas PD: PANTHER version 6: protein sequence and function evolution data with expanded representation of biological pathways. Nucleic Acids Res. 2007, D247-52. 35 Database.Google Scholar
- Cho RJ, Campbell MJ: Transcription, genomes, function. Trends Genet. 2000, 16: 409-415. 10.1016/S0168-9525(00)02065-5View ArticlePubMedGoogle Scholar
- Benjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Statist Soc B. 1995, 57: 289-300.Google Scholar
- Cinatl J, Vogel JU, Cinatl J, Weber B, Rabenau H, Novak M, Kornhuber B, Doerr HW: Long-term productive human cytomegalovirus infection of a human neuroblastoma cell line. Int J Cancer. 1996, 65: 90-96. 10.1002/(SICI)1097-0215(19960103)65:1<90::AID-IJC16>3.0.CO;2-MView ArticlePubMedGoogle Scholar
- Kotchetkov R, Cinatl J, Blaheta R, Vogel JU, Karaskova J, Squire J, Hernáiz Driever P, Klingebiel T, Cinatl J: Development of resistance to vincristine and doxorubicin in neuroblastoma alters malignant properties and induces additional karyotype changes: a preclinical model. Int J Cancer. 2003, 104: 36-43. 10.1002/ijc.10917View ArticlePubMedGoogle Scholar
- Kotchetkov R, Driever PH, Cinatl J, Michaelis M, Karaskova J, Blaheta R, Squire JA, Von Deimling A, Moog J, Cinatl J: Increased malignant behavior in neuroblastoma cells with acquired multi-drug resistance does not depend on P-gp expression. Int J Oncol. 2005, 27: 1029-1037.PubMedGoogle Scholar
- Michaelis M, Cinatl J, Anand P, Rothweiler F, Kotchetkov R, von Deimling A, Doerr HW, Shogen K, Cinatl J: Onconase induces caspase-independent cell death in chemoresistant neuroblastoma cells. Cancer Lett. 2007, 250: 107-116. 10.1016/j.canlet.2006.09.018View ArticlePubMedGoogle Scholar
- Michaelis M, Bliss J, Arnold SC, Hinsch N, Rothweiler F, Deubzer HE, Witt O, Langer K, Doerr HW, Wels WS, Cinatl J: Cisplatin-resistant neuroblastoma cells express enhanced levels of epidermal growth factor receptor (EGFR) and are sensitive to treatment with EGFR-specific toxins. Clin Cancer Res. 2008, 14: 6531-6537. 10.1158/1078-0432.CCR-08-0821View ArticlePubMedGoogle Scholar
- Cinatl J, Michaelis M, Driever PH, Cinatl J, Hrabeta J, Suhan T, Doerr HW, Vogel JU: Multimutated herpes simplex virus g207 is a potent inhibitor of angiogenesis. Neoplasia. 2004, 6: 725-735. 10.1593/neo.04265PubMed CentralView ArticlePubMedGoogle Scholar
- Cinatl J, Margraf S, Vogel JU, Scholz M, Cinatl J, Doerr HW: Human cytomegalovirus circumvents NF-kappa B dependence in retinal pigment epithelial cells. J Immunol. 2001, 167: 1900-1908.View ArticlePubMedGoogle Scholar
- Papoutsi M, Sleeman JP, Wilting J: Interaction of rat tumor cells with blood vessels and lymphatics of the avian chorioallantoic membrane. Microsc Res Tech. 2001, 55: 100-107. 10.1002/jemt.1161View ArticlePubMedGoogle Scholar
- Schadendorf D, Kern MA, Artuc M, Pahl HL, Rosenbach T, Fichtner I, Nürnberg W, Stüting S, von Stebut E, Worm M, Makki A, Jurgovsky K, Kolde G, Henz BM: Treatment of melanoma cells with the synthetic retinoid CD437 induces apoptosis via activation of AP-1 in vitro, and causes growth inhibition in xenografts in vivo. J Cell Biol. 1996, 135: 1889-1898. 10.1083/jcb.135.6.1889View ArticlePubMedGoogle Scholar
- Fichtner I, Slisow W, Gill J, Becker M, Elbe B, Hillebrand T, Bibby M: Anticancer drug response and expression of molecular markers in early-passage xenotransplanted colon carcinomas. Eur J Cancer. 2004, 40: 298-307. 10.1016/j.ejca.2003.10.011View ArticlePubMedGoogle Scholar
- Gerber HP, Dixit V, Ferrara N: Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem. 1998, 273: 13313-13316. 10.1074/jbc.273.21.13313View ArticlePubMedGoogle Scholar
- Gupta K, Kshirsagar S, Li W, Gui L, Ramakrishnan S, Gupta P, Law PY, Hebbel RP: VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on MAPK/ERK and SAPK/JNK signaling. Exp Cell Res. 1999, 247: 495-504. 10.1006/excr.1998.4359View ArticlePubMedGoogle Scholar
- Michaelis M, Suhan T, Michaelis UR, Beek K, Rothweiler F, Tausch L, Werz O, Eikel D, Zörnig M, Nau H, Fleming I, Doerr HW, Cinatl J: Valproic acid induces extracellular signal-regulated kinase 1/2 activation and inhibits apoptosis in endothelial cells. Cell Death Differ. 2006, 13: 446-453. 10.1038/sj.cdd.4401759View ArticlePubMedGoogle Scholar
- Zachary I: VEGF signalling: integration and multi-tasking in endothelial cell biology. Biochem Soc Trans. 2003, 31: 1171-1177. 10.1042/BST0311171View ArticlePubMedGoogle Scholar
- Angelo LS, Kurzrock R: Vascular endothelial growth factor and its relationship to inflammatory mediators. Clin Cancer Res. 2007, 13: 2825-2830. 10.1158/1078-0432.CCR-06-2416View ArticlePubMedGoogle Scholar
- Jiang BH, Liu LZ: AKT signaling in regulating angiogenesis. Curr Cancer Drug Targets. 2008, 8: 19-26. 10.2174/156800908783497122View ArticlePubMedGoogle Scholar
- Eggert A, Ikegaki N, Kwiatkowski J, Zhao H, Brodeur GM, Himelstein BP: High-level expression of angiogenic factors is associated with advanced tumor stage in human neuroblastomas. Clin Cancer Res. 2000, 6: 1900-1908.PubMedGoogle Scholar
- Rössler J, Taylor M, Geoerger B, Farace F, Lagodny J, Peschka-Süss R, Niemeyer CM, Vassal G: Angiogenesis as a target in neuroblastoma. Eur J Cancer. 2008, 44: 1645-1656. 10.1016/j.ejca.2008.05.015View ArticlePubMedGoogle Scholar
- Tsuchida R, Das B, Yeger H, Koren G, Shibuya M, Thorner PS, Baruchel S, Malkin D: Cisplatin treatment increases survival and expansion of a highly tumorigenic side-population fraction by upregulating VEGF/Flt1 autocrine signaling. Oncogene. 2008, 27: 3923-3934. 10.1038/onc.2008.38View ArticlePubMedGoogle Scholar
- Hatzi E, Murphy C, Zoephel A, Rasmussen H, Morbidelli L, Ahorn H, Kunisada K, Tontsch U, Klenk M, Yamauchi-Takihara K, Ziche M, Rofstad EK, Schweigerer L, Fotsis T: N-myc oncogene overexpression down-regulates IL-6; evidence that IL-6 inhibits angiogenesis and suppresses neuroblastoma tumor growth. Oncogene. 2002, 21: 3552-3561. 10.1038/sj.onc.1205440View ArticlePubMedGoogle Scholar
- Schramm A, von Schuetz V, Christiansen H, Havers W, Papoutsi M, Wilting J, Schweigerer L: High activin A-expression in human neuroblastoma: suppression of malignant potential and correlation with favourable clinical outcome. Oncogene. 2005, 24: 680-687. 10.1038/sj.onc.1208087View ArticlePubMedGoogle Scholar
- Kang J, Rychahou PG, Ishola TA, Mourot JM, Evers BM, Chung DH: N-myc is a novel regulator of PI3K-mediated VEGF expression in neuroblastoma. Oncogene. 2008, 27: 3999-4007. 10.1038/onc.2008.15PubMed CentralView ArticlePubMedGoogle Scholar
- Teodoro JG, Evans SK, Green MR: Inhibition of tumor angiogenesis by p53: a new role for the guardian of the genome. J Mol Med. 2007, 85: 1175-1186. 10.1007/s00109-007-0221-2View ArticlePubMedGoogle Scholar
- Amoh Y, Li L, Yang M, Jiang P, Moossa AR, Katsuoka K, Hoffman RM: Hair follicle-derived blood vessels vascularize tumors in skin and are inhibited by doxorubicin. Cancer Res. 2005, 65: 2337-2343. 10.1158/0008-5472.CAN-04-3857View ArticlePubMedGoogle Scholar
- Amoh Y, Li L, Katsuoka K, Hoffman RM: Chemotherapy targets the hair-follicle vascular network but not the stem cells. J Invest Dermatol. 2007, 127: 11-15. 10.1038/sj.jid.5700486View ArticlePubMedGoogle Scholar
- Gustafson DL, Rastatter JC, Colombo T, Long ME: Doxorubicin pharmacokinetics: Macromolecule binding, metabolism, and excretion in the context of a physiologic model. J Pharm Sci. 2002, 91: 1488-1501. 10.1002/jps.10161View ArticlePubMedGoogle Scholar
- Gustafson DL, Merz AL, Long ME: Pharmacokinetics of combined doxorubicin and paclitaxel in mice. Cancer Lett. 2005, 220: 161-169. 10.1016/j.canlet.2004.09.007View ArticlePubMedGoogle Scholar
- Breistøl K, Hendriks HR, Fodstad O: Superior therapeutic efficacy of N-L-leucyl-doxorubicin versus doxorubicin in human melanoma xenografts correlates with higher tumour concentrations of free drug. Eur J Cancer. 1999, 35: 1143-1149. 10.1016/S0959-8049(99)00074-XView ArticlePubMedGoogle Scholar
- Lev DC, Onn A, Melinkova VO, Miller C, Stone V, Ruiz M, McGary EC, Ananthaswamy HN, Price JE, Bar-Eli M: Exposure of melanoma cells to dacarbazine results in enhanced tumor growth and metastasis in vivo. J Clin Oncol. 2004, 22: 2092-2100. 10.1200/JCO.2004.11.070View ArticlePubMedGoogle Scholar
- Kato Y, Okollie B, Raman V, Vesuna F, Zhao M, Baker SD, Bhujwalla ZM, Artemov D: Contributing factors of temozolomide resistance in MCF-7 tumor xenograft models. Cancer Biol Ther. 2007, 6: 891-897. 10.1158/1535-7163.MCT-06-0125PubMed CentralView ArticlePubMedGoogle Scholar
- Levina V, Marrangoni AM, DeMarco R, Gorelik E, Lokshin AE: Drug-selected human lung cancer stem cells: cytokine network, tumorigenic and metastatic properties. PLoS ONE. 2008, 3: e3077- 10.1371/journal.pone.0003077PubMed CentralView ArticlePubMedGoogle Scholar
- Mita AC, Mita MM, Nawrocki ST, Giles FJ: Survivin: key regulator of mitosis and apoptosis and novel target for cancer therapeutics. Clin Cancer Res. 2008, 14: 5000-5005. 10.1158/1078-0432.CCR-08-0746View ArticlePubMedGoogle Scholar
- Kawasaki H, Toyoda M, Shinohara H, Okuda J, Watanabe I, Yamamoto T, Tanaka K, Tenjo T, Tanigawa N: Expression of survivin correlates with apoptosis, proliferation, and angiogenesis during human colorectal tumorigenesis. Cancer. 2001, 91: 2026-2032. 10.1002/1097-0142(20010601)91:11<2026::AID-CNCR1228>3.0.CO;2-EView ArticlePubMedGoogle Scholar
- Tu SP, Jiang XH, Lin MC, Cui JT, Yang Y, Lum CT, Zou B, Zhu YB, Jiang SH, Wong WM, Chan AO, Yuen MF, Lam SK, Kung HF, Wong BC: Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer. Cancer Res. 2003, 63: 7724-7732.PubMedGoogle Scholar
- Tu SP, Cui JT, Liston P, Huajiang X, Xu R, Lin MC, Zhu YB, Zou B, Ng SS, Jiang SH, Xia HH, Wong WM, Chan AO, Yuen MF, Lam SK, Kung HF, Wong BC: Gene therapy for colon cancer by adeno-associated viral vector-mediated transfer of survivin Cys84Ala mutant. Gastroenterology. 2005, 128: 361-375. 10.1053/j.gastro.2004.11.058View ArticlePubMedGoogle Scholar
- Zhao Y, Shen S, Guo J, Chen H, Greenblatt DY, Kleeff J, Liao Q, Chen G, Friess H, Leung PS: Mitogen-activated protein kinases and chemoresistance in pancreatic cancer cells. J Surg Res. 2006, 136: 325-335. 10.1016/j.jss.2006.06.031View ArticlePubMedGoogle Scholar
- Mirmohammadsadegh A, Mota R, Gustrau A, Hassan M, Nambiar S, Marini A, Bojar H, Tannapfel A, Hengge UR: ERK1/2 is highly phosphorylated in melanoma metastases and protects melanoma cells from cisplatin-mediated apoptosis. J Invest Dermatol. 2007, 127: 2207-2215. 10.1038/sj.jid.5700870View ArticlePubMedGoogle Scholar
- Ding Q, Huo L, Yang JY, Xia W, Wei Y, Liao Y, Chang CJ, Yang Y, Lai CC, Lee DF, Yen CJ, Chen YJ, Hsu JM, Kuo HP, Lin CY, Tsai FJ, Li LY, Tsai CH, Hung MC: Down-regulation of myeloid cell leukemia-1 through inhibiting Erk/Pin 1 pathway by sorafenib facilitates chemosensitization in breast cancer. Cancer Res. 2008, 68: 6109-6117. 10.1158/0008-5472.CAN-08-0579PubMed CentralView ArticlePubMedGoogle Scholar
- Carroll VA, Ashcroft M: Regulation of angiogenic factors by HDM2 in renal cell carcinoma. Cancer Res. 2008, 68: 545-552. 10.1158/0008-5472.CAN-06-4738View ArticlePubMedGoogle Scholar
- West KA, Castillo SS, Dennis PA: Activation of the PI3K/Akt pathway and chemotherapeutic resistance. Drug Resist Updat. 2002, 5: 234-248. 10.1016/S1368-7646(02)00120-6View ArticlePubMedGoogle Scholar
- Caporali S, Levati L, Starace G, Ragone G, Bonmassar E, Alvino E, D'Atri S: AKT is activated in an ataxia-telangiectasia and Rad3-related-dependent manner in response to temozolomide and confers protection against drug-induced cell growth inhibition. Mol Pharmacol. 2008, 74: 173-183. 10.1124/mol.107.044743View ArticlePubMedGoogle Scholar
- Gagnon V, Van Themsche C, Turner S, Leblanc V, Asselin E: Akt and XIAP regulate the sensitivity of human uterine cancer cells to cisplatin, doxorubicin and taxol. Apoptosis. 2008, 13: 259-271. 10.1007/s10495-007-0165-6View ArticlePubMedGoogle Scholar
- Mitsiades CS, Mitsiades N, Koutsilieris M: The Akt pathway: molecular targets for anti-cancer drug development. Curr Cancer Drug Targets. 2004, 4: 235-256. 10.2174/1568009043333032View ArticlePubMedGoogle Scholar
- Wen XF, Yang G, Mao W, Thornton A, Liu J, Bast RC, Le XF: HER2 signaling modulates the equilibrium between pro- and antiangiogenic factors via distinct pathways: implications for HER2-targeted antibody therapy. Oncogene. 2006, 25: 6986-6996. 10.1038/sj.onc.1209685View ArticlePubMedGoogle Scholar
- Xia C, Meng Q, Cao Z, Shi X, Jiang BH: Regulation of angiogenesis and tumor growth by p110 alpha and AKT1 via VEGF expression. J Cell Physiol. 2006, 209: 56-66. 10.1002/jcp.20707View ArticlePubMedGoogle Scholar
- Anfosso L, Efferth T, Albini A, Pfeffer U: Microarray expression profiles of angiogenesis-related genes predict tumor cell response to artemisinins. Pharmacogenomics J. 2006, 6: 269-278.PubMedGoogle Scholar
- Levina V, Su Y, Nolen B, Liu X, Gordin Y, Lee M, Lokshin A, Gorelik E: Chemotherapeutic drugs and human tumor cells cytokine network. Int J Cancer. 2008, 123: 2031-2040. 10.1002/ijc.23732PubMed CentralView ArticlePubMedGoogle Scholar
- Zaghloul N, Hernandez SL, Bae JO, Huang J, Fisher JC, Lee A, Kadenhe-Chiweshe A, Kandel JJ, Yamashiro DJ: Vascular endothelial growth factor blockade rapidly elicits alternative proangiogenic pathways in neuroblastoma. Int J Oncol. 2009, 34: 401-407.PubMed CentralPubMedGoogle Scholar
- Fernando NT, Koch M, Rothrock C, Gollogly LK, D'Amore PA, Ryeom S, Yoon SS: Tumor escape from endogenous, extracellular matrix-associated angiogenesis inhibitors by up-regulation of multiple proangiogenic factors. Clin Cancer Res. 2008, 14: 1529-1539. 10.1158/1078-0432.CCR-07-4126View ArticlePubMedGoogle Scholar
- Miller K, Wang M, Gralow J, Dickler M, Cobleigh M, Perez EA, Shenkier T, Cella D, Davidson NE: Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer. N Engl J Med. 2007, 357: 2666-2676. 10.1056/NEJMoa072113View ArticlePubMedGoogle Scholar
- Meitar D, Crawford SE, Rademaker AW, Cohn SL: Tumor angiogenesis correlates with metastatic disease, N-myc amplification, and poor outcome in human neuroblastoma. J Clin Oncol. 1996, 14: 405-414.PubMedGoogle Scholar
- Ribatti D, Surico G, Vacca A, De Leonardis F, Lastilla G, Montaldo PG, Rigillo N, Ponzoni M: Angiogenesis extent and expression of matrix metalloproteinase-2 and -9 correlate with progression in human neuroblastoma. Life Sci. 2001, 68: 1161-1168. 10.1016/S0024-3205(00)01030-4View ArticlePubMedGoogle Scholar
- Katzenstein HM, Cohn SL, Crawford S, Meitar D: Angiogenesis in neuroblastoma. J Clin Oncol. 2000, 18: 2789-2791.PubMedGoogle Scholar
- Cañete A, Navarro S, Bermúdez J, Pellín A, Castel V, Llombart-Bosch A: Angiogenesis in neuroblastoma: relationship to survival and other prognostic factors in a cohort of neuroblastoma patients. J Clin Oncol. 2000, 18: 27-34.PubMedGoogle Scholar
- Oberthuer A, Berthold F, Warnat P, Hero B, Kahlert Y, Spitz R, Ernestus K, König R, Haas S, Eils R, Schwab M, Brors B, Westermann F, Fischer M: Customized oligonucleotide microarray gene expression-based classification of neuroblastoma patients outperforms current clinical risk stratification. J Clin Oncol. 2006, 24: 5070-5078. 10.1200/JCO.2006.06.1879View ArticlePubMedGoogle Scholar
- Asgharzadeh S, Pique-Regi R, Sposto R, Wang H, Yang Y, Shimada H, Matthay K, Buckley J, Ortega A, Seeger RC: Prognostic significance of gene expression profiles of metastatic neuroblastomas lacking MYCN gene amplification. J Natl Cancer Inst. 2006, 98: 1193-1203. 10.1093/jnci/djj330View ArticlePubMedGoogle Scholar
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