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Supplementary Table S3. Related to Figure 3. Gene ontology for genes with higher expression in patient tumors vs. PDOXs. Supplementary Table S4. Related to Figure 3. List of the gene clusters defined across PDOXs #1-5 obtained from serial orthotopic passaging. Supplementary Table S5. Related to Figure 3. List of enriched Gene Ontology terms by cluster defined in PDOXs #1-5.

Supplementary Table S8. Related to Figure 5. List of top varying peptides based on SD across all samples derived from Patient/PDOX #5. Supplementary Table S9. Related to Figure 5. List of top varying phospho-peptides based on SD across all samples derived from Patient/PDOX #5. Supplementary Table S10. Related to Figure 5. List of enriched Gene Ontology terms by cluster derived from the protein clusters defined in Suppl. Table S8. Supplementary Table S11. Related to Figure 5. List of enriched Gene Ontology terms by cluster derived from the phospho-protein clusters defined in Suppl. Table S9. Supplementary Table S12. Related to Figure 5. List of differentially expressed proteins from group 1-3 of PDOX #5. Supplementary Table S13. Related to Figure 5. List of differentially expressed phosphorylated proteins from group 1-3 of PDOX #5. Supplementary Table S14. Related to Figure 5. List of peptides measured in the targeted PRM assay.

Supplementary Figure S2. Related to Figure 4. Intratumor heterogeneity is less prominent than intertumor heterogeneity.

Supplementary Table S6. Related to Figure 4. Top 1000 varying genes based on SD across all samples derived from Patient/PDOX #5. Supplementary Table S7. Related to Figure 4. List of enriched Gene Ontology terms by cluster derived from the gene clusters defined in Suppl. Table S6.

Supplementary Table S1. Related to Figure 1. List of all somatic mutations detected in patient tumors, and in PDOXs from low and high in vivo generations. Supplementary Table S2. Related to Figure 2. List of all chromosomal copy number changes in patient tumors, and in PDOXs from low and high in vivo generations.

Patient-derived xenografts (PDX) and the Avatar, a single PDX mirroring an individual patient, are emerging tools in preclinical cancer research. However, the consequences of intratumor heterogeneity for PDX modeling of biomarkers, target identification, and treatment decisions remain underexplored. In this study, we undertook serial passaging and comprehensive molecular analysis of neuroblastoma orthotopic PDXs, which revealed strong intrinsic genetic, transcriptional, and phenotypic stability for more than 2 years. The PDXs showed preserved neuroblastoma-associated gene signatures that correlated with poor clinical outcome in a large cohort of patients with neuroblastoma. Furthermore, we captured spatial intratumor heterogeneity using ten PDXs from a single high-risk patient tumor. We observed diverse growth rates, transcriptional, proteomic, and phosphoproteomic profiles. PDX-derived transcriptional profiles were associated with diverse clinical characteristics in patients with high-risk neuroblastoma. These data suggest that high-risk neuroblastoma contains elements of both temporal stability and spatial intratumor heterogeneity, the latter of which complicates clinical translation of personalized PDX–Avatar studies into preclinical cancer research.Significance: These findings underpin the complexity of PDX modeling as a means to advance translational applications against neuroblastoma. Cancer Res; 78(20); 5958–69. ©2018 AACR.

Supplementary Data from HIF-1α and HIF-2α Are Differentially Regulated In vivo in Neuroblastoma: High HIF-1α Correlates Negatively to Advanced Clinical Stage and Tumor Vascularization

Supplementary methods and legends to supplementary figures and tables

Patient-derived xenografts (PDX) and the Avatar, a single PDX mirroring an individual patient, are emerging tools in preclinical cancer research. However, the consequences of intratumor heterogeneity for PDX modeling of biomarkers, target identification, and treatment decisions remain under-explored. In this study, we undertook serial passaging and comprehensive molecular analysis of neuroblastoma orthotopic PDXs, which revealed strong intrinsic genetic, transcriptional, and phenotypic stability for more than 2 years. The PDXs showed preserved neuroblastoma-associated gene signatures that correlated with poor clinical outcome in a large cohort of patients with neuroblastoma. Furthermore, we captured spatial intratumor heterogeneity using ten PDXs from a single high-risk patient tumor. We observed diverse growth rates, transcriptional, proteomic, and phosphoproteomic profiles. PDX-derived transcriptional profiles were associated with diverse clinical characteristics in patients with high-risk neuroblastoma. These data suggest that high-risk neuroblastoma contains elements of both temporal stability and spatial intratumor heterogeneity, the latter of which complicates clinical translation of personalized PDX-Avatar studies into preclinical cancer research. Significance: These findings underpin the complexity of PDX modeling as a means to advance translational applications against neuroblastoma. (C) 2018 AACR.

Purpose: Hypoxia is considered to be a major driving force behind tumor angiogenesis. The stabilization and activation at hypoxia of the hypoxia-inducible factors HIF-1α and HIF-2α and the concomitant induction of expression of vascular endothelial growth factor (VEGF) and other proangiogenic factors provide a molecular frame for hypoxia-driven tumor angiogenesis. This study has investigated how HIF and VEGF protein levels relate to each other with regard to vascularization, tumor stage, and overall survival in neuroblastoma. Experimental Design: Tissue cores taken from tumor specimens representing 93 children with neuroblastoma were arranged on a microarray and stained for HIF-1α, HIF-2α, VEGF, and CD31 proteins. Both fraction of positive cells and staining intensity were evaluated and protein levels were correlated with each other and with clinical variables. Results: Although high levels of both HIF-1α (P < 0.001) and HIF-2α (P < 0.001) correlated positively to VEGF expression, they did not fully correlate with each other. Moreover, HIF-1α (P = 0.002) and VEGF (P < 0.001), but not HIF-2α, correlated negatively to vascularization as determined by CD31 staining abundance. VEGF expression or degree of vascularization did not correlate with tumor stage or overall survival. High HIF-1α levels correlated with low tumor stage (P < 0.001) and were associated with a favorable patient prognosis (P = 0.08). Conclusions: The discordant results on expression of HIF-1α and HIF-2α suggest that these two proteins are differentially regulated in vivo, thus reflecting distinctive protein expression/stabilization mechanisms. The association between HIF-1α and favorable outcome stresses the importance of discriminating HIF-2α from HIF-1α expression and has implications for using HIFs as treatment targets. (Clin Cancer Res 2009;15(23):7130–6)

High hypoxia-inducible factor-2α (HIF-2α) protein levels predict poor outcome in neuroblastoma, and hypoxia dedifferentiates cultured neuroblastoma cells toward a neural crest-like phenotype. Here, we identify HIF-2α as a marker of normoxic neural crest-like neuroblastoma tumor-initiating/stem cells (TICs) isolated from patient bone marrows. Knockdown of HIF-2α reduced VEGF expression and induced partial sympathetic neuronal differentiation when these TICs were grown in vitro under stem cell-promoting conditions. Xenograft tumors of HIF-2α-silenced cells were widely necrotic, poorly vascularized, and resembled the bulk of tumor cells in clinical neuroblastomas by expressing additional sympathetic neuronal markers, whereas control tumors were immature, well-vascularized, and stroma-rich. Thus, HIF-2α maintains an undifferentiated state of neuroblastoma TICs. Because low differentiation is associated with poor outcome and angiogenesis is crucial for tumor growth, HIF-2α is an attractive target for neuroblastoma therapy.

Adaptation to low oxygen conditions is essential for maintaining homeostasis and viability in oxygen-consuming multi-cellular tissues, including solid tumors. Central in these processes are the hypoxia-inducible transcription factors, HIF-1 and HIF-2, controlling genes involved in e.g. glucose metabolism and neovascularization. Tumor hypoxia and HIF expression have also been associated with a dedifferentiated phenotype and increased aggressiveness. In this report we show that the MAX interactor-1 (MXI1) gene is directly regulated by HIF proteins in neuroblastoma and breast cancer cells. HIF-binding and transactivation were detected within MXI1 gene regulatory sequences in the vicinity of the MXI1-0 promoter, leading to rapid induction of the alternate MXI1-0 isoform followed by a long-term induction of both the MXI1-0 and MXI1 isoforms. Importantly, knock-down of MXI1 had limited effect on MYC/MYCN activity under hypoxia, an observation that might be related to the different functional attributes of the two MXI1 isoforms.

Loss of the tumor suppressor gene von Hippel-Lindau (VHL) plays a key role in the oncogenesis of clear cell renal cell carcinoma (CCRCC). The loss leads to stabilization of the HIF transcription complex, which induces angiogenic and mitogenic pathways essential for tumor formation. Nonetheless, additional oncogenic events have been postulated to be required for the formation of CCRCC tumors. Here, we show that the Notch signaling cascade is constitutively active in human CCRCC cell lines independently of the VHL/HIF pathway. Blocking Notch signaling resulted in attenuation of proliferation and restrained anchorage-independent growth of CCRCC cell lines. Using siRNA targeting the different Notch receptors established that the growth-promoting effects of the Notch signaling pathway were attributable to Notch-1 and that Notch-1 knockdown was accompanied by elevated levels of the negative cell-cycle regulators p21 Cip1 and/or p27 Kip1. Treatment of nude mice with an inhibitor of Notch signaling potently inhibited growth of xenotransplanted CCRCC cells. Moreover, Notch-1 and the Notch ligand Jagged-1 were expressed at significantly higher levels in CCRCC tumors than in normal human renal tissue, and the growth of primary CCRCC cells was attenuated upon inhibition of Notch signaling. These findings indicate that the Notch cascade may represent a novel and therapeutically accessible pathway in CCRCC.

High HIF-2alpha protein levels in the sympathetic nervous system-derived childhood tumour neuroblastoma as well as immature phenotype correlate to unfavourable outcome. Here we show that a small subset of perivascularly located, strongly HIF-2alpha-positive tumour cells (MYCN amplified) lacks expression of differentiation markers, but expresses neural crest and early sympathetic progenitor marker genes such as Notch-1, HES-1, c-Kit, dHAND, and vimentin. HIF-2alpha- and CD68-positive tumour-associated macrophages were frequently found close to the immature and HIF-2alpha-positive neuroblastoma cells and as VEGF levels are high in the perivascular niche, we hypothesize that neuroblastoma neural crest-like cells and macrophages cooperate to facilitate angiogenesis and thereby contribute to the aggressive neuroblastoma phenotype. Copyright (c) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. (Less)

Arsenic trioxide (As(2)O(3)) is toxic to multidrug-resistant neuroblastoma cells in vivo and in vitro. In neuroblastoma, As(2)O(3) does not exert its cell death-promoting effects via a classical apoptotic pathway. A death mechanism involving proteolytic cleavage of Bax to a p18 form seems to be of importance, because inhibition of Bax cleavage coincides with diminished cell death. As existing models of cell death implicate Bax in the intrinsic apoptotic pathway, triggering death after Bax translocation to the mitochondria, we investigated the cellular localization of p18 Bax by subcellular fractionation. After As(2)O(3) treatment, p18 Bax was only present in nuclei-enriched, mitochondria-depleted fractions. Cytoplasmic p21 Bax levels decreased, whereas total (p21 and p18) nuclear Bax increased. Overexpressed p21 Bax localized to the cytoplasm and nuclei, whereas overexpressed p18 Bax localized to extra-nuclear structures only. The inability of overexpressed p18 Bax to locate to the nucleus, and the As(2)O(3)-induced reduction of p21 Bax in the cytosol, suggest an As(2)O(3)-induced mechanism where p18 Bax gets cleaved and 'trapped' in the nucleus. This model is strengthened by the observation that calpain, the protease responsible for p18 Bax generation, is present in the nuclei, and that nuclear calpain is induced by increasing As(2)O(3) and Ca(2+) levels.

Treatment of high-risk childhood neuroblastoma is a clinical challenge which has been hampered by a lack of reliable neuroblastoma mouse models for preclinical drug testing. We have previously established invasive and metastasising patient-derived orthotopic xenografts (PDXs) from high-risk neuroblastomas that retained the genotypes and phenotypes of patient tumours. Given the important role of the tumour microenvironment in tumour progression, metastasis, and treatment responses, here we analysed the tumour microenvironment of five neuroblastoma PDXs in detail. The PDXs resembled their parent tumours and retained important stromal hallmarks of aggressive lesions including rich blood and lymphatic vascularisation, pericyte coverage, high numbers of cancer-associated fibroblasts, tumour-associated macrophages, and extracellular matrix components. Patient-derived tumour endothelial cells occasionally formed blood vessels in PDXs; however, tumour stroma was, overall, of murine origin. Lymphoid cells and lymphatic endothelial cells were found in athymic nude mice but not in NSG mice; thus, the choice of mouse strain dictates tumour microenvironmental components. The murine tumour microenvironment of orthotopic neuroblastoma PDXs reflects important hallmarks of aggressive and metastatic clinical neuroblastomas. Neuroblastoma PDXs are clinically relevant models for preclinical drug testing.

Background: Cancer cell lines have traditionally been established in serum-containing medium though it has been questioned whether serum-grown cell lines faithfully represent the tumors they are derived from. Potential serum-induced differentiation of in vitro cultured cells could partly explain why xenografted cell lines often lack invasive growth and robust metastasis. Direct implantation of patient tumor material into mice, i.e. patient derived xenografts (PDXs), has shown to more closely mimic the original tumor growth pattern. By isolating cells from neuroblastoma PDXs and culturing the PDX cells under serum-free conditions, we expect to preserve a less differentiated phenotype, enabling us to use PDX cells as a source for identifying potential neuroblastoma stem-like cells. Moreover, to facilitate future drug screening, we investigated conditions that would allow us to grow neuroblastoma PDX cells adherently, without compromising their tumorigenic or metastatic capacity. Materials & Methods: PDX cells were cultured in stem cell medium or serum-containing medium for up to 7 days. IHC, RT-qPCR and WB were used to investigate the expression of sympathetic neuronal differentiation markers. For monolayer culture, cells were grown on laminin. Results: PDX cells can routinely be established as neurospheres in serum-free medium with retained tumorigenic and metastatic capacity. Addition of serum induces loss of sphere formation, adherent growth and a robust increase in sympathetic differentiation markers, both at mRNA and protein level. Serum-cultured cells also show a decreased cell proliferation (without increased apoptotic rate, as determined by sub-G0/G1 analyses). The serum-induced differentiation was not irreversible since transferring serum-grown cells back to serum-free medium resulted in a phenotypic switch, with recovered proliferation and decreased expression of differentiation markers. Growing cells on laminin achieved adherent culture of PDX cells. This resulted only in slight morphological differentiation but did not affect growth rate or the cells tumorigenic and metastatic capacity. Conclusion: Culturing neuroblastoma PDX cells in serum-containing medium results in a more differentiated phenotype. Thus, avoiding serum appears to be a key strategy in order to preserve the phenotypic origin of these cells. The phenotypic effects of serum on tumor cells have largely been overlooked and might be a general effect on tumor cells explaining why serum-established cell lines do not metastasize and grow invasively. Citation Format: Camilla U. Persson, Kristoffer von Stedingk, Daniel Bexell, My Merselius, David Gisselsson, Siv Beckman, Sofie Mohlin, Sven Påhlman, Caroline Wigerup. Serum induces differentiation in aggressive MYCN-amplified neuroblastoma patient-derived xenograft cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5834. doi:10.1158/1538-7445.AM2017-5834

Neuroblastoma is a childhood tumour with heterogeneous characteristics and children with metastatic disease often have a poor outcome. Here we describe the establishment of neuroblastoma patient-derived xenografts (PDXs) by orthotopic implantation of viably cryopreserved or fresh tumour explants of patients with high risk neuroblastoma into immunodeficient mice. In vivo tumour growth was monitored by magnetic resonance imaging and fluorodeoxyglucose–positron emission tomography. Neuroblastoma PDXs retained the undifferentiated histology and proliferative capacity of their corresponding patient tumours. The PDXs expressed neuroblastoma markers neural cell adhesion molecule, chromogranin A, synaptophysin and tyrosine hydroxylase. Whole genome genotyping array analyses demonstrated that PDXs retained patient-specific chromosomal aberrations such as MYCN amplification, deletion of 1p and gain of chromosome 17q. Thus, neuroblastoma PDXs recapitulate the hallmarks of high-risk neuroblastoma in patients. PDX-derived cells were cultured in serum-free medium where they formed free-floating neurospheres, expressed neuroblastoma gene markers MYCN, CHGA, TH, SYP and NPY, and retained tumour-initiating and metastatic capacity in vivo. PDXs showed much higher degree of infiltrative growth and distant metastasis as compared to neuroblastoma SK-N-BE(2)c cell line-derived orthotopic tumours. Importantly, the PDXs presented with bone marrow involvement, a clinical feature of aggressive neuroblastoma. Thus, neuroblastoma PDXs serve as clinically relevant models for studying and targeting high-risk metastatic neuroblastoma. What's new? Neuroblastoma is a childhood tumour with heterogeneous characteristics and children with metastatic disease have a poor outcome. Here, the authors established neuroblastoma patient-derived xenografts (PDXs) by orthotopic implantation of viably cryopreserved or fresh tumour explants of patients with high-risk neuroblastoma into immunodeficient mice. The PDXs retained the genotype and phenotype of patient tumours and exhibited substantial infiltrative growth and metastasis to distant organs including bone marrow. PDX-derived neuroblastoma cells were expanded in vitro and retained tumourigenic and metastatic capacity in vivo. The PDXs may thus represent an important tool for investigating neuroblastoma growth and metastasis as well as drug targeting.

Widespread metastasis is a major problem for the treatment of high-risk neuroblastoma. Relevant neuroblastoma animal models are hence needed to study and target high-risk metastatic neuroblastoma. We developed neuroblastoma patient-derived orthotopic xenografts (PDXs) using viably cryopreserved or fresh patient neuroblastoma fragments which were implanted orthotopically into immunodeficient NSG mice. Immunohistochemistry showed that PDXs retain neuroblastoma markers and a highly infiltrative growth pattern. Importantly, we found distant metastasis to lungs, liver and bone marrow. Single nucleotide polymorphism array analysis confirmed that PDXs maintain patient-specific chromosomal aberrations 1p del, MYCN amp and 17q gain. In vitro cultures established from the PDXs express neuroblastoma markers and retain their tumorigenic and metastatic ability in vivo after orthotopic injection. PDXs were highly vascularized with mouse endothelial cells and one PDX model formed tumor vasculature through co-engrafted human tumor endothelial cells. Tumors also contained pericytes, cancer-associated fibroblasts, macrophages and extracellular components resembling the patient disease. PDXs established in athymic nude mice additionally developed intratumoral lymphatic vessels and contained mouse-derived CD45+ lymphoid cells. Thus, PDXs maintain important tumor microenvironment hallmarks for high stage neuroblastoma. Furthermore, we demonstrate the feasibility of using short-term in vitro cultured PDX-derived cells as a drug-testing model, and show that the HIF2A oncogene is transcriptionally regulated at hypoxia via the PI3K/mTORC2 pathway in these cells. The results establish the PI3K and mTORC2 pathways as potential therapeutic targets of aggressive neuroblastoma. In conclusion, neuroblastoma orthotopic PDXs resemble many clinical aspects of the patient disease, e.g., distant metastases, and PDXs are valuable models for preclinical drug testing. Citation Format: Noémie Braekeveldt, Caroline Wigerup, David Gisselsson, Sofie Mohlin, My Merselius, Siv Beckman, Irene Tadeo, Anna P. Berbegall, Ingrid Öra, Samuel Navarro, Rosa Noguera, Sven Påhlman, Daniel Bexell. Neuroblastoma patient-derived orthotopic xenografts: Clinically relevant models for drug testing. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A02.

Small cell lung carcinoma (SCLC) is extremely aggressive and frequently metastasizes widely in its early stage. Because tumor hypoxia is related to aggressive tumor behavior and the hypoxic adaptation of SCLC is poorly documented, we stained SCLC tumors arranged in a tissue microarray for hypoxia-inducible factor (HIF)-1α and HIF-2α proteins. We found an overall lack of HIF-2α protein expression, which was confirmed in large tumor sections. HIF-1α protein was strongly expressed in most tumors, frequently adjacent to necrotic regions. In concordance, cultured SCLC but not non-small cell lung carcinoma cells showed no or extremely low levels of HIF-2α mRNA and no HIF-2α protein at hypoxia. HIF-1α was stabilized after 4 hours at hypoxia, and its accumulation increased up to 96 hours. SCLC cells survived well and showed net proliferation and low cell death in modest (1% oxygen) and severe (0.1% oxygen) hypoxia. HIF-1α repression virtually did not influence cell death or viability despite reduced levels of hypoxia-inducible genes, such as BNIP3 and BNIP3L. At 1% oxygen no increased autophagy (LC3B-II activation) or NF-κB signaling were detected, whereas the unfolded protein response was activated at severe hypoxia. Our data indicate that HIFs are not exclusively required for SCLC cell survival at modest or severe hypoxia and that additional, yet uncharacterized, hypoxia-driven adaptation pathways may become activated.

A correction to Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes by K De Preter, J Vandesompele, P Heimann, N Yigit, S Beckman, A Schramm, A Eggert, RL Stallings, Y Benoit, M Renard, A De Paepe, G Laureys, S Påhlman and F Speleman. Genome Biology 2006 7:R84

Transcriptome profiling of neuroblasts and neuroblastoma tumor cells provides strong support for a neuroblast origin of neuroblastoma and highlights new candidate neuroblastoma genes, Background Neuroblastoma tumor cells are assumed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. Because these precursor cells are not detectable in postnatal life, their transcription profile has remained inaccessible for comparative data mining strategies in neuroblastoma. This study provides the first genome-wide mRNA expression profile of these human fetal sympathetic neuroblasts. To this purpose, small islets of normal neuroblasts were isolated by laser microdissection from human fetal adrenal glands. Results Expression of catecholamine metabolism genes, and neuronal and neuroendocrine markers in the neuroblasts indicated that the proper cells were microdissected. The similarities in expression profile between normal neuroblasts and malignant neuroblastomas provided strong evidence for the neuroblast origin hypothesis of neuroblastoma. Next, supervised feature selection was used to identify the genes that are differentially expressed in normal neuroblasts versus neuroblastoma tumors. This approach efficiently sifted out genes previously reported in neuroblastoma expression profiling studies; most importantly, it also highlighted a series of genes and pathways previously not mentioned in neuroblastoma biology but that were assumed to be involved in neuroblastoma pathogenesis. Conclusion This unique dataset adds power to ongoing and future gene expression studies in neuroblastoma and will facilitate the identification of molecular targets for novel therapies. In addition, this neuroblast transcriptome resource could prove useful for the further study of human sympathoadrenal biogenesis.

Solid tumors are frequently necrotic and hypoxic due to poor vascularization. Tumor cells adapt to hypoxia by modulating their phenotype. Key players in this process are the hypoxia-inducible factors (HIF-1alpha to 3alpha). HIFs are also expressed during normal development; for example, HIF-2alpha is specifically expressed and appears to be involved in the development of the murine sympathetic nervous system (SNS). Here, we demonstrate that HIF-2alpha protein is selectively present in human fetal week 8.5 SNS paraganglia. Neuroblastoma is derived from SNS precursors. In a subset of neuroblastomas, a spontaneous neuronal to neuroendocrine differentiation occurs in areas adjacent to necrotic zones. As HIF-2alpha activity has been associated not only with hypoxic but also with hypoglycemic conditions, we have investigated putative effects of hypoxia, glucose depletion, and HIF-2alpha on the neuroblastoma phenotype. HIF-2alpha was detected in hypoxic and in well-oxygenized neuroblastoma cells and tissue, presumably reflecting their embryonic features. With regard to differentiation, hypoxic cells lost their neuronal/neuroendocrine features and gained marker gene expression associated with an immature, neural crest-like phenotype. Low glucose potentiated the effect of hypoxia. These findings suggest that poorly vascularized neuroblastomas become immature and maintain a more aggressive phenotype, which possibly could involve a sustained stabilization and activation of HIF-2alpha.

In cultured neuroblastoma cells, hypoxia induces a dedifferentiated phenotype. We tested whether hypoxia-induced dedifferentiation also occurs in vivo in mammary ductal carcinoma in situ with its well-defined lesions and distinct areas of necrosis. Ductal carcinoma in situ cells surrounding the central necrosis have high hypoxia inducible factor-1alpha protein levels, down-regulated estrogen receptor-alpha, and increased expression of the epithelial breast stem cell marker cytokeratin 19; lose their polarization; and acquire an increased nucleus/cytoplasm ratio, hallmarks of poor architectural and cellular differentiation. The hypoxia-induced changes were confirmed in cultured breast cancer cells. We propose that hypoxia-induced dedifferentiation is a mechanism that promotes tumor progression in breast cancer.

Neuroblastoma is a childhood tumor located in the sympathetic nervous system and most often found in the adrenal gland. Half of the patients have metastases already at diagnosis. There is a need for improved preclinical in vivo models of human high-stage neuroblastoma. Aims: 1) To establish relevant xenograft in vivo models for studying neuroblastoma orthotopic growth and metastatic growth in bone marrow, 2) to detect tumor growth by the two imaging modalities utilized in the clinic, i.e. magnetic resonance imaging (MRI) and I-123-metaiodobenzylguanidine (MIBG)-Single photon emission computed tomography (SPECT), and 3) to characterize orthotopic and bone marrow tumor growth at a pathohistological level. Methods: SK-N-Be2c and KCN-69 neuroblastoma cells were injected into adrenal gland of immunodeficient NSG mice to establish orthotopic tumors. SK-N-Be2c cells were injected directly into femur bone marrow of NSG mice to model bone marrow metastatic growth in a reproducible way. Tumor growth was monitored by MRI or by MIBG-SPECT. Histopathological assessment was performed on formalin-fixed paraffin-embedded tissue sections. Conclusion: We have established a model for orthotopic neuroblastoma growth and characterized the model by MRI and MIBG-SPECT. Orthotopic tumors grow infiltratively into surrounding tissues and resemble the histopathological hallmarks of patient neuroblastoma. We also describe a reproducible in vivo model for neuroblastoma bone marrow metastatic growth. The in vivo models will be important tools for studying mechanisms of neuroblastoma growth and for preclinical drug testing. Citation Format: Daniel Bexell, Noémie Braekeveldt, Siv Beckman, Sofie Mohlin, David Gisselsson, Sven Påhlman. Modeling human metastatic neuroblastoma in immunodeficient mice. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B57.

Small cell lung carcinoma (SCLC) is an extremely aggressive tumor that is widely metastasized in early stages of the disease and the 5-year survival rate for these patients is only approximately 9-15%. Here we study the adaptive response of SCLC cells to lower oxygen levels, since tumor hypoxia (∼1% oxygen) is related to aggressive tumor behaviour and poor outcome in several tumor forms. Areas of hypoxia are a common characteristic in solid tumors and in response to hypoxia tumor cells induce expression of several genes involved in a variety of processes. This adaptive response is primarily regulated by the transcription factors hypoxia inducible factor (HIF)-1α and HIF-2α, which become stable at low oxygen levels. Studies using among others lung adenocarcinoma, neuroblastoma and breast carcinoma cells have demonstrated that HIF-1α primarily mediates the acute hypoxic response, whereas HIF-2α dominates during the more chronic phase of hypoxia. We investigated the viability of SCLC cells cultured at hypoxia and characterized the expression levels of HIF transcription factors in SCLC cells and specimens using quantitative real-time PCR, western blot and immunohistochemistry. Expression levels of well known hypoxia-driven genes were studied and to further characterize the hypoxic adaptive response, a gene expression profile of hypoxic SCLC cells was performed using microarray analyses. In all experiments a panel of non-SCLC cells and neuroblastoma cells were included for comparison. Our results show that SCLC cells have a high adaptive capacity to hypoxia, even though a modest induction of well known hypoxia-driven genes is observed. Interestingly, the HIF-1α and HIF-2α are differentially regulated in SCLC cells as compared to non-SCLC. In SCLC cells, no HIF-2α protein is detectable whereas HIF-1α protein levels are sustained and increased up to 96 hours. In addition, knock-down of HIF-1α at 0.1% oxygen are not affecting the viability of the SCLC cells and our gene expression profile of hypoxic SCLC cells has identified genes important for SCLC cells survival at hypoxia. Our data suggest that SCLC cells have an adaptive response to hypoxia that is partially HIF-independent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2058. doi:10.1158/1538-7445.AM2011-2058

We study the effect of hypoxia on mammary epithelial cell differentiation at a non-malignant stage as a putative step in early malignant progression. Due to inadequate vasculature solid tumors often contain regions with low oxygen levels (hypoxia). Hypoxia is associated with increased metastatic potential and worse patient outcome. The hypoxic response is in large driven by the two transcription factors hypoxia inducible factor (HIF) 1 and HIF 2. We have reported that hypoxia leads to a less differentiated phenotype in breast cancer and in neuroblastoma, and in the clinic low tumor cell differentiation is related to poor prognosis in these diseases. To investigate the effect of hypoxia on mammary epithelial cell differentiation we employed the model of acinar morphogenesis. In this model immortalized mammary epithelial cells in three-dimensional (3D) culture on a laminin-rich matrix differentiate and form acini-like structures with evacuated lumen surrounded by polarized cells. We have previously seen that hypoxia (1% oxygen) impairs the process of acinar morphogenesis. The hypoxic cells failed to obtain polarity and markers of differentiation were affected at expression level as well as cellular localization. Addressing the mechanism behind the hypoxia-impaired differentiation, we find that hypoxia impairs the global histone deacetylation that is associated with changes in cell morphology and differentiation of the mammary epithelial cells. Three days post-seeding, during the proliferation stage, both normoxic and hypoxic cells stained positive for acetylated histone 4 (AcH4). When the normoxic cells go through morphological changes and acinar formation the acetylation of histone 4 is reduced whereas it is maintained in the hypoxic cell-clusters 10 days after seeding. Transfer of acini from normoxia to hypoxia at this time-point leads to an increase in AcH4. Thus, the hypoxic condition prevents global deacetylation, which is generally associated with high level of transcription and an undifferentiated proliferative cell state, rendering the cells an open chromatin state and hindering acinar formation. These data indicate that hypoxia promotes loss of differentiation also at a non-malignant stage. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 463.

Background: Neuroblastoma tumor cells are assumed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. Because these precursor cells are not detectable in postnatal life, their transcription profile has remained inaccessible for comparative data mining strategies in neuroblastoma. This study provides the first genome-wide mRNA expression profile of these human fetal sympathetic neuroblasts. To this purpose, small islets of normal neuroblasts were isolated by laser microdissection from human fetal adrenal glands. Results: Expression of catecholamine metabolism genes, and neuronal and neuroendocrine markers in the neuroblasts indicated that the proper cells were microdissected. The similarities in expression profile between normal neuroblasts and malignant neuroblastomas provided strong evidence for the neuroblast origin hypothesis of neuroblastoma. Next, supervised feature selection was used to identify the genes that are differentially expressed in normal neuroblasts versus neuroblastoma tumors. This approach efficiently sifted out genes previously reported in neuroblastoma expression profiling studies; most importantly, it also highlighted a series of genes and pathways previously not mentioned in neuroblastoma biology but that were assumed to be involved in neuroblastoma pathogenesis. Conclusion: This unique dataset adds power to ongoing and future gene expression studies in neuroblastoma and will facilitate the identification of molecular targets for novel therapies. In addition, this neuroblast transcriptome resource could prove useful for the further study of human sympathoadrenal biogenesis.