Neuroblastoma Tumor-initiating Cells Research Articles

Verteporfin induces apoptosis and reduces the stem cell-like properties in Neuroblastoma tumour-initiating cells through inhibition of the YAP/TAZ pathway

Neuroblastoma is an embryonal malignancy of early childhood arising from the embryonic sympatho-adrenal lineage of the neural crest. About half of all cases are currently classified as high-risk of disease recurrence, with an overall survival rate of less than 40% at 5 years despite intensive therapy. Recent studies on matched primary tumours and at the relapse revealed downregulation of genes transcriptionally silenced by YAP as significant association with neuroblastoma relapse. Here, we evaluated the pharmacological targeting of YAP/TAZ with the YAP/TAZ-TEAD inhibitor Verteporfin (VP) in Tumour Initiating Cells (TICs) derived from High-Risk Neuroblastoma patients. VP treatment suppresses YAP/TAZ expression, induces apoptosis and causes the re-organization of the cytoskeleton reducing cells migration and clonogenic ability. Moreover, VP reduces the percentage of side population cells and ABC transporters involved in drug resistance, and the percentage of stem cell subpopulations CD133+ and CD44+ of TICs. Finally, we demonstrated that VP sensitizes TICs to the standard drugs used for neuroblastoma therapy etoposide and cis-platin opening the way to use VP as drug repositioning candidate for recurrent neuroblastoma.

Induced differentiation inhibits sphere formation in neuroblastoma

Neuroblastoma arises from the neural crest, the precursor cells of the sympathoadrenal axis, and differentiation status is a key prognostic factor used for clinical risk group stratification and treatment strategies. Neuroblastoma tumor-initiating cells have been successfully isolated from patient tumor samples and bone marrow using sphere culture, which is well established to promote growth of neural crest stem cells. However, accurate quantification of sphere-forming frequency of commonly used neuroblastoma cell lines has not been reported. Here, we show that MYCN-amplified neuroblastoma cell lines form spheres more frequently than non-MYCN-amplified cell lines. We also show that sphere formation is directly sensitive to cellular differentiation status. 13-cis-retinoic acid is a clinically used differentiating agent that induces a neuronal phenotype in neuroblastoma cells. Induced differentiation nearly completely blocked sphere formation. Furthermore, sphere formation was specifically FGF-responsive and did not respond to increasing doses of EGF. Taken together, these data suggest that sphere formation is an accurate method of quantifying the stemness phenotype in neuroblastoma.

Heterogeneity of neuroblastoma.

Neuroblastoma (NB) is the most common pediatric solid tumor and represents 15% of all childhood cancer deaths. High cellular heterogeneity is a hallmark of NB, which may account for the wide range of clinical presentations and non-uniform response to treatment. Compelling evidence indicates that cancer cells in solid tumors may not necessarily be hierarchically organized, but rather, also following the clonal evolution model in which cancer cells may evolve from one subtype to the others, generating tumors with distinct features. Cancer cells may even transiently acquire stemness properties depending on the tumor context [1–3]. Importantly, these two models are not mutually exclusive in cancers, accounting for the tumor heterogeneity [4]. To date, there is still no defined surface marker available for the identification of tumor initiating cells (TICs) from NB. TICs isolated from the high staged NBs were mainly based on their stem cell characteristics, including sphere-formation capacity and plasticity with respect to differentiation along several lineages [5, 6]. Transcriptome analysis of NB TICs further highlighted the complexity of these cells and suggested that NB TICs may exist as a dynamic and heterogeneous cell population strongly dependent on the particular niche environment [5]. In our recent manuscript by Liu et. al. [7], we made use of various lines of NB-TICs isolated from the primary and bone-marrow metastasized NB, and NB cell lines to generate in vivo models for studying tumor heterogeneity. We discovered that a population of NB cells, which express high level of c-KIT (c-KIThigh), a tyrosine-kinase receptor for stem cell factor (SCF), is de novo generated and dynamically maintained within the tumors to sustain tumor progression. c-KIThigh NB cells possess the stem cell characteristics: they expressed high levels of neural crest and stem cell markers (SLUG, SOX2, NANOG), possess high clonogenic capacity, differentiation plasticity and drug resistance. Even though c-KIT expression is not required for the tumor formation, serial transplantation assays showed that c-KIThigh cells are more aggressive and induce tumors 9-fold more efficiently than c-KIT−/low cells. More importantly, c-KIThigh cells exhibited a long-term in vivo self-renewal capacity to sustain the formation of the secondary and tertiary tumors in mice. All these data support the notion that NB cells change dynamically, they evolve and generate new progenies during tumor progression. The progenies would be more tumorigenic than the parental cells to sustain the tumor progression. Since c-KIT−/low cells can give rise to c-KIThigh cells and vice versa, targeting c-KIT may not be sufficient to effectively eradicate the tumor. Thus, we proposed to target a growth factor, namely Prokineticin, where its receptors are ubiquitously expressed in both c-KIThigh and c-KIT−/low cells and supports the formation and growth of c-KIThigh cells. We showed that knocking down the receptors for Prokineticin provides a more effective way to inhibit the tumor formation than directly targeting c-KIT, by abolishing the de novo generation of c-KIThigh cells. In summary, we have employed NB-TIC lines isolated from primary and bone-marrow metastasized tumors to establish an in vivo cancer model for understanding how NB TICs give rise to heterogeneous derivatives and how these derivatives are organized and influence disease progression or treatment response. We also provided evidence that targeting the growth factor which supports the formation of moving target represents an effective way to eradicate NB. Obviously, NB is highly heterogeneous, comprising cancer cells with very different molecular features. The next important questions are to understand how the clonal composition of a tumor influences its progression and treatment response, how the molecular features determine the behavior of tumor cells, and what are the common features or molecular signatures shared by the most aggressive tumor cells of various tumors. NB-TIC derived tumor model would be a useful platform for understanding tumor formation, progression and tumor heterogeneity. Recent advances in single cell sequencing technology allow a precise examination of the expression profile of the heterogeneous cancer populations at a single cell level. Comparisons of the molecular signatures of the tumor cells derived from various NB-TICs will allow a systematic analysis of heterogeneous populations of a tumor and help identify candidate genes for future drug intervention. Nevertheless, the most formidable challenge of the field is to know which population of cancer cells within the individual tumor we should target: TIC or its most aggressive progenies, in order to translate our knowledge of cancer biology to establish the personalized treatment strategy for the patients.

Genotype analysis of tumor‐initiating cells expressing CD133 in neuroblastoma

Neuroblastoma (NB) is the most common and lethal extracranial solid tumor of childhood. Despite aggressive therapy, more than half of the children with advanced NB will die of uncontrolled metastatic disease. After chemotherapy, tumor-initiating cells (TICs) could persist, cause relapses and metastasis. The aim of this study is to demonstrate the tumor-initiating properties of CD133high NB cells and to identify new specific genetic abnormalities. Isolation of the CD133high cell population from NB cell lines was followed by neurosphere formation, soft agar assays, and orthotopic injections in NOD/SCID/IL2Rγc-null mice. A differential genotyping analysis was performed with Affymetrix SNP 6.0 arrays on CD133low and CD133high populations and the frequency of the abnormalities of 36 NB tumors was determined. Our results show that CD133high NB cells possess tumor-initiating properties, as CD133high cells formed significantly more neurospheres and produced significantly more colonies in soft agar than CD133low. Injection of 500 CD133high cells was sufficient to generate primary tumors and frequent metastases in mice. Differential genotyping analysis demonstrated two common regions with gains (16p13.3 and 19p13.3) including the gene EFNA2 in the CD133high population, and two with loss of heterozygosity (16q12.1 and 21q21.3) in the CD133low population. The gain of EFNA2 correlated with increased expression of the corresponding protein. These abnormalities were found in NB samples and some were significantly correlated with CD133 expression. Our results show that CD133high NB cells have TICs properties and present different genotyping characteristics compared to CD133low cells. Our findings reveal insights into new therapeutic targets in NB TICs.

Rab15 alternative splicing is altered in spheres of neuroblastoma cells

Neuroblastoma is an aggressive pediatric tumor that accounts for 15% of cancer-related deaths in children. More than half of high-risk neuroblastoma patients develop tumor relapse that is lethal in most cases. A small population of tumor-initiating cells (TICs), recently identified from high-risk neuroblastoma patients as spheres, is believed to be responsible for tumor relapse. Rab family small G proteins are essential in controlling membrane traffic and their misregulation results in several cancers. Rab15 was originally isolated as a brain-specific Rab protein regulating the endocytic recycling pathway and was recently identified as a downstream target of the neural transcription factor Atoh1. Previously, we identified two alternatively spliced Rab15 isoforms in neuroblastoma cells and showed a significant correlation between Rab15 expression and neuronal differentiation. As aberrant alternative splicing is intimately associated with an increasing number of cancers, its use as a new diagnostic and/or prognostic biomarker has attracted considerable attention. In the present study, we explored cancer-associated changes of Rab15 alternative splicing in neuroblastoma TICs. We found that Rab15 alternative splicing generated two novel isoforms designated as Rab15(AN2) and Rab15(AN3) in addition to two known isoforms designated as Rab15(CN) and Rab15(AN1). Although both Rab15(AN2) and Rab15(AN3) contained premature termination codons, they were detected in not only neuroblastoma cells but also in normal human tissues. One isoform was predominantly expressed in the brain and testis, while the other isoform was more specifically expressed in the brain. In neuroblastoma, Rab15 isoform balance measured by the Rab15(CN)/Rab15(AN1+AN2+AN3) ratio was significantly decreased in spheres compared to parental cells. These results suggest that Rab15 alternative splicing may serve as a biomarker to discriminate TICs from non-TICs in neuroblastoma.

Abstract 4336: Oncolytic reovirus as a novel therapy for neuroblastoma

Abstract Background: Neuroblastoma (NB), a tumour of neurocrest progenitor cells of the sympathetic nervous system, is the most common extracranial pediatric tumour and accounts for approximately 15 percent of childhood cancer mortality. Even with surgery, radiation therapy, aggressive cytotoxic chemotherapy including autologous stem cell transplantation and more recently immunotherapy, high risk neuroblastoma has only a 30 percent predicted survival rate. Reovirus, a double-stranded RNA virus, has been shown to be effective against a myriad of cancers through its ability to preferentially lyse cancer cells with aberrant Ras pathway signaling. In this study, we investigate the potential of reovirus (serotype 3, strain Dearing) as a novel treatment for neuroblastoma and neuroblastoma tumour initiating cells (nbTIC). Experimental Design/Results: Reovirus induces dramatic cytotoxic effects at a multiplicity of infection (MOI) of 40 within 48h as assessed by WST-1 viability assays for the IMR-32, IMR-5, SK-N-AS, SK-N-SH, LAN-1, LAN-5, and SHEP human neuroblastoma cell lines in vitro. Interestingly the human neuroblastoma tumor-initiating cell lines, NB-12, NB88 and NB122 (kind gift from Dr David Kaplan) were also found to be very sensitive to reovirus-induced cytotoxicity, suggesting a role for reovirus treatment of refractory neuroblastoma. In order to further study reovirus, immunotherapy and neuroblastoma, a syngeneic, immunocompetent murine model (Neuro2a, A/J neuroblastoma model) was utilized to test reovirus-induced cytotoxicity and RV-directed immunotherapy of Neuro2A in vitro and in vivo. Preliminary data suggest that the Neuro2a cells are exquisitely sensitive to reovirus in vitro. Updated results will be presented. Conclusion: These preclinical results suggest that reovirus holds promise as a novel therapeutic for neuroblastoma and that it warrants further investigation in early phase clinical trials. 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 4336. doi:10.1158/1538-7445.AM2011-4336

Abstract LB-366: Patient-derived EBV-immortalized B-lymphocytes are a dominant contaminant of in vitro cultured human neuroblastoma tumor-initiating cells isolated from bone marrow

Abstract Neuroblastoma is one of the most common solid tumors of infancy and is believed to originate from immature sympathetic nervous system progenitor cells. Tumor stem cells from different cancer forms have during the last decade been isolated and propagated in vitro in stem cell-promoting medium. These cells have given the opportunity to study the population within the tumor that is believed to be responsible for relapse and metastatic disease. Three years ago, putative neuroblastoma tumor-initiating cells (TICs) were isolated from bone marrow of patients with high stage disease. Several findings concerning these cells have been difficult to explain in a neuroblastoma context, i.e. that initially only cells from MYCN non-amplified tumors could be established as proliferating cultures in vitro, that they have a B-cell phenotype with VDJ rearrangement as presented at the 2010 Advances in Neuroblastoma Research (ANR) meeting, and as presented here, that they lack genomic imbalances and fail to recapitulate neuroblastoma morphology when xenografted. We show that these widely studied patient-derived TIC cultures (NB12, NB88 and NB122R), also at low passages, are highly contaminated by proliferating cells of a B-lymphocyte phenotype, with the absolute majority of cells expressing multiple B-cell antigens while lacking expression of neuroblastoma-associated markers. We further show that these lymphoblastoid cells are Epstein-Barr virus (EBV)-infected with robust expression of the two EBV nuclear antigens EBNA2 and EBNA3A. In addition, orthotopic and subcutaneous TIC-derived xenograft tumors all have EBNA2 and B-cell marker CD19 positive cells at a high frequency. Published and unpublished results based on the use of the proposed TIC lines as a model for neuroblastoma development should be considered with caution, as these lines, at least with time in culture, mainly consist of EBV-transformed B-lymphoblastoid cells. 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 LB-366. doi:10.1158/1538-7445.AM2011-LB-366

Abstract 4344: Membrane-type 1 matrix metalloproteinase-mediated pro-invasive properties of neuroblastoma initiating cells

Abstract Introduction. Neuroblastoma is the most common and deadly extracranial solid tumour of childhood. Unlike localized neuroblastomas which have generally good prognosis, metastatic stages are associated with poor outcome. In this context, increasing evidences suggest that neuroblastoma may contain tumor-initiating cells (TICs) that cause relapse and metastasis. Our recent work indicates that CD133, a putative marker of TICs in various cancers, is associated with chemoresistance and poor outcome in neuroblastoma. In addition, we and others have demonstrated that membrane-type 1 matrix metalloproteinase (MT1-MMP) is associated with neuroblastoma progression and metastasis. Purpose. The purpose of this study was to characterize the tumor-initiating properties of CD133high neuroblastoma cells and determinate the role of MT1-MMP in pro-invasive properties of these cells. Methods/Results. Following CD133high cells selection from neuroblastoma cell lines (SK-N-DZ, SK-N-FI, SK-N-SH and SJ-NB10) with FACSAria cell sorter, we have examined their tumor-initiating features with neurosphere-forming assay in serum-free medium, colony-forming assay in soft agar and orthotopic transplantation into the adrenal gland (major site of primary neuroblastoma) of severe immunocompromised NOD/SCID/IL2Rγ- mice (NSG). Contrary to their counterpart CD133low, CD133high cells formed more colonies in soft agar and develop more neurospheres. Interestingly, orthotopic transplantation of as few as 500 CD133high cells into NSG mice resulted in tumor formation into the adrenal gland and dissemination to liver, lungs, brain and bone marrow, while matched control CD133low presented no tumor, suggesting that CD133high cells are TICs in neuroblastoma. Further western blot analyses of neuroblastoma cell lines revealed that CD133 expression correlated with that of MT1-MMP. In addition, CD133high cells growth within three-dimensional type I collagen matrices was markedly higher than CD133low cells and was significantly reduced by anti-MT1-MMP neutralizing antibodies. In the same vein, we performed cell migration assay in Boyden chamber and observed that CD133high cells exhibit more migratory abilities that CD133low cells, this migration being inhibited by anti-MT1-MMP neutralizing antibodies. Conclusions. Altogether, these findings strongly suggest that CD133-expressing TICs of neuroblastoma exhibit high pro-invasive capacities, requiring the involvement of MT1-MMP. Targeting pro-invasive capacities of neuroblastoma TICs with MMPs-activaed pro-drugs in combination with conventional therapy, represents an attractive therapeutic strategy aimed at eliminating both neuroblastoma TICs and the bulk of the tumour. Acknowledgment. Fondation Centre de cancérologie Charles-Bruneau. 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 4344. doi:10.1158/1538-7445.AM2011-4344

Neuroblastoma tumors have a hierarchy of tumor initiating cells reflecting neural crest development

A number of different tumors have been shown to contain subsets of cells that have tumor forming capability, known as tumor initiating cells (TICs). We have established neuroblastoma TICs (NTIC) from primary neuroblastoma tumors and from bone marrow metastases. These cell lines genetically resemble the tumor of origin. A number of these NTIC cell lines express markers of committed neuroendocrine sympathoadrenal cells such as TH and DBH. We found that other cell lines are partially or completely devoid of these neuroendocrine markers and instead show expression of markers of the early neural crest including NESTIN, SOX10/SOX9 (SOX-E proteins), CD24, CD133 and NOTCH. Some combinations of the markers are reflective of cells of the pre-migratory neural crest. Our data suggests that the developmental hierarchy of the neural crest is retained in the tumor. We will present data on the developmental classification, the developmental plasticity and response of the NTICs to external differentiation clues.

Correction: Systems-Level Analysis of Neuroblastoma Tumor-Initiating Cells Implicates AURKB as a Novel Drug Target for Neuroblastoma

In this article (Clin Cancer Res 2010;16:4572–82), which was published in the September 15, 2010, issue of Clinical Cancer Research (1), there was an error in Table 1. The data in the Description column for the NB121 row should read: Bone marrow metastasis, diagnosis.

System-Level Analysis of Neuroblastoma Tumor–Initiating Cells Implicates AURKB as a Novel Drug Target for Neuroblastoma

Neuroblastoma (NB) is an aggressive tumor of the developing peripheral nervous system that remains difficult to cure in the advanced stages. The poor prognosis for high-risk NB patients is associated with common disease recurrences that fail to respond to available therapies. NB tumor-initiating cells (TICs), isolated from metastases and primary tumors, may escape treatment and contribute to tumor relapse. New therapies that target the TICs may therefore prevent or treat tumor recurrences. We undertook a system-level characterization of NB TICs to identify potential drug targets against recurrent NB. We used next-generation RNA sequencing and/or human exon arrays to profile the transcriptomes of 11 NB TIC lines from six NB patients, revealing genes that are highly expressed in the TICs compared with normal neural crest-like cells and unrelated cancer tissues. We used gel-free two-dimensional liquid chromatography coupled to shotgun tandem mass spectrometry to confirm the presence of proteins corresponding to the most abundant TIC-enriched transcripts, thereby providing validation to the gene expression result. Our study revealed that genes in the BRCA1 signaling pathway are frequently misexpressed in NB TICs and implicated Aurora B kinase as a potential drug target for NB therapy. Treatment with a selective AURKB inhibitor was cytotoxic to NB TICs but not to the normal neural crest-like cells. This work provides the first high-resolution system-level analysis of the transcriptomes of 11 primary human NB TICs and identifies a set of candidate NB TIC-enriched transcripts for further development as therapeutic targets.

HIF-2α maintains an undifferentiated state in neural crest-like human neuroblastoma tumor-initiating cells

High hypoxia-inducible factor-2alpha (HIF-2alpha) protein levels predict poor outcome in neuroblastoma, and hypoxia dedifferentiates cultured neuroblastoma cells toward a neural crest-like phenotype. Here, we identify HIF-2alpha as a marker of normoxic neural crest-like neuroblastoma tumor-initiating/stem cells (TICs) isolated from patient bone marrows. Knockdown of HIF-2alpha 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-2alpha-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-2alpha maintains an undifferentiated state of neuroblastoma TICs. Because low differentiation is associated with poor outcome and angiogenesis is crucial for tumor growth, HIF-2alpha is an attractive target for neuroblastoma therapy.