Ludivine Coudière Morrison

In vivo generation of neural tumors from neoplastic pluripotent stem cells models early human pediatric brain tumor formation.

Recent studies have identified gene signatures in malignant tumors that are associated with human embryonic stem cells, suggesting a molecular relationship between aggressive cancers and pluripotency. Here, we characterize neural precursors (NPs) derived from transformed human embryonic stem cells (N‐t‐hESCs) that exhibit neoplastic features of human brain tumors. NPs derived from t‐hESCs have enhanced cell proliferation and an inability to mature toward the astrocytic lineage, compared with progeny derived from normal human embryonic stem cells (N‐hESCs) independent of adherent or neurosphere outgrowth. Intracranial transplantation of NPs derived from N‐t‐hESCs and N‐hESCs into NOD SCID mice revealed development of neuroectoderm tumors exclusively from the N‐t‐hESCs NPs and not from normal N‐hESCs. These tumors infiltrated the ventricles and the cerebellum of recipient mice and displayed morphological, phenotypic, and molecular features associated with classic medulloblastoma including retention of a pluripotent signature. Importantly, N‐t‐hESCs did not exhibit cytogenetic changes associated with medulloblastoma, suggesting that aberrant cellular and molecular properties precede the acquisition of karyotypic changes thus underscoring the value of this model system of human medulloblastoma. Our study demonstrates that NPs from a starting population of neoplastic human pluripotent parent cells possess brain tumor‐initiating cell capacity, thereby providing a model system to investigate initiation and progression of primitive human neural cancers that are difficult to assess using somatic sources. STEM CELLS 2012;30:392–404

Differential cellular responses induced by dorsomorphin and LDN-193189 in chemotherapy-sensitive and chemotherapy-resistant human epithelial ovarian cancer cells

Inherent or acquired drug resistance is a major contributor to epithelial ovarian cancer (EOC) mortality. Novel drugs or drug combinations that produce EOC cell death or resensitize drug resistant cells to standard chemotherapy may improve patient treatment. After conducting drug tolerability studies for the multikinase inhibitors dorsomorphin (DM) and it is structural analogue LDN‐193189 (LDN), these drugs were tested in a mouse intraperitoneal xenograft model of EOC. DM significantly increased survival, whereas LDN showed a trend toward increased survival. In vitro experiments using cisplatin (CP)‐resistant EOC cell lines, A2780‐cp or SKOV3, we determined that pretreatment or cotreatment with DM or LDN resensitized cells to the killing effect of CP or carboplatin (CB). DM was capable of blocking EOC cell cycle and migration, whereas LDN produced a less pronounced effect on cell cycle and no effect on migration. Subsequent analyses using primary human EOC cell samples or additional established EOC cells lines showed that DM or LDN induced a dose‐dependent autophagic or cell death response, respectively. DM induced a characteristic morphological change with the appearance of numerous LC3B‐containing acidic vacuoles and an increase in LC3BII levels. This was coincident with a decrease in cell growth and the altered cell cycle consistent with DM‐induced cytostasis. By contrast, LDN produced a caspase 3‐independent, reactive oxygen species‐dependent cell death. Overall, DM and LDN possess drug characteristics suitable for adjuvant agents used to treat chemotherapy‐sensitive and ‐resistant EOC.

Characterization of novel biomarkers in selecting for subtype specific medulloblastoma phenotypes.

Major research efforts have focused on defining cell surface marker profiles for characterization and selection of brain tumor stem/progenitor cells. Medulloblastoma is the most common primary malignant pediatric brain cancer and consists of 4 molecular subgroups: WNT, SHH, Group 3 and Group 4. Given the heterogeneity within and between medulloblastoma variants, surface marker profiles may be subtype-specific. Here, we employed a high throughput flow cytometry screen to identify differentially expressed cell surface markers in self-renewing vs. non-self-renewing SHH medulloblastoma cells. The top 25 markers were reduced to 4, CD271/p75NTR/NGFR, CD106/VCAM1, EGFR and CD171/NCAM-L1, by evaluating transcript levels in SHH tumors relative to samples representing the other variants. However, only CD271/p75NTR/NGFR and CD171/NCAM-L1 maintain differential expression between variants at the protein level. Functional characterization of CD271, a low affinity neurotrophin receptor, in cell lines and primary cultures suggested that CD271 selects for lower self-renewing progenitors or stem cells. Moreover, CD271 levels were negatively correlated with expression of SHH pathway genes. Our study reveals a novel role for CD271 in SHH medulloblastoma and suggests that targeting CD271 pathways could lead to the design of more selective therapies that lessen the broad impact of current treatments on developing nervous systems.

OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells

ABSTRACT Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into four subtypes based on different genomic alterations, gene expression profiles and response to treatment: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example, expression of the transcription factor Orthodenticle homeobox2 (OTX2) is frequently dysregulated in multiple MB variants; however, its role may be subtype specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs), but not their normal counterparts (hENs), resemble Groups 3 and 4 MB in vitro and in vivo. Here, we tested the utility of this model system as a means of dissecting the role of OTX2 in MB using gain- and loss-of-function studies in hENs and trans-hENs, respectively. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and SHH MB cells by regulating growth, self-renewal and migration in vitro and tumor growth in vivo. This was accompanied by decreased expression of pluripotent genes, such as SOX2, and was supported by overexpression of SOX2 in OTX2+ SHH MB and hENs that resulted in significant rescue of self-renewal and cell migration. By contrast, OTX2 is oncogenic and promotes self-renewal of trans-hENs and Groups 3 and 4 MB independent of pluripotent gene expression. Our results demonstrate a novel role for OTX2 in self-renewal and migration of hENs and MB cells and reveal a cell-context-dependent link between OTX2 and pluripotent genes. Our study underscores the value of human embryonic stem cell derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression. Summary: Human embryonic stem cell neural derivatives can be used to model the molecular and cellular properties of medulloblastoma.

Embryonic Stem Cell Models of Human Brain Tumors

Utilization of human embryonic stem cells (hESCs) as a model system to study highly malignant pediatric cancers has led to significant insight into the molecular mechanisms governing tumor progression and has revealed novel therapeutic targets for these devastating diseases. Here, we describe a method for generating heterogeneous populations of neural precursors from both normal and neoplastic hESCs and the subsequent injection of neoplastic human embryonic neural cells (hENs) into intracerebellar or intracranial xenograft models. Histopathologically, neural tumors derived from neoplastic hENs exhibit features similar to more aggressive medulloblastoma, the most common malignant primary pediatric brain tumor. In this chapter, we will outline the detailed methods for culturing normal and neoplastic neural precursor cells in both adherent and tumorsphere format and the full characterization of the brain tumors generated from these cells in non-obese diabetic severe combined immunodeficiency (NOD SCID) mice.

Characterization of a novel OTX2‐driven stem cell program in Group 3 and Group 4 medulloblastoma

Medulloblastoma (MB) is the most common malignant primary pediatric brain cancer. Among the most aggressive subtypes, Group 3 and Group 4 originate from stem/progenitor cells, frequently metastasize, and often display the worst prognosis, yet we know the least about the molecular mechanisms driving their progression. Here, we show that the transcription factor orthodenticle homeobox 2 (OTX2) promotes self‐renewal while inhibiting differentiation in vitro and increases tumor initiation from MB stem/progenitor cells in vivo. To determine how OTX2 contributes to these processes, we employed complementary bioinformatic approaches to characterize the OTX2 regulatory network and identified novel relationships between OTX2 and genes associated with neuronal differentiation and axon guidance signaling in Group 3 and Group 4 MB stem/progenitor cells. In particular, OTX2 levels were negatively correlated with semaphorin (SEMA) signaling, as expression of 9 SEMA pathway genes is upregulated following OTX2 knockdown with some being potential direct OTX2 targets. Importantly, this negative correlation was also observed in patient samples, with lower expression of SEMA4D associated with poor outcome specifically in Group 4 tumors. Functional proof‐of‐principle studies demonstrated that increased levels of select SEMA pathway genes are associated with decreased self‐renewal and growth in vitro and in vivo and that RHO signaling, known to mediate the effects of SEMA genes, is contributing to the OTX2 KD phenotype. Our study provides mechanistic insight into the networks controlled by OTX2 in MB stem/progenitor cells and reveals novel roles for axon guidance genes and their downstream effectors as putative tumor suppressors in MB.

Novel glycolipid agents for killing cisplatin-resistant human epithelial ovarian cancer cells

BackgroundChemotherapy resistance is one of the major factors contributing to mortality from human epithelial ovarian cancer (EOC). Identifying drugs that can effectively kill chemotherapy-resistant EOC cells would be a major advance in reducing mortality. Glycosylated antitumour ether lipids (GAELs) are synthetic glycolipids that are cytotoxic to a wide range of cancer cells. They appear to induce cancer cell death in an apoptosis-independent manner.MethodsHerein, the effectiveness of two GAELs, GLN and MO-101, in killing chemotherapy-sensitive and –resistant EOC cells lines and primary cell samples was tested using monolayer, non-adherent aggregate, and non-adherent spheroid cultures.ResultsOur results show that EOC cells exhibit a differential sensitivity to the GAELs. Strikingly, both GAELs are capable of inducing EOC cell death in chemotherapy-sensitive and –resistant cells grown as monolayer or non-adherent cultures. Mechanistic studies provide evidence that apoptotic-cell death (caspase activation) contributes to, but is not completely responsible for, GAEL-induced cell killing in the A2780-cp EOC cell line, but not primary EOC cell samples.ConclusionsStudies using primary EOC cell samples supports previously published work showing a GAEL-induced caspase-independent mechanism of death. GAELs hold promise for development as novel compounds to combat EOC mortality due to chemotherapy resistance.

Neural derivatives from human embryonic stem cells: modelling early cellular and molecular events contributing to Depediatric brain tumorigenesis

Suppl 2 – S5 Understanding the molecular mechanism of invasion could help in designing novel therapeutic approaches in order to prevent the need for repeat surgery, decrease morbidity and improve patient survival. The aim of this study was to identify the key factors and underlying mechanisms which govern invasive properties of meningiomas. Methods: Towards this end, we performed gene expression profiling of invasive and non-invasive meningioma tumors. Matrix metalloproteinases (MMPs) 16 and 19 were among the genes associated with cell movement and invasion. Results & Discussion: We establish that the expression level of MMP16 was significantly elevated in both bone-invasive and brain invasive meningiomas. Gainand loss-of-function experiments indicated a role for MMP16 in meningioma cell movement, invasion and tumor cell growth. Furthermore, MMP16 was shown to positively regulate MMP2, suggesting this mechanism may modulate meningioma invasion in invasive meningiomas. Conclusions: Overall, the results support a role for MMP16 in promoting invasive properties of the meningioma tumors. Further studies to explore the potential value for clinical use of matrix metalloproteinases inhibitors, perhaps specifically targeting MMP16 are warranted.