Hypoxia and Stem Cells in Normal and Tumor Development

Sammanfattning: Neuroblastoma is a childhood malignancy of the sympathetic nervous system and accounts for 15% of all cancer-related deaths in children. To understand the biology behind neuroblastoma, clarify timing of cellular events and expression profiles of proteins preceding neuroblastoma initiation, we need more relevant preclinical models.In the first part of this thesis, we established two preclinical in vitro models of neuroblastoma: patient-derived xenograft (PDX) cells derived from an orthotopic PDX model of high-risk neuroblastoma and chick embryo derived trunk crestospheres. Wedemonstrate that PDX cells can be cultured as spheres in stem cell promoting medium with retained patient tumor characteristics and maintained tumorigenic and metastatic capacity. However, addition of serum to the culture media resulted in loss of theirimmature phenotype and induced neuronal differentiation, while adherent culture on laminin maintained cells in an undifferentiated state. Further, we also isolated and optimized culture conditions for chick embryo derived trunk crestospheres,comprised of both neural crest stem and progenitor cells. We demonstrate that these crestospheres are multipotent, display selfrenewal capacity over several weeks in vitro and can be manipulated via lentiviral transduction.In the second part of this thesis, we first demonstrate that immature mesenchymal-type neuroblastoma cells are resistant to retinoic acid (RA), a differentiating agent used as a component for treatment of high-risk neuroblastoma. We further demonstrate that mesenchymal-type neuroblastoma cells had endogenous synthesis of RA, is dependent on RA for their proliferation and migration and clustered closely with normal peripheral glia stem cells called Schwann cell precursors (SCPs). Together these data indicate that the endogenous dependency on RA in mesenchymal-type neuroblastoma cells might play a role in the acquired resistance towards RA treatment in the clinic. We finally demonstrate that hypoxia-inducible factor (HIF)-2α, a transcription factor involved in cellular adaption to low oxygen levels, is highly expressed at oxygenated conditions both in vitro an in vivo in neuroblastoma, particularly in the cytoplasmic fraction. We further show that treatment with the HIF-2α transcriptional inhibitor PT2385 had no effects on HIF-2 downstream targets, in contrast to HIF-2α protein knockdown, suggesting that HIF-2α possesses additional, non-canonical functions in neuroblastoma.