D. Bashllari2, R. Van Noord2, A. Opipari2, E. Lawlor2, V. Castle2, E. Newman2 2University Of Michigan,Pediatrics, Obstetrics And Gynecology, And Pediatric Surgery,Ann Arbor, MI, USA
Introduction: Neuroblastoma (NB) is a developmental cancer that arises from neural crest stem cells (NCSC). The amplification of the transcription factor MYCN predicts poor prognosis and correlates with an undifferentiated cell phenotype. MYCN is expressed during normal NCSC development, regulating neural progenitor cell proliferation and differentiation at distinct time points. Our preliminary work has shown that deregulation of MYCN during normal human NCSC differentiation increases cell proliferation, motility, migration, and invasion in-vitro. To further determine how MYCN contributes to NB tumorigenesis, we generated a novel in-vivo human model to test our hypothesis that MYCN deregulation during early NCSC development is a critical component of tumor initiation.
Methods: Human embryonic stem cells (hESC) were plated on a mouse embryonic fibroblasts feeder layer and grown in stromal-derived inducing activity (SDIA) media to differentiate into NCSC. NCSC were collected using staining for p75 and HNK1+ neural markers by FACS. Double positive sorted cells were then transduced with a doxycycline inducible V5-tagged MYCN cDNA lentiviral construct. MYCN expression was confirmed by Western blot and immunocytochemistry (ICC). NCSC-MYCN+ cells in a 1:1 mix with matrigel were flank-injected into NOD/SCID mice, while NCSC-MYCN- cells were injected into the opposite flanks. Mice were fed doxycycline water to induce MYCN expression. Animals were monitored daily for health and tumor formation. Resultant tumors were excised, characterized, and examined for tumor markers using paraffin-embedded slides stained for H/E, MYCN, tyrosine hydroxylase (TH), Chromogranin A, and TrkB.
Results: NCSC-MYCN+ cells formed robust tumors at flank injection sites that were metastatic to the abdominal cavity, while NCSC-MYCN- flanks did not form tumors. Primary and metastatic tumors were locally invasive and morphologically consistent with poorly differentiated NB, with immature neuroecterdermal cells that palisaded around blood vessels, forming pseudorosettes and brisk mitotic rates. Resultant tumors stained positive for TH, Chromogranin A, TrkB, and MYCN. Sections of lymph nodes and abdominal spread sites were consistent with NB and stained positive for all markers as well.
Conclusion: Our results are progress towards establishing the first human model of NB using hESC and human NCSC, and further implicate MYCN in early NCSC tumor initiation. These data demonstrate that forced constitutive expression of MYCN is sufficient for NCSC tumor initiation in-vivo that is capable of metastatic spread, and pave the way for studies to determine how NB tumors develop.
