Neuroblastoma and tumor cell differentiation There are few tumor forms that present such a tight link between clinical behavior and tumor cell differ- entiation stage as the childhood cancer neuroblas- toma. Being derived from sympathetic nervous system precursor cells or immature neuroblasts, neu- roblastoma cells are arrested at varying stages of differentiation, and, based on histopathology and degree of morphological differentiation, three tumor variants have been deﬁned: 1) the benign ganglio- neuromas exclusively containing ganglion-like cells and stroma, 2) ganglioneuroblastomas containing neuroblast-like immature cells with small nuclei and scant cytoplasms intermixed with a smaller or larger proportion of more differentiated tumor cells with larger nucleus and cytoplasm, and 3) neuroblas- toma proper with neuroblast-like cells and lacking tumor cells that show apparent signs of morphological ganglionic differentiation. More intriguing, neuro- blastomas show one of the highest rates of spontane- ous differentiation, a phenomenon ﬁrst reported in 1927 by Cushing and Wolbach who described a child with a sympaticoblastoma (later termed neuroblas- toma) that spontaneously developed into a more differentiated, non-aggressive tumor with a differen- tiated sympathetic phenotype (3). At the molecular level, the association between high expression of sym- pathetic ganglionic marker genes and favorable dis- ease was established 80 years later based on global gene expression analyses (4). Through the pioneering work by June Biedler, Robert Seeger, and others, neuroblastoma was one of the ﬁrst human tumors to be established in culture, and some of these cell lines were shown to have retained the capacity to differentiate along a ganglionic lineage in vitro in response to external stimuli such as phorbol esters and retinoic acid (5,6). These ﬁndings suggested that aggressive neuroblastomas might become treatable by inducing a differentiation response as part of the treatment protocol. Today 13-cis-retinoic acid at pharmacological levels is used as adjuvant treatment following myeloablative therapy; whether the observed clinical effects are related to retinoic acid- induced changes in the stage of tumor cell differen- tiation has not been established. Neuroblastoma and phenotypic heterogeneity In a subset of neuroblastomas, tumor cells are orga- nized in lobular structures with zones of necrotic cells in the lobule center (7). In these lobules a neuronal- to-neuroendocrine lineage conversion occurs with the neuroendocrine cells located adjacent to the necrosis (8). The sympathetic nervous system lineage markers Differentiation and cancer 219 used in these studies could not distinguish between sympathetic paraganglionic and sympathetic SIF (small intensely ﬂuorescent) phenotypes. Hence, the exact nature of the neuroendocrine cells detected in lobular neuroblastomas has not been established, although the adrenal chromafﬁn marker gene PNMT is not expressed, ruling out a neuroblastoma-to- pheochromocytoma conversion pathway. These old data exemplify the frequently occurring intra-tumoral phenotypic heterogeneity in solid cancers and do suggest that such heterogeneity is not exclusively a result of mosaicism of genetic aberrations. Our data further imply the existence of intra-tumoral mechanisms regulating the differentiation stage of tumor cells. Hypoxia promotes an immature, stem cell-like neuroblastoma phenotype Concurrently with our report that peri-necrotic neu- roblastoma cells differ in phenotype compared to cells located closer to the ﬁbrovascular stroma, the corre- lation between tumor hypoxia and aggressive disease was demonstrated (9), and the molecular basis for cellular adaptation to hypoxia began to be resolved. Semenza and co-workers identiﬁed hypoxia inducible factor HIF-1 (10), which together with HIF-2 is the central transcription factor governing cellular adap- tation to hypoxia (11). These dimeric transcription factors have a b-subunit (also called ARNT) in com- mon and an oxygen-sensitive a-subunit unique for HIF-1 and HIF-2, respectively. At hypoxia the a-subunits become stabilized, transported into the nucleus to dimerize with ARNT, and by additional mechanisms activated to induce the transcription of a number of hypoxia-driven genes (12). Based on the potential impact by which the HIF transcription fac- tors can affect the tumor cell phenotype, we asked if hypoxic cells of established neuroblastoma cell lines gained a neuroendocrine phenotype similar to that of peri-necrotic, lobular, neuroblastoma cells. As reported, none of the tested cell lines showed a hypoxia-induced neuronal-to-neuroendocrine lineage shift, but instead hypoxia promoted an immature, neural crest-like phenotype (13,14). As classical neu- roblastoma cell lines are all derived from high-stage, most often MYCN-ampliﬁed tumors, and not tumors of the lobular type described above, our current hypothesis is that we did not use the adequate cells for testing in vitro the capacity of lobular neuroblas- toma cells to convert into a neuroendocrine cell under hypoxic conditions. The important outcome of these experiments was the observation that hypoxia can dedifferentiate tumor cells, a ﬁnding now corrobo- rated in many different tumor forms (15), including