BMC Cancer 2008, 8:30 http://www.biomedcentral.com/1471-2407/8/30 addition, SH-SY5Y cells showed a slight reduction in migratory capability whereas invasiveness was signifi- cantly reduced after 3 days of RA treatment [24]. In this regard, it is important to underline that our results on SK- N-SH motility have been obtained by scratching the cell monolayer after 6 days RA treatment, when DCX expres- sion reaches the lowest values. We have also observed that after RA treatment the cell motility is retained by the cells expressing both DCX and LIS1 but not by those cells expressing LIS1 alone. Interestingly, we found that DCX and VIM signals may colocalize in the same N-type cell during short exposure to RA; however, after prolonged exposure, differentiated N-type cells loose DCX immunoreactivity and acquire a VIM positive phenotype. Although this effect is clearly evi- dent by IF analysis, WB quantitation performed on the whole SK-N-SH cell population does not show VIM increase. This might probably be due to the concomitant slight decrease of VIM expression in S-type cells as observed by IF. Actually, using a NB cell line deprived of S-type cells, other authors have documented a significant increase of VIM expression after RA treatment [4]. Given the fact that motility and invasiveness are characteristics of DCX+/VIM- cells, it is possible to postulate that VIM could be a marker not only of S-type cells [2,3], but also of those N-type cells that assume a DCX-/VIM+ phenotype in response to RA treatment. This hypothesis is supported by the proposed role of VIM for axonal elongation in dif- ferentiating NB cells [33]. Finally, we have also observed that after RA treatment all the three I-cell subtypes (DCX-/VIM-; DCX+/VIM-; DCX-/ VIM+) are still present. The presence of DCX+ cells might indicate a resistance of these cells to RA treatment or the existence of a subpopulation of cells transiently express- ing this protein during their differentiation process. Actu- ally, it has been demonstrated that during adult hippocampal neurogenesis DCX is not expressed by stem cells, but only by neural progenitor cells and disappears when progenitor cells differentiate to mature neurons [34]. We may therefore suppose that RA could induce the differentiation of DCX-/VIM- I-type cells toward a DCX-/ VIM+ phenotype through an intermediate phase character- ized by DCX expression. Further studies will be addressed to specifically test this hypothesis. Besides the observation that DCX is a marker for the min- imal residual disease in human neuroblastoma [6], our results provides the new evidence that this protein is not present in all cell subtypes composing SK-N-SH cell line but it is express only in the more aggressive neuroblast- oma cell subpopulation and in the retinoic acid-resistant cells that possess high motility and invasiveness. Conclusion We report the first demonstration that DCX is differen- tially expressed only in some (N and I) cell types compos- ing SK-N-SH human NB cell-line. All the DCX+ cells show high migratory capability and invasiveness; moreover, we show that RA treatment induces a significant reduction of these parameters and down-regulates DCX expression. RA is unable to completely block DCX expression in all the cells, and the few DCX+ cells that are still present after the treatment show high motility and invasiveness. We found that the some DCX+ RA-resistant cells have the morphological features of I-type cells, and since I-type cells are indicated as the more undifferentiated and aggressive cellular component of human NB, the presence of these cells after the treatment might support the emerg- ing concept about the existence of cancer stem cells liable to tumor relapse [35]. This last hypothesis is presently under investigation. Irrespective of a direct role of DCX in NB cell motility, the present data suggest that the detection of DCX+ cells in untreated and treated tumors in neuroblastoma affected patients may be prognostic of tumor aggressiveness, as well as of a severe tumor relapse after treatment. Abbreviations NB: Neuroblastoma; DCX: Doublecortin; LIS1: Lissen- cephaly-1; VIM: Vimentin; NF-68: Neurofilaments-68; IF: Immunofluorescence; PC: Phase contrast; CIP: Calf intes- tine alkaline phosphatase; WB: Western blot; RA: Retinoic Acid. Competing interests The author(s) declare that they have no competing inter- ests. Authors' contributions EM and MCF carrier out all the experiments. CC per- formed sequence analysis of the DCX gene. MZ and RM were responsible for the progress of the work. All contrib- utors participated in the conception and design of the study and have been involved in the writing of the manu- script. Acknowledgements We thank our friend Athina Samara for helping us with her wise sugges- tions, and Franco Taroni (Istituto Neurologico Carlo Besta, Milan) for the support for DCX gene analysis. This work was supported by Italian Ministry for Research, FIRB: RBAU01FXRT, and PRIN2005 # 2005051740. Page 11 of 12 (page number not for citation purposes)