Study of the phenotypic relationship in the IMR-32 human neuroblastoma cell line by Sedimentation Field Flow Fractionation

Abstract

Abstract. Neuroblastoma (NB) is the most common childhood solid tumor. Although spontaneous regression can occur in patients <1-year old, 70% of patients over the age of 1 year have a high-risk and difficult-to-treat NB. Cell type heterogeneity is observed either in the morphological appearance of NB tumors or in cell lines isolated from tumor specimens. NB consists of two principal neoplastic cell types: i) neuroblastic or N-type (undifferentiated cells); and ii) stromal or S-type (differentiated cells). As NB cells seem to have the capacity to differentiate spontaneously in vivo and in vitro, their heterogeneity could affect treatment outcome, in particular the response to apoptosis induced by chemotherapy. Therefore, it is important to understand the underlying process governing changes in differentiation in order to improve treatment response and NB patient outcome and the neoplastic population in IMR-32 represented a good model for such a study. Results showed that this cell line was extremely heterogeneous and highly variable in its stage of differentiation and we demonstrated that sedimentation field flow fractionation (SdFFF) permitted the isolation of 2 N-phenotypes and contributed to the understanding of the IMR-32 cell population dynamics. The first N-phenotype forms a pool of quiescent undifferentiated cells while the second one was able to proliferate (incorporation of BrdU) and also give rise to adherent S-type cells (PSA-N-CAM + and N-CAM + ). The results could also suggest a close interaction between these different cellular phenotypes to create the IMR-32 cell lineage.

Cancer stem cells from human glioma cell line are resistant to Fas-induced apoptosis

“…A previous study on the neuroblastoma cell line IMR-32 (26) showed that SdFFF could separate two phenotypes from the same cell lineage: the first N-phenotype forming a pool of quiescent undifferentiated cells and the second able to proliferate and to give rise also adherent S-type cells (26). In this way, we demonstrated that SdFFF could separate two phenotypes from U-87 MG: large adherent cells in PF1 and small sphere cells in PF3.…”

“…4), leading, as for neuroblastoma cell line (26), to complex fractograms (Fig. 3) in contrast to many other cell lines (22,27,42,43).…”

“…According to results concerning: i) the particle elution away from the accumulation wall (s>r); ii) field dependence of the Robs value; and iii) the limited interactions between cells and accumulation wall leading to low reversible cellular release (RP , Fig. 3); we can suppose that U-87 MG cells were eluted under Hyperlayer elution mode as it has been demonstrated for other cell models (22,26,27,44,45).…”

“…Hyperlayer elution mode allowed, by limiting cell-accumulation wall interactions, a cell elution which: i) respected repeatability, reproducibility and recovery; ii) respected cell functional integrity; iii) provided high levels of short and long-term viability (no apoptosis induction); iv) respected the maturation and differentiation stages; and finally; v) enhanced better sub-population sorting and characterization (20,22,26,27).…”

“…SdFFF separation device used in this study derived from those previously described and schematized (26,27). The apparatus consists of two 938x40x2 mm polystyrene plates, separated by a mylar ® spacer in which the channel was curved.…”