Figure 7.

HDAC activity blockade downgrades GBM aggressiveness in synergy with the developing microenvironment. U87-MG oncopheres were cultured in the presence or absence of iHDACs (100 nM TSA) for 72 hours. After the xenograft, the embryos were allowed to develop until stage E5. In situ hybridization and immunostaining for HNK-1 revealed that when the oncospheres remained placed in the lumen of the developing neural tube, the outermost layer of cells expressed HNK-1 only in the iHDAC group: control oncospheres: (A) Alu in situ hybridization; (B) immunostaining for HNK-1 and iHDAC oncospheres: (C) Alu in situ hybridization; (D) immunostaining for HNK-1. The cells that were integrated into the embryonic mesenchyme were able to migrate and to associate with developing embryonic structures. In the control group, only embryonic cells expressed HNK-1 since Alu ⁺ nuclei did not colocalize with the HNK-1 (E–N, arrow heads). In iHDAC oncospheres, however, it was possible to observe the colocalization of Alu ⁺ nuclei and HNK-1 staining (G–P, arrow heads). In fact, control (Q, R) and iHDAC (S, T) treated oncospheres did not constitutively express HNK-1 since their placement in the developing neural tube lumen, followed immediately by fixation did not reveal HNK-1 staining in the oncospheres. Positive control for HNK-1 staining was done with migrating neural crest cells (Q–T, asterisks).