Figure 2.
OTX2 knockdown decreases tumor growth and tumor‐initiating capacity in vivo and increases levels of neuronal differentiation and axon guidance genes. (A) Immunoblot validation of stable OTX2 knockdown in D283 cells using two shRNA sequences relative to scramble control. β‐Actin serves as a loading control. (B) Representative images of tumors derived from D283 scramble or D283 OTX2 knockdown cells following injection into the cerebellum of NOD SCID mice. Scale bar: 1000 μm. Arrows denote intracerebellar tumors from each. (C) Quantification of tumor area following intracerebellar injection of 2 × 105 D283 scramble (N = 8) or OTX2 knockdown (N = 7) tumorsphere cells. Error bars: SEM. P < 0.05*. (D) Limiting dilution analysis of tumors derived from D283 scramble relative to D283 OTX2 knockdown tumorspheres following intracerebellar injection. OTX2 knockdown cells exhibit a decrease in tumor‐initiating capacity as indicated by the lower number of animals displaying evidence of tumor growth. Scale bar: 1000 μm. (E) Representative images of Ki67 staining in tumors derived from D283 scramble or D283 OTX2 knockdown cells following injection into NOD SCID mice. Scale bar: 50 μm. (F,G) IPA analysis showing major pathways (F) affected by OTX2 knockdown in D283 tumorspheres and the frequency of genes within these pathways (G) that are upregulated (black) and downregulated (gray). (H) Gene set enrichment analysis (GSEA) demonstrating enrichment of genes associated with cell cycle in the control scramble D283 tumorspheres. (I) GSEA demonstrating that neuronal differentiation and axon guidance genes are enriched in genes sets that are downregulated in the scramble and upregulated in the OTX2 KD D283 tumorspheres. (J) Representative heat map of genes that are significantly downregulated (blue) and upregulated (red) following OTX2 knockdown in D283 tumorspheres. Note that the majority of genes are upregulated following OTX2 knockdown.