Knockdown of L1CAM in CD133+ glioma cells reduces self-renewal due to increased apoptosis. A, Knockdown of L1CAM expression in CD133+ cells isolated from the primary anaplastic astrocytoma T3565 and glioblastoma T3691 patient specimens was performed using L1CAM lentiviral shRNA (L1) without effects by non-targeting control shRNA (NT) as determined by Western blotting. B, Targeting L1CAM expression disrupted neurosphere formation of CD133+ brain tumor cells derived from a T3691 glioblastoma patient specimen or a D456MG pediatric glioblastoma xenograft. CD133+ cells infected with lentivirus expressing non-targeting shRNA (NT) formed neurospheres, whereas infection with lentivirus expressing L1CAM shRNA attenuated neurosphere formation. C, Representative images of CD133+ and CD133− brain tumor cells isolated from the primary glioblastoma T3691 and normal neural progenitors stained with Annexin V-FITC (green) and DAPI (blue) after treatment with lentivirus expressing non-targeting shRNA or L1CAM shRNA are shown. D, FACS analysis of apoptosis with Annexin V-FITC staining in CD133+ and CD133− brain tumor cells isolated from the primary glioblastoma T3691 and normal neural stem cells confirmed that CD133+ brain tumor cells have a greater dependence on L1CAM for cell survival. *, p < 0.01; **, p < 0.001 with comparison to non-targeting shRNA.

Lentiviral shRNA targeting of L1CAM suppresses tumor growth and increased survival of mice bearing intracranial brain tumor xenografts. A, Representative images of brains of immunocompromised mice implanted with CD133+ cells isolated from the glioblastoma specimen T3691 and infected with non-targeting shRNA (NT) or L1CAM shRNA prior to intracranial implantation are shown. 48 hours after infection, identical numbers of viable cells (10⁵ cells/mouse) were implanted and brains examined on day 17 after injection. Gross images and coronal sections from representative brains bearing glioma xenografts are displayed. B, Knockdown of L1CAM in CD133+ cells isolated from the glioblastoma patient specimen T3691 prior to intracranial implantation increased survival. * p < 0.002 with comparison to non-targeting control. CD133+ cells isolated from brain tumor patient specimens and infected with non-targeting shRNA (NT) or L1CAM shRNA prior to injection were implanted into the brains of immunocompromised mice as in (A). C, Representative images of coronal sections of brains of mice implanted with CD133+ brain tumor cells isolated from the T3837 glioblastoma patient specimen and then infected with lentivirus expressing non-targeting shRNA (NT) or L1CAM shRNA are shown. Identical numbers of CD133+ brain tumor cells (10⁵ cells/mouse) were implanted into mice brains to establish intracranial tumors. After 5 days to allow tumor engraftment, lentivirus expressing non-targeting shRNA (NT) or shRNA directed against L1CAM were delivered to the tumor site through direct injection (once every other day for 12 days). D, Targeting L1CAM in vivo through lentiviral mediated shRNA suppressed intracranial tumor growth of the established brain tumor xenograft and significantly increased the survival of mice bearing intracranial glioma xenografts (*, p < 0.004).

L1CAM is highly expressed on the surface of CD133+ glioma cells. A, Flow cytometry demonstrated most CD133+ glioma cells isolated from surgical biopsy specimens were also L1CAM+. Total population fractions of CD133+ cells and L1CAM+ cells were very similar. Unsorted tumor cells from primary brain tumor samples were labeled with anti-CD133-APC and anti-L1CAM-PE, and then subjected to FACS analysis to determine CD133+ and L1CAM+ subpopulations. The total fractions of CD133+ cells and L1CAM+ cells in unsorted tumor cells were very similar in each case and there was significant overlap between the populations. B, Immunofluorescent staining with L1CAM antibody demonstrated that L1CAM was overexpressed on the cell surface of CD133+ glioma stem cells in comparison to matched CD133− cells. C, CD133+ cells from glioma surgical biopsy specimens including the anaplastic astrocytoma T3565 and the glioblastomas T3691, T3750, and T3359 expressed L1CAM protein at higher levels than matched CD133− cells by Western blot analysis. D, CD133+ glioma cells from the glioblastoma patient tumor specimens T3832, T3691, and T3359 as well as the pediatric glioblastoma xenograft D456MG expressed L1CAM protein at higher levels than CD133+ normal neural progenitor cells (designated by lot number).

L1CAM regulates Olig2 and p21^WAF1/CIP1 expression to maintain CD133+ glioma cell growth and survival. A, Reduction of L1CAM down-regulated expression of Olig2 transcription factor specific in glioma stem cells (CD133+). i, L1CAM knockdown with lentiviral mediated shRNA reduced Olig2 expression in CD133+ glioma cells isolated from the anaplastic astrocytoma T3565 and glioblastoma T3691 surgical specimens as well as the pediatric glioblastoma xenograft D456MG. Whole cell lysates from CD133+ cells infected with lentivirus expressing no shRNA (VC), non-targeting shRNA (NT) or L1CAM-targeting shRNA (L1) for 48 hours were resolved by SDS-PAGE and immunoblotted for L1CAM, Olig2, and tubulin as a loading control. ii, Knockdown of L1CAM dramatically and specifically down-regulated Olig2 protein in CD133+ glioma cells isolated from the glioblastoma patient specimen T3982. Olig2 was not detectable in CD133− cells before or after treatment with L1CAM-targeting lentivirus (L1) in comparison to non-targeting shRNA (NT) for 48 hours. B, L1CAM knockdown in CD133+ cells isolated from the glioblastoma patient specimen T3982 up-regulated p21^WAF1/CIP1 but not p27^KIP1 protein in comparison to non-targeting control shRNA (NT). Cells were infected with the non-targeting or L1CAM-targeting shRNA lentivirus for the indicated time. C, Knockdown of L1CAM decreased Olig2 and increased p21^WAF1/CIP1 mRNA expression. RNA was purified from CD133+ cells isolated from the glioblastoma patient specimen T3982 infected with lentivirus encoding either non-targeting shRNA or L1CAM shRNA for 48 hours, and then reverse transcribed to cDNA used for Real Time PCR with sequences specific for L1CAM, Olig2, and p21 and results normalized to GAPDH levels. *, p < 0.01. D, Olig2 overexpression rescued CD133+ cells from L1CAM knockdown induced cell death. CD133+ cells isolated from the primary glioblastoma patient specimen T3982 were transfected with vector control or vector expressing Olig2 for 24 hours, and then infected with lentivirus expressing either non-targeting shRNA (NT) or L1CAM shRNA for 48 hours. L1CAM knockdown significantly reduced numbers of live cells when CD133+ cells were transfected with control vector (*, p < 0.01), but this effect was prevented by Olig2 overexpression (** , p < 0.001 with comparison to vector control with L1CAM shRNA).