ACSVL3 knockdown decreased GBM neurosphere cell growth and tumor initiation capacity of GBM neurosphere cells. A. GBM neurosphere cells (HSR-GBM1B and GBM-DM14602) were transiently transfected with ACSVL3 siRNAs for 72 hours and cultured for 5–7 days. Neurosphere cell growth was determined by counting total cell number in cultures. Compared to control, there was a ∼ 45-55% and ∼37-45% cell number decrease in HSR-GBM1B and GBM-DM cells receiving ACSVL3 siRNAs, respectively. B. GBM neurosphere cells were transiently transfected with ACSVL3 siRNAs and cultured continuously for 14 days in neurosphere medium. Neurospheres were immobilized in agar and the number of neurospheres measuring bigger than 100 μm in diameter per low powered microscopic field was counted by computer-assisted morphometry MCID. ACSVL3 siRNA significantly inhibited neurosphere-forming ability of GBM neurosphere cells. C. Control or ACSVL3 knockdown GBM neurosphere cells were cultured in soft agar for 14 days before quantifying neurospheres number and size with MCID. ACSVL3 down-regulation significantly decreased clonogenicity of GBM neurosphere cells in soft agar. D. GBM1B neurosphere cells were incubated with scrambled siRNA and ACSVL3 siRNA3 for 48 h followed by serial dilution neurosphere assay. After counting live cells with trypan blue exclusion, single suspension neurosphere cells were plated at 25, 50 and 100 cells per plate into 48wells/plates. Wells containing neurospheres were counted after 3 days. E. ACSVL3 knockdown reduced tumor propagation of GBM stem cell enriched neurospheres. HSR-GBM1A or HSR-GBM1B cells were transfected with scrambled siRNA (con) or ACSVL3 siRNA for 3 days in vitro. Equal numbers of viable cells (1× 104) were implanted into the caudate/putamen region of mouse brains (n = 10). After 10 weeks, the mice were sacrificed. Histological analysis (H & E staining) revealed that all the animals receiving control transfected cells developed intracranial tumors. In animals receiving ACSVL3 knockdown GBM neurosphere cells, only 40-50% of them developed detectable tumors.