Neuroblastoma Stem Cells: Identifying Two New Classes of Drugs with in-Vitro Activity in Cell Populations with High Side-Population Profiles

Background: Neuroblastoma (NB) is a pediatric tumor of neural crest origin. It is one of several small round blue cell tumors. Patients who have MYC-N amplified tumors that respond sub-optimally to initial therapies have poor outcomes with 30-40 % long-term survival.  These tumors may have a higher content of therapy resistant cancer stem cells that utilize autophagy as an enhanced survival mechanism. In the following study we identified a stem cell phenotype in an MYC-N amplified neuroblastoma cell line from ATCC (BE-2) and  investigated  the activity of two new classes of drugs (GLUT-1 inhibitors and  bisquinolone antimalarials). Methods: Flow cytometry was utilized to identify stem cell fractions within an unselected BE-2 population.  Briefly, in side population analysis 1 x 10⁶ cells were incubated with Hoescht IMDM staining media (1 mcg/ml) and incubated for 30 minutes with Hoechst 33342 dye.  Cells were then kept on ice until flow cytometry analysis.  Flow cytometry was performed using a BD FACSAria.   Autophagy was measured using a flow cytometry based assay measuring membrane bound LC3 (Milipore Inc). Screening assays for drug activity used a 96 well platform with MTT to measure cytotoxicity of bisquinolone and GLUT-1 cytotoxicity. Neurospheres were grown in non-adherent plates to assess activity in this stem cell enriched screening assay.  Results: Flow cytometry demonstrated a high percent of side population cells on three separate analyses (11%, 38%, 28% of live cells). In comparison two murine breast cancer cells lines (Clone 66 and 4T1) and human mantle cell line (Granta) side population fractions were approximately 1% of live cells.  A commercially available bisquinolone, hydroxychloroquine, demonstrated the ability to inhibit autophagy as measured by the flow cytometry assay. Using the two in-vitro screening assays described above, a number of bisquinolone antimalarials and GLUT-1 inhibitors were assessed for activity and three compunds were identified with high activity (Q2-15 antimalarial and STF-04 and STF-05 GLUT-1 inhibitor).   Conclusion: In the following study we describe a large cancer stem cell population as measured by side population assay in BE-2 human neuroblastoma cells. Bisquinolone antimalarial drugs demonstrated the ability to inhibit autophagy. Both bisquinolone antimalarials and GLUT-1 inhibitors demonstrated cytotoxic activity in an MTT assay and activity in a stem cell enriched neurosphere assay. These two classes of drugs have the potential to improve therapy in poor prognostic neuroblastoma and studies combining candidate compounds are ongoing both in cell culture and an NSG animal model.