Abstract 1934: Retinoic acid and DFMO induce differentiation and inhibit tumor formation in neuroblastoma
Authors: Voydanoff Austin , Zhao Ping , Nagulapally Abhinav , Bond Jeff , Sholler Giselle L. 
Published: July 2017

Abstract
Background: A key component of high risk neuroblastoma (NB) therapy involves cis-retinoic acid (RA) for differentiation of minimal residual disease. DFMO induces differentiation and inhibition of tumor formation through the targeting of cancer stem cell (CSC) pathways via reversal of the Lin28/Let7 axis. Preventatitve DFMO therapy is currently in a phase II clinical trial at the end of therapy and in a Pilot study in combination with RA and ch14:18 antibody. We hypothesize that the combination of cis-RA and DFMO will induce greater differentiation, inhibition of tumor formation, and reduction of cell proliferation of NB.
Methods: NB cell lines SMSKCNR, BE2C and CHLA90 were incubated in 96 well plates for 24 and 48 hours with low doses of DFMO (2.5 and 5 mM), RA (5 and 10 μM), and the four combinations of dosages. A Calcein AM Cell Viability Assay and BrdU Cell Proliferation Assay Kit were used to determine cell viability and cell proliferation, respectively. Western blot analysis was used to measure protein levels of CSC and differentiation markers. A neurosphere assay was used to assess inhibition of CSCs and tumor formation within wells. Cells were plated 2 cells/well in 96 well plates, drugged with single agents, cominbation, or DMSO and the percentage of wells per plate that formed neurospheres was determined after 1 and 2 weeks. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of neurite length to assess differentiation of NB cells.
Results: Low dose RA and DFMO combination treatment (2.5-5mM DFMO and 5-10 μM RA) resulted in decreased cell viability as demonstrated through calcein AM. DFMO and RA combination treatments reduced cell viability by 60-71%, 75-78%, and 83-91%, in Be2C, CHLA90, and SMSR cells, respectively. BrdU incorporation demonstrated a reduction in cell proliferation at 48 hours of was 69-70.%, 60.2-64.5%, 62.7-71.1% in Be2c, CHLA90, and SMSR, respectively . Western blot analysis showed that DFMO, RA, and their combination reduced the CSC and increased the differentiation markers at 48 hours compared to control. The combination treatment also decreased tumor formation; the relative reduction in neurosphere formation at 2 weeks was 34.5% with 2.5 mM DFMO, 48% with 5 µM RA, and 73.2% with combination treatment. Lastly, differentiation was shown by neurite length increased by a factor of 1.4-1.6 and 5, in SMSR and BE2C cells, respectively with combination treatment.
Conclusion: This study indicates that the combination retinoic acid and DFMO effectively cause a decrease in cell viability with a reduction in cell proliferation. Further, the combination results in differentiation of NB cells as well as targeting of CSC pathways and inhibition of tumor formation. Preventative DFMO therapy has been initiatied with RA in a pilot study for the treatment of high-risk neuroblastoma patients.
Citation Format: Austin Voydanoff, Ping Zhao, Abhinav Nagulapally, Jeff Bond, Giselle L. Sholler. Retinoic acid and DFMO induce differentiation and inhibit tumor formation in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1934. doi:10.1158/1538-7445.AM2017-1934