Abstract 2474: DFMO targets cancer stem cells in neuroblastoma
Authors: Avequin Tracey , Zhao Ping , Nagulapally Abhinav , Bond Jeffrey , Azizi Ebrahim , Wicha Max , Saulnier Sholler Giselle L. 
Published: July 2016

Abstract
Background: Difluoromethylornithine (DFMO) has shown promise in recent clinical trials as a therapeutic agent in treating neuroblastoma (NB). High-risk NB patients have an approximately 50% relapse rate, after which survival rate drops to lower than 10%. By targeting the cancer stem cells (CSC) we could decrease relapse rate in high-risk patients and improve long-term survival. DFMO is known to inhibit ornithine decarboxylase (ODC) impeding key polyamine biosynthesis as well as the LIN28/Let7 pathway and disrupting the glycolytic metabolism pathways both of which have been shown to be important in CSC models. We propose that DFMO may be an effective treatment against neuroblastoma CSCs.
Methods: BE(2)C and SMS-KCNR NB cell lines were studied. Limiting dilution assays of xenograft studies were performed. Untreated, 10-day 5mM DFMO-pretreated, or 20-day 5mM DFMO-pretreated cells were injected into mice between 10-5000 cells/mouse and followed for tumor formation. Xenograft Be2C mice were treated with either vehicle or 2%DFMO in drinking water and harvested at 7-14 days for tumor analysis. Tumors and cell cultures of untreated and DFMO-treated cells were analyzed by RT-qPCR, Western Blot analysis, and GeneChip gene expression analysis measuring levels of ODC1, LIN28B, mirLet7, MYCN, SLC2A4 (a glucose transporter), and CSC markers such as CXCR4, POU5F1, NANOG, SOX2, and KLF4. Riboflavin induced autofluorescence (AF) was used to identify CSC. This method detected cells after a 24-hour bath in 30μM riboflavin and excitement at 488nm in a MoFlo cell sorter at the University of Michigan Flow Core, after which cells are sorted into AF- and AF+ sub-populations for imaging, neurosphere assays with or without DFMO, RT-qPCR and Western Blot analysis. Knock-down of ODC1 is achieved by siRNA transfection via Lipofectamine in cells and analyzed by RT-qPCR, Western Blot, and neurosphere assays with or without DFMO.
Results: Pretreatment with DFMO in BE(2)C and SMS-KCNR cells in vitro and in vivo inhibits tumor formation in neurosphere assays and in limiting dilution xenograft models respectively. Tumors harvested from xenografts show a decrease in expression of CSC biomarkers and neurosphere formation capability in those treated with DFMO. AF+ cells showed an increase in stem cell marker expression and increase neurosphere formation relative to AF- cells, identifying them as cancer stem cells. In addition, AF+ cells are more sensitive to DFMO treatment with a greater decrease in neurosphere formation. Cells transfected with ODC siRNA or subjected to DFMO show a reduction in CSC biomarker RNA and protein and a decrease in neurospheres formation.
Conclusion: These results suggest that DFMO targets the CSC subpopulation in neuroblastoma, disrupting cell growth pathways and inhibiting tumor formation. This concept is under further investigation in a Phase II clinical trial to prevent relapse in high risk neuroblastoma patients.
Citation Format: Tracey Avequin, Ping Zhao, Abhinav Nagulapally, Jeffrey Bond, Ebrahim Azizi, Max Wicha, Giselle L. Saulnier Sholler. DFMO targets cancer stem cells in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2474.