Abstract 1800: 4-HPR (fenretinide) sensitizes human neuroblastoma cells for antibody-independent and ch14.18-mediated NK cell killing
Authors: Shibina Anastasia , Seidel Diana , Somanchi Srinivas , Lode Holger N. , Lee Dean A. , Reynolds C.Patrick , Huebener Nicole 
Published: June 2012

Abstract
Introduction: Neuroblastoma (NB) is the most common extracranial solid tumor in children. Recently, the addition of passive immunotherapy with the anti-GD2 antibody ch14.18 provided an increased survival of children with high-risk disease. However, still one third of the patients die from their disease emphasizing the need for improved therapy protocols. 4-HPR is a synthetic retinoid known to induce apoptosis in cancer cells mainly via the accumulation of ceramides. We tested the hypothesis whether 4-HPR and ch14.18 would work additively in NK cell-mediated killing (antibody-dependent and independent cellular cytotoxicity, ADCC and AICC) when applied in combination.
Methods: We employed 6 GD2+, drug-resistant human NB cell lines. First, we tested whether 4-HPR treatment would result in an increase of the GD2 binding index by using flow cytometry (FC). Second, we determined the effect of 4-HPR on death receptor (DR) expression on human NB cells (Fas, TRAIL-R1 and TRAIL-R2) by FC. Third, cytotoxicity assays were performed in which 4-HPR-treated, live cells were used with expanded natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs) isolated from healthy donors as effector cells. Forth, we blocked ganglioside synthesis by using the glucosylceramide synthase inhibitor PPPP and determined the effects on ADCC and AICC and GD2 and DR expression. Fifth, we induced s.c. tumors (cell line CHLA-136) in NOD/SCID mice and treated them with 240mg/kg/d 4-HPR by oral gavage for 12 days. Then, we isolated tumor cells and measured GD2 and Fas expression levels by FC. Further, tumor cells were employed ex vivo in cytotoxicity assays using expanded NK cells as effector population.
Results: Pre-treatment of drug-resistant NB cells with 4-HPR significantly enhances ch14.18-mediated ADCC as well as AICC by both human NK cells and PBMCs. Interestingly, we also found an increase of the GD2 binding index as well as an increase of DRs in all cell lines tested. Blocking of ganglioside synthesis results in an abrogation of the increased ADCC response by human NK cells but has no effect on the enhanced AICC after 4-HPR pre-treatment. Finally, we could show that tumor xenografts isolated from mice that were treated with 4-HPR exhibit an increased GD2 binding index as well as Fas expression compared to tumors isolated from vehicle treated animals. Importantly, this correlates well with a significantly higher ex vivo ADCC and AICC response of human NK cells towards 4-HPR treated tumors than towards vehicle treated controls.
Conclusion: New therapy protocols are needed in order to optimize treatment of children with high-risk NB. We can show here for the first time that 4-HPR treatment increases ADCC and AICC by human NK cells and PBMCs towards drug-resistant human NB cells, thereby providing an important baseline for the combination of 4-HPR and passive immunotherapy with ch14.18 in future clinical trials.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1800. doi:10.1158/1538-7445.AM2011-1800