C. Subramanian1, P. T. White1, R. Kuai2, J. Moon2,5, V. P. Opipari6, B. N. Timmermann4, A. Schwendeman2, M. S. Cohen1,3 5University Of Michigan,Department Of Biomedical Engineering,Ann Arbor, MI, USA 6University Of Michigan,Department Of Pediatrics,Ann Arbor, MI, USA 1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2University Of Michigan,College Of Pharmacy,Ann Arbor, MI, USA 3University Of Michigan,Department Of Pharmacology,Ann Arbor, MI, USA 4University Of Kansas,Department Of Medicinal Chemistry,Lawrence, KS, USA
Introduction: Only 40% of patients with high-risk neuroblastoma (NB) survive beyond 5 yrs despite current treatments. We have demonstrated that 4,19,27-triacetyl withalongolide A (WGA-TA) is a novel inhibitor of the Hsp90 molecular chaperone, targeting the PI3K/Akt/NFΚ B pathways in NB. These pathways are critical for NB cancer stem cell (CSC) function and these CSCs have been ascribed the capacity to recapitulate a tumor from a few cells, enhance tumor drug resistance or its metastatic potential. While withanolides are potent against NB, they do not directly target NB cells and are difficult to solubilize in vivo. Synthetic HDL mimetic (sHDL) targets the SR-B1 receptor on the cell surface and solubilizes lipophilic drugs like WGA-TA. Since NB cells and their CSCs highly overexpress SR-B1, we hypothesize that nanoparticle conjugation of a novel mimetic sHDL nanoparticle with WGA-TA will improve targeting and inhibition of NB CSC function in vitro and in vivo to decrease tumor growth, self-renewal and invasion.
Methods: Validated human NB cells (IMR32,SH-EP,SH-SY5Y,SK-N-BE(2),and SK-N-AS) were evaluated for the expression of SR-B1 by qPCR and western blot (WB). Drug uptake in vitro was measured by fluorescence microscopy, and in vivo uptake in NB tumors (IMR32) was measured on an IVIS system using fluorescent labeled drug. Self renewal and migration/invasion were assessed by sphere formation and Boyden chamber assays, respectively. Percentage viability was analyzed by Cell-Titer Glow(CTG) assay.
Results: qPCR and WB analysis revealed a 4-6 fold higher-level expression of SR-B1 in N-myc amplified NB cells compared to N-myc non-amplified or Jurkat cells(p<0.01). In vitro uptake of sHDL indicated significantly higher levels of uptake in high SR-B1 expressing (IMR32, SK-N-BE(2)) cells compared to low expressers(SH-EP, SH-SY5Y), which was almost completely blocked by excess sHDL(p<0.001). IVIS imaging of IMR32 tumors indicated highest uptake of sHDL in the tumor and liver (where it is metabolized) whereas uptake in other organs was negligible. After treatment of both IMR32 and SK-N-BE(2) cells, there was a dose dependent decrease in sphere formation starting from 8 nM [with complete blockage of self renewal at 62.5 nM (p<0.001 vs control)], and invasion and migration starting at 250 nM vs. untreated control (p<0.001). There was no change in cell viability for sHDL up to 20 µ M, whereas the sHDL-WGA-TA and WGA-TA had an IC50 value of 15-100nM which was 15-51 fold higher than the IC50 of control MRC5 cells(p<0.001).
Conclusion: Mimetic sHDL-WGA-TA is a novel treatment strategy for NB to improve drug delivery to NBs and their CSCs through SR-B1 targeting in vitro and in vivo. sHDL conjugation of withanolides enhances their ability to prevent NB tumor self-renewal and migration/invasion in vitro. Further in vivo efficacy studies are warranted to evaluate the translation effect of NB CSC inhibition on tumor growth, invasion, and metastatic spread.