Abstract LB-159: Bisphosphonates are internalised by myeloid derived suppressor cells (MDSCs) in vivo in a mouse model of breast cancer
Authors: Shay Gemma , Hornick Mary , Heys Steven , Weilbaecher Katherine , Rogers Michael 
Published: June 2012

Abstract
Zoledronic acid (Zometa; ZOL), a nitrogen-containing bisphosphonate (N-BP), has become the standard treatment for metastatic bone disease due to its ability to inhibit osteoclast-mediated resorption. The molecular target of ZOL in osteoclasts is FPP synthase, an enzyme of the mevalonate pathway. Inhibition of this enzyme prevents the synthesis of isoprenoid lipids necessary for the prenylation of small GTPases such as Rho, Rac and Rap, thereby causing the accumulation of the unprenylated form of these proteins and fundamentally affecting cell function and survival. ZOL and other N-BPs have anti-tumour effects in some preclinical models of skeletal and non-skeletal tumours, and, more recently, have tantalising effects on patient survival in some clinical trials. However, the exact mechanisms responsible for these anti-tumour effects in vivo are still unknown, and the extent to which N-BPs are internalised by cells other than osteoclasts (such as immune cells) is unclear. We have sought to address this question in the 4T1 murine breast cancer model using a novel, fluorescently-labeled N-BP (AF647-RIS). 24 hours after a subcutaneous injection, using flow cytometry we have demonstrated for the first time the cellular uptake of this N-BP by Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs), as well as by CD14+F4/80+ monocytes/macrophages (TAMs) in mammary tumours, whole blood, liver, spleen and bone marrow. Uptake of N-BP was not detectable in the tumour cells or other cell types. Furthermore, 7 days after a single injection of a clinically relevant dose of ZOL into tumour-bearing mice, we detected a significant reduction in MDSC number in the spleen.
It remains unknown whether ZOL affects protein prenylation in MDSCs or TAMs in vivo. Currently available methods to detect changes in protein prenylation, using western blotting to detect unprenylated small GTPases, are too insensitive to detect subtle effects of N-BPs on prenylation in cells in vivo. Importantly, we have now optimised a new, highly sensitive enzyme-based approach to detect the accumulation of unprenylated proteins in cell and tissue extracts from N-BP-treated animals. Additional studies are now in the very final stages to determine conclusively whether a clinically-relevant dose of ZOL inhibits protein prenylation in MDSCs and TAMs in 4T1 mice.
In summary, we provide the first conclusive evidence that N-BPs can be internalized in vivo by MDSC and other myeloid cells such as TAMs. Given the important role of these cells in the progression of tumour growth and metastasis, our studies will finally clarify the mechanism underlying the preclinical, and potentially clinical, anti-tumour activity of these agents.
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 LB-159. doi:10.1158/1538-7445.AM2011-LB-159