Abstract 2233: MARCKS-ED peptide targets highly curved vesicles: Potential probe for exosome detection.

Malignant tumor cells overexpress and release nanosized, lipid-enclosed vesicles referred to as exosomes into body fluids for the primary function of facilitating cell-to-cell communication and transferring vital proteins necessary for survival to other parts of the body, resulting in metastasis. It was found that an increased secretion of exosomes in the peripheral blood is correlated to lung cancer and melanoma metastasis. The hallmarks of exosomes are their highly curved membrane surface (d = ∼30-100 nm) and their enrichment in the anionic lipid phosphatidylserine (PS) in the outer leaflet of the membrane bilayer. These properties may provide unique strategies in designing peptides to selectively target exosomes as potential cancer biomarkers. Some current clinical methods that are being used to classify metastatic patients, such as the biopsy and assays measuring circulating tumor cells, are not reproducible and do not always indicate whether or not treatment is reducing metastasis. Furthermore, these methods involve invasive procedures. We aim to develop a novel, minimally invasive, peptide-based diagnostic tool to detect and measure exosome overexpression in cancer patients. The successful creation of this diagnostic tool for cancer metastasis will help in the proper prognosis and therapy for cancer patients. We rationally chose the effector domain of the intracellular membrane protein myristoylated alanine-rich C-kinase substrate (MARCKS) as our core molecule based on recent reports that this peptide differentially interacts with nanometer size synthetic lipid vesicles. It has also been shown that MARCKS inserts its five hydrophobic phenylalanine (Phe) residues into the membrane of highly curved synthetic lipid vesicles, and its binding affinity increases with increasing vesicle curvature, presumably corresponding to a larger number of defects on the membrane surface. We hypothesize that as vesicle curvature increases, the Phe residues will insert deeper into the membrane due to more lipid defects exposed on the membrane surface in highly curved vesicles. Five analogs of the MARCKS peptide were synthesized, substituting one Phe residue with a cysteine residue for all 5 Phe positions. The cysteine was then labeled with 1-oxyl-2,2,5,5-tetramethyl-▵3-pyrroline-3-methyl) methanethiosulfonate (MTSL), which will be used as a paramagnetic spin label for electron paramagnetic resonance spectroscopy (EPR). The depth parameter of insertion of the MTSL into the lipid vesicle membrane at each of the five cysteine positions will be quantified using EPR and will be reported herein. Citation Format: Sara K. Coulup, Leslie A. Morton, Hang Yin. MARCKS-ED peptide targets highly curved vesicles: Potential probe for exosome detection. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2233. doi:10.1158/1538-7445.AM2013-2233 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.