Microvesicles and exosomes, herein collectively referred to as vesicles, are submicron sized lipid containers released by cells that play important roles in extracellular communication in normal and pathologic processes. The vesicles have an aqueous, cargo containing core surrounded by a spherical membrane bilayer. Arrival of the vesicles at distant cells allows transport of cargo as diverse as nucleic acids (DNA, mRNA, and microRNA), proteins, or lipids. Cancer cells have been shown to release vesicles that modulate the extracellular environment around the tumor to promote growth and metastasis. Cancer cell derived vesicles have been detected in culture, but also in the blood of patients with prostate cancer, lung adenocarcinoma, melanoma, and gastric cancers. This work has suggested that circulating vesicle levels might have application as a novel, non-invasive biomarker for multiple types of cancer. Two factors have hindered the implementation of circulating vesicles as clinical biomarkers: 1) they are submicroscopic in size and therefore cannot be detected with conventional optical methods, and 2) their multifaceted physiologic functions and origins mean there is no general surface marker that can be used for antibody based detection. In the proposed work we aim to prove a new application of rationally designed peptides as microvesicle and exosome cancer biomarker detectors. Synthetic peptides have tremendous potential as clinical probes;they are easily modifiable, resistant to degradation, and low in cost to produce.
In Aim 1, the binding mechanism of existing vesicle binding peptides will be determined using molecular dynamics simulations. Computational simulations are uniquely suited to this problem because they can provide the atomistic detail and femtosecond time resolution necessary to resolve binding. Using an innovative method for modeling peptides interacting with curved membranes, the mechanism of binding of current vesicle binding peptides will be examined. This understanding will then be used to rationally design second generation of peptide probes with improved affinity and specificity by simultaneously targeting the membrane curvature and composition. Completion of Aim 1 will provide a library of highly selective curvature binding peptides.
In Aim 2, circular dichroism spectroscopy, isothermal titration calorimetry, and fluorescent enhancement assays will be conducted using synthetic vesicles to experimentally validate the designed peptides. Promising peptides candidates will then be used to test cancer patient blood samples for vesicle levels. Using the synthesized peptides, we aim to establish statistical correlations between circulating vesicle levels and cancer presence or progression in patients. If successful, there is a significant potential to advance the applications of synthetic peptides and reduce future patient mortality.
Early detection and treatment of cancer is one of the best ways to improve cancer patient prognoses. Pre- metastatic cancers release increased levels of small vesicles;detection of these vesicles in circulation may help physicians decide if cancer is present or progressing in the body. Circulating vesicles are difficult to detect, however rationall designed peptide probes have the potential to quickly, easily, and inexpensively identify these vesicles in the blood, leading to earlier diagnoses and improved patient outcomes.
|Kastelowitz, Noah; Yin, Hang (2014) Exosomes and microvesicles: identification and targeting by particle size and lipid chemical probes. Chembiochem 15:923-8|