(193d) Simultaneous Capture and Characterization of Circulating Exosomes/Microvesicles Using Tethered Lipoplex Nanoparticles for Lung Cancer Diagnosis and Surveillance

Lung cancer is the leading cause of cancer deaths worldwide with a disappointing 15% overall 5-year survival rate. A patient-friendly early detection and surveillance method would substantially reduce the mortality in this serious disease. Circulating exosomes/microvesicles based diagnostic platform, as a non-invasive and sensitive detection method, may achieve this goal. Many studies have shown that circulating exosomes/microvesicles are potential biosignatures because the tumor is the primary source of circulating exosomes/microvesicles in cancer patients. In lung cancer, it has been shown that total exosomes and exosomal microRNA/mRNA levels in lung cancer patients are significantly higher than healthy controls, and the circulating exosomal microRNA/mRNA patterns may be a potential predictor of the overall survival in lung cancer patients.

Current technologies, such as qRT-PCR and NanoString nCounter®, need to first isolate total RNA and then measure the target microRNA/mRNA, which require large quantity of samples and are labor intensive and time consuming.  We have developed a novel and simple tethered lipoplex nanoparticles (tLN) device that is capable to simultaneously capture and characterize circulating exosomes/microvesicles, and only consumes as less as 10uL of samples.  In this study, microRNA-21 (miR-21) and lung specific TTF-1 mRNA were selected as the biomarkers for lung cancer early detection. In the tLN device, intracellular molecular probes, such as molecular beacons (MBs) that detect miR-21 and TTF-1 mRNA, were encapsulated in cationic lipoplex nanoparticles and tethered on the glass substrate.

The tLN device was first demonstrated using non-small cell lung cancer cells (A549) and normal human bronchial epithelial cells (HBEC). Exosomes secreted by A549 and HBEC cells were collected using ultracentrifugation and applied on tLN device to detect circulating miR-21 and TTF-1 mRNA. Conventional qRT-PCR was also conducted to quantitatively correlate the expressions of miR-21 and TTF-1 mRNA with those measured by tLN device. Exosomes produced by A549 showed higher miR-21 and TTF-1 mRNA expression compared to the exosomes secreted by normal HBEC cells, which was consistent with qRT-PCR results. Preliminary results using lung cancer patient blood samples also demonstrated promising results. Serum and serum exosomes were isolated from the whole blood, and then applied to tLN device for miR-21 and TTF-1 mRNA detection. Compared with normal donors, patient samples showed higher miR-21 and TTF-1 mRNA expression, which agreed with qRT-PCR results. The biological atomic force microscopy (Bio-AFM) and the total internal reflection fluorescence (TIRF) microscopy were used to investigate the interactions between exosomes/microvesicles and tethered lipoplex nanoparticles.

Our tLN device represents an innovative approach to characterizing the peripheral blood signature in lung cancer. This facile technique holds great promise in our fight against lung cancer. It may also be applied to many other cancers and deadly diseases.