Abstract 3800: Analysis of circulating tumor exosomes: their ability to serve as a biomarker for recurrence in lung cancer
Authors: Serrano Nicholas , Blue John , Alam Asim , Rabender Christopher S. , Weiss Elisabeth , Anscher Mitchell S. , Mikkelsen Ross B. , Yakovlev Vasily A. 
Published: July 2017

Abstract
Objectives: Exosomes (Exos) are 30-150 nm wide nanovesicles originating from the endosomal network and are found in most body fluids. Production of Exos increases in cancer, making Exos potential biomarkers. Exos are a fundamental driver of intercellular communication by transferring proteins, lipids and miRNA. Analysis of Exos and their interaction with the microenvironment may uncover cellular pathways involved with disease progression. We hypothesized that Exos may serve as a liquid biopsy for tumor recurrence.
Materials and Methods: Six lung cancer patients (LCPs) were prospectively enrolled on an IRB approved study. Exos were precipitated and purified with a Norgen kit from 0.7 ml of blood samples collected prior to, during and 6 months after radiation therapy (RT). Exos numbers were quantified by Exocet Exosome Quantitation Kit (System Biosciences). Total exosomal RNA was isolated by the Norgen RNA/DNA/Protein Purification kit. For the miRNAs expression profiling we used the miRCURY LNA™ Universal RT microRNA PCR assay (Exiqon) and ExiLENT SYBR® Green Maser Mix (Exiqon). Expressions of 10 different miRNAs (let-7a-5p, miR-20a-5p, miR-21-5p, miR-30b-5p, miR-106a-5p, miR-146a-5p, miR-155-5p, miR-200b-5p, miR-203a, miR-208a) for all samples were determined. Human lung fibroblasts (MRC-5s) in serum-free medium for 24 hrs were treated with Exos, TGF-β (positive control) or PBS (negative control) to determine their relative effects on MRC-5 proliferation.
Results: Of the 6 LCPs, 2 had limited stage small cell and 4 had non-small cell at diagnosis. Three patients were diagnosed with recurrence at the time of their post-RT blood draw. Six miRNAs demonstrated significantly different expressions between recurrent and non-recurrent groups of LCPs: let-7a-5p, miR-20a-5p, miR-21-5p, miR-106a-5p, miR-155-5p, and miR-203a. MRC-5 proliferation increases 2-fold when treated with Exos from blood collected prior to RT compared to PBS (p = 0.01). There is &lt;2-fold increase compared to TGF-β (p = 0.03). Plasma Exos levels decrease during RT as do their relative effects on MRC-5 proliferation when compared at equivalent numbers. Exos collected at 6 months from patients with recurrence significantly stimulated MRC-5 proliferation (p = 0.03), whereas Exos from patients without recurrence do not. Western blot analyses of MRC-5 cells stimulated with Exos revealed decreased PTEN but enhanced CDC25A expression and increased AKT and SMAD phosphorylation, regardless of when the Exos were collected.
Conclusions: Recurrent and non-recurrent groups of LCPs demonstrated significant differences in exosomal miR profile before and after RT. Exos from untreated LCPs, and recurrent LCPs after RT, stimulate MRC-5 proliferation and possible conversion to CAFs. Our results suggest that Exos could serve as a “liquid biopsy” to assess the potential for recurrence when imaging modalities are equivocal.
Citation Format: Nicholas Serrano, John Blue, Asim Alam, Christopher S. Rabender, Elisabeth Weiss, Mitchell S. Anscher, Ross B. Mikkelsen, Vasily A. Yakovlev. Analysis of circulating tumor exosomes: their ability to serve as a biomarker for recurrence in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3800. doi:10.1158/1538-7445.AM2017-3800