Abstract 1947: Tumor-derived circulatory exosomal miRNAs as biomarkers of recurrent lung tumors.
Authors: Munagala Radha , Aqil Farrukh , Taylor Douglas D. , Gupta Ramesh 
Published: August 2014

Abstract
More people in the U.S. (157,300 per year) die of lung cancers than of prostate cancer, breast cancer and colon cancer combined. Eighty five percent of NSCLC patients die within 5 years. Even after resection of an early stage NSCLC, many patients die of relapse. Despite 2 decades of cisplatin-based chemotherapy for NSCLC, the survival benefit remains modest. Poor outcomes and relapse indicate need for new screening and early biomarkers for detection of recurrent lung tumors. In this study, first, we determined the frequency of lung tumor recurrence in female athymic nude mice (4-5 wk-old; n=25) using H1299 cells (1.5 x 106 cells). When tumors reached 300-400 mm3, the tumors were completely excised under anesthesia from 18/25 mice; the primary tumors in the remaining animals (n=7) were allowed to grow. We recorded the appearance of first tumor recurrence, 1 week after the surgery and the incidence rate of recurrent tumors was 60% (11/18). Average recurrent tumor volume at the end of the study was 431 ± 236 mm3. Second, we determined the circulating miRNA profiles by analyzing exosomal miRNAs in serum from control and H1299 lung tumor-bearing mice. Exosomes, nano-vesicles that carry load of miRNAs and mRNAs, were isolated from 1ml serum by pooling blood from five mice, using ExoQuick reagent (System Biosciences). Exosomes were visualized and their sizes measured by NanoSight (Wiltshire) and confirmed by transmission electron microscopy. Total RNA was isolated from exosomes with Trizol and was analyzed by qPCR using cancer-pathway finder miRNA array, containing 84 miRNAs (Qiagen). Our data showed i) the presence of circulatory exosomes with particle size 34-97 nm, ii) circulatory exosomal miRNAs iii) differences &gt;2-fold in the up- and downregulated miRNAs in the H1299 primary tumors and recurrent tumors versus normal human bronchial epithelial cells, and serum exosomes from tumor-bearing versus control mice, iv) similarity of all 25 miRNAs expressed in the recurrent tumors with 25/34 miRNAs in primary tumors, suggesting that recurrent tumors originated from microscopic primary tumors, and v) similarity of 10 miRNAs expressed in serum exosomes with those expressed in primary tumors (e.g., miR-132 and let-7c), suggesting that serum exosomal miRNAs originated from the lung tumors and suggest their biomarker significance. Further, we performed immunoaffinity enrichment of tumor-specific exosomes with anti-EpCAM magnetic beads using serum from control and lung tumor-bearing mice. Western blot analysis revealed that the enriched exosomes were positive for the tumor marker EpCAM, and was significantly higher in exosomes from tumor-bearing vs. control nude mice. Thus, our initial observations suggest that the miRNAs carried by the tumor-derived exosomes can be a true representation of tumor-profile and a more stringent validation of miRNAs as circulatory biomarkers. (Supported by Agnes Brown Duggan Endowment and Helmsley Endowment).
Citation Format: Radha Munagala, Farrukh Aqil, Douglas D. Taylor, Ramesh Gupta. Tumor-derived circulatory exosomal miRNAs as biomarkers of recurrent lung tumors. [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 1947. doi:10.1158/1538-7445.AM2013-1947