Abstract 948: Microenvironmental origin of circulating miRNAs in lung cancer
Authors: Fortunato Orazio , Borzi Cristina , Conte Davide , Centonze Giovanni , Boeri Mattia , Verri Carla , Calzolari Linda , Pastorino Ugo , Sozzi Gabriella 
Published: July 2016

Abstract
INTRODUCTION We reported the identification of diagnostic miRNA signatures in plasma samples of lung cancer patients detected by low dose computed tomography (LDCT) screening. Circulating miRNAs are released into the bloodstream by different mechanisms such as passive leakage from broken cells or active secretion through microvesicles or protein complexes.
MATERIAL and METHODS To evaluate the origin and the release of the 24 miRNAs we analyzed their expression by real-time PCR in different lung cell types including fibroblasts, hematopoietic, endothelial, bronchial epithelial (HBECs) and tumor cells and in the respective culture medium (CM) as well as in plasma samples from heavy smokers and patients.
RESULTS and DISCUSSION The analysis of lung cancer plasma samples in patients showed that miR-106a, miR-140, miR-142-3p, miR-17, miR-660, miR-320, miR-197 and mir-28-3p were those significantly returning to a physiological level after surgery (p&lt;0.05). On the other hand, mir-126 and mir-451 remained deregulated after tumor resection suggesting an “host-related” origin and likely indicating the persistence of a risk profile. To evaluate which lung cell type express these miRNAs we performed in situ hybridization and observed that mir-451 is specifically expressed in lung interstitial alveolar walls by cells of the fibro-monocytic infiltrate whereas mir-126 seems to be expressed by endothelial cells.
The analysis of the 24-miRNAs composing the signature in the various lung cell types showed an increased expression in specific cellular components such as that of mir-16-17-19b-106-145 in fibroblasts, mir-126 in endothelial cells, mir-451 and mir-142-3p in hematopoietic cells. The analysis of miRNAs released in CM showed that miRNAs were secreted differently and independently from their cellular levels and that tumor cells were the less involved in this release. Interestingly, we identified miRNAs that were expressed and released from blood cells only such as mir-451, mir-133 and mir-142-3p. To better characterize the origin of plasma miRNAs, we isolated exosomes from CM of the different cell types. We detected the presence of the 24 miRNAs in exosomes and identified exosomal miRNAs that were secreted by specific cellular compartments (mir-101 from blood cells, mir-145 from fibroblasts and mir-126 from endothelial cells). Furthermore, by analyzing the exosome fraction of 29 plasma samples of 21 smokers and 8 patients we confirmed the presence of all the 24 miRNAs inside exosomes and comparing miRNAs content in plasma and in their respective exosome fraction we observed a good degree of correlation (Pearson's R = 0.89, p for Pearson's R &lt;0.001) indicating that miRNAs expression in exosomes may reflect miRNA levels in total plasma.
CONCLUSION Our findings on the origin of circulating miRNAs support the conclusion that plasma miRNAs are heterogeneous and are actively secreted inside exosomes by different cellular components and not specifically by tumor cells.
Citation Format: Orazio Fortunato, Cristina Borzi, Davide Conte, Giovanni Centonze, Mattia Boeri, Carla Verri, Linda Calzolari, Ugo Pastorino, Gabriella Sozzi. Microenvironmental origin of circulating miRNAs in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 948.