Abstract 4981: Circulating mir-320 promotes immunosuppressive macrophages M2 phenotype associated with lung cancer progression

Abstract

Abstract INTRODUCTION miRNAs play a role in the complex network of signaling between cancer cells and tumor microenvironment. We previously reported the identification of diagnostic miRNA signatures (MSC) based on 24-miRNAs in plasma samples of lung cancer patients detected by low dose computed tomography (LDCT) screening. MATERIAL and METHODS To evaluate the potential origin of the miRNAs of the diagnostic signature, we analyzed their expression by real-time or digital PCR in both cells and conditioned medium (CM) from different cell types of the lung microenvironment as well as in plasma samples of heavy smokers and patients. Lung tissues and cell-blocks were analyzed by miRNAs in situ hybridization. Modulation of miRNAs after in vitro treatments, known to induce changes associated with cancer progression, in different cell types was assessed and correlated to changes observed in circulating miRNAs signatures. RESULTS and DISCUSSION The analysis of 100 pre and post surgery plasma samples from 31 patients suggested a tumor-related origin of those miRNAs which significantly declined to basal levels after curative tumor resection. Other miRNAs (miR-126, miR-92a, miR-320, miR-28-3p, miR-486, miR-451, miR-16) remained deregulated afer surgery likely indicating a “host-related” origin and the persistence of a risk profile. Specific expression of mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133 in skeletal muscle cells, mir-451 and 142-3p in hematopoietic cells was observed with a good degree of correlation between cellular and secreted miRNAs levels in each cell types (Pearson correlation range: 0.59-0.81). In vitro experiments comparing activated vs. resting neutrophils, showed that 17 out of the 24 miRNAs were concordantly modulated as in lung cancer patients' plasma (Pearson r=0.60, p<0.01), indicating a role of activated neutrophils in determining plasma miRNAs profile associated with lung cancer . Interestingly, we found increased miR-320 levels in cells and CM of neutrophils isolated from MSC positive compared to MSC negative subjects (fold increase: 1.7 and 2.4, respectively, p<0.05). Treatment of cultured macrophages with CM of neutrophils resulted in higher levels of the mature form of miR-320 but not of its pri-miRNA expression levels. Exogenous miR-320 induced M2 polarization, as assessed by the increase of IL-10 expression and of CD163 and CD68 positive cells. These effects of mir-320 were abolished when anti-mir-320 was transfected in recipient cells. Mir-320 over-expressing macrophages were able to stimulate cancer growth both in vitro and in vivo models. Furthermore, we demonstrated that STAT4 was a direct target of miR-320 and responsible of the M2-like switch. CONCLUSION These findings suggest that circulating miRNAs are non cell-autonomous, may act in paracrine signalling and have a causative role in lung carcinogenesis and immunesuppression. Citation Format: Orazio Fortunato, Cristina Borzi, Massimo Milione, Giovanni Centonze, Davide Conte, Mattia Boeri, Massimo Moro, Federica Facchinetti, Luca Roz, Veronica Huber, Chiara Camisaschi, Chiara Castelli, Licia Rivoltini, Valeria Cancila, Claudio Tripodo, Ugo Pastorino, Gabriella Sozzi. Circulating mir-320 promotes immunosuppressive macrophages M2 phenotype associated with lung cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4981.