Circulating miRNAs in Lung Cancer Are Associated to Pro-Tumorigenic and Immunosuppressive Microenvironment

We previously 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 damaged cells or active secretion through extracellular-vesicles or protein complexes. To evaluate the potential origin and the release of the 24 miRNAs of the diagnostic signature we analyzed their expression by real-time or digital PCR in both cells and conditioned medium (CM) from cancer cell and different cell types of the lung microenvironment. Lung tissues and cell-blocks were analyzed by miRNAs in situ hybridization (ISH). 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. 24-miRNAs analysis showed higher abundance in specific cellular components such as mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133a in skeletal muscle cells or mir-451 and 142-3p in hematopoietic cells. Generally, tumor cells showed lower levels of miRNAs compared to bronchial epithelial cells. MiRNAs specific localization in lung tissue was confirmed by ISH. We observed that mir-451 is specifically expressed in lung interstitial alveolar walls while mir-126 by endothelial cells outside the tumor bulk; miR-145 is characteristic of fibroblast and muscle cells and miR-142-3p of hematopoietic cells, fibroblast and muscle whereas mir-21 is over-expressed in the tumor. The analysis of miRNAs in CM showed that miRNAs secretion is correlated with cellular expression for most cell types (Pearson correlation range: 0.41-0.80). Interestingly, platelets and granulocytes were the components that mostly secreted miRNAs. In vitro experiments showed that endothelial cells under hypoxic condition up-regulate mir-126 and that mir-145 was up-regulated and secreted in lung cancer-associated compared to normal fibroblasts. Interestingly, during conversion of T lymphocytes into T regulatory cells up-regulation of mir-15b, mir-19b and mir-320 was observed whereas mir-15b and mir-197 were up-regulated in the conversion of macrophages into M2 phenotype. Modulation of miRNAs in immune and stromal cells was consistent with up-regulation of the same miRNAs observed in plasma samples. Our findings on the origin of circulating miRNAs support the conclusion that plasma miRNAs are heterogeneous and secreted by different cellular components of lung microenvironment rather than by tumor cells. In particular, we demonstrated that a pro-tumorigenic and immunosuppressive microenvironment contributes to the de-regulation of miRNAs observed in plasma of lung cancer patients.