Smoking Induced Extracellular Vesicles Release and Their Distinct Properties in Non-Small Cell Lung Cancer

Abstract

Purpose: Smoking is a strong relative risk factor for lung cancer. Extracellular vesicles (EVs), particularly exosomes, have been implicated in cancers. In this study, we characterized smoking induced extracellular vesicles in smokers with non-small cell lung cancer (NSCLC). Methods: EVs were isolated from bronchoalveolar lavage (BAL) from smokers and NSCLC patients. EV microRNAs (miRNAs) were analyzed by using a TaqMan microRNA assays. Vesicle mRNAs and long non-coding RNAs (lncRNAs) were measured with quantitative RT-PCR. Tumor associated antigens were examined by Western Blot. Results: Higher levels of local site EVs are found in the lung of smokers and NSCLC patients. Further, over 90% of lung EVs are round vesicles of approximately 50-200 nm, ie., exosomes. There are 21 EV miRNAs up regulated, while 10 miRNAs under regulated, in smokers when compared to controls (relative fold > 2, p < 0.05). These miRNAs were further observed to be dysregulated in NSCLC patients when compared to smokers. Bioinformatic analysis demonstrated that Proteoglycans, Fatty acid biosynthesis, ErbB, Hippo, TGF-beta, Wnt, Rap1, AMPK and Ras pathways were the most prominent pathways enriched in NSCLC EV miRNA signatures. In addition, messenger RNA transcripts including EGFR, KRAS, ALK, MET, LKB1, BRAF, PIK3CA, RET, and ROS1 were significantly higher expressed in lung EVs in smokers and NSCLC patients compared to controls. Long non-coding RNAs, including MALAT1, HOTAIR, HOTTIP, AGAP2-AS1, ATB, TCF7, FOXD2-AS1, HOXA11-AS, PCAF1, and BCAR4, were over expressed in EVs from smokers and NSCLC patients. Furthermore, protein levels of tumor associated antigens including BAGE, PD-L1, MAGE-3, and AKAP4 were significantly dysregulated in EVs of smokers and NSCLC patients compared to healthy controls. Conclusions: In conclusion, these data demonstrated an intrinsic relationship of smoking dysregulated EVs and EVs contained RNA, proteins which may involve in the development of NSCLC.

Keywords: NSCLC; extracellular vesicles; smoker; tumor associated antigens.

Figure 1

Higher EV concentration with smoking. A. Exosomal phenotyped EVs were examined by flow cytometric analysis. B. Cross-sectional cohort analysis of EV concentration in 14 smokers and 17 NSCLC patients compared to 14 healthy subjects. C. EVs concentrations when isolated by differential ultracentrifugation protocols.

Figure 2

Heatmap of BAL exosomal differential miRNA profiles in smokers and NSCLC patients. Heatmap representation of the mean fold change in smokers and NSCLC patients related differential miRNA signature. Two-dimensional grid matrix displaying 31 exosomal miRNAs in BAL was obtained by the functional heat-map in R. Columns refer to time course comparison: 48 healthy controls, 26 smokers and 26 NSCLC patients. Rows stand for the 31 differential miRNAs. Each entry of the grid refers to relative fold (log2) between the expression level of a given exosomal miRNA. The color of each entry is determined by the value of that fold difference, ranging from green (negative values) to red (positive values).

Figure 3

Quantitative RT-PCR experiments were performed on EGFR, KRAS, ALK, MET, LKB1, BRAF, PIK3CA, RET, and ROS1 mRNAs by using BAL exosomal RNA isolated from 26 smokers, 26 NSCLC patients, and 48 healthy controls.

Figure 4

Quantitative RT-PCR experiments were performed on nine lncRNAs including MALAT1, HOTAIR, HOTTIP, AGAP2-AS1, ATB, TCF7, FOXD2-AS1, HOXA11-AS, PCAF1, and BCAR4 by using BAL exosomal RNA isolated from 26 smokers, 26 NSCLC patients, and 48 healthy controls.

Figure 5

Western blot to test HLA-class I, AKAP4, BAGE, PD-L1, MAGE-3, and Annexin-A2 protein levels in lung exosomes isolated from 5 healthy controls, 5 smokers, and 5 NSCLC patients. Annexin-V was loaded as a positive control.