fig7

Figure 7. MiRNA analyses of EBC and exhaled EVs collected from control and lung tumor-bearing mice. (A) Western blot evaluation of anti-human and anti-mouse CD63 antibodies using EVs purified from tissue culture media of human breast cancer MDA-MB-231 cells, human kidney cancer HEK293 cells, and normal mouse bone marrow endothelial cells (BMECs); (B) Heatmap analysis of the top 142 most differentially detectable miRNAs between small-RNA extracted from whole EBC (green), and sequentially purified from human exhaled EVs using the anti-human anti-CD63 EV-CATCHER assay from whole EBC (orange), and mouse exhaled EVs using the anti-mouse anti-CD63 EV-CATCHER assay from the same whole EBC samples (purple), collected at weeks 21 (light grey), 22 (brown), and 23 (dark grey) from female control (blue) and lung tumor-bearing (red) mice detectable at study end (week 24). We conducted our analyses in triplicate (i.e., three repeats per RNA purification) on 9 control female mice and 9 lung tumor-bearing female mice. The EBC collected three times a week from the same 3 females was combined (~ 300 µL) to conduct the three different analyses (whole EBC, human exh-EVs, mouse exh-EVs) in triplicate [i.e., 3 sets of 3 EBC collections per control (n = 9) or lung tumor-bearing group (n = 9)]; (C) Venn Diagram displaying the overlap in the identity of the miRNAs detected between whole EBC of lung tumor-bearing mice (yellow), human exh-EVs in lung tumor-bearing mice (orange), mouse exh-EVs in control mice (purple), and human exh-EVs in controls mice (green, non-specific signal). The miRNAs that were selected for these analyses were detected by NGS but had at least 5 reads in 6 of the 9 samples analyzed and were reproducibly detected at least two of the three weeks (weeks 20, 21, and 22). The Venn diagram indicates that 21 miRNAs were non-specifically detected by use of the anti-human anti-CD63 EV-CATCHER assay with EBC of control mice and represented 9% of all selected miRNAs; (D) Small RNAs extracted from whole EBC samples collected at weeks 2, 6, and 11 were evaluated for expression of miR-222, miR-210, miR-374a, and miR-584 by Taqman^TM quantitative PCR analyses using the 2^ΔΔCt method to evaluate fold change by comparison to the control sample at week 2. All 4 miRNAs were selected because they were found to be upregulated in the whole EBC of lung tumor-bearing mice compared to control mice by NGS analyses.