Abstract: The present disclosure provides a single mouse exhaled breath collection device and proof-of-concept analyses utilizing an ultra-sensitive and customizable EV purification assay, EV-CATCHER® (Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release) assay. Preliminary studies show that this assay work well for the isolation of exhaled extracellular vesicles (exh-EVs) from exhaled breath condensates (EBC) to capture and characterize miRNAs contained in exh-EVs. Because assembling a large enough cohort of asymptomatic subjects requires time, a complementary approach of using humanized animal models has been chosen to comprehensively assess the robustness and sensitivity of transcriptomic (miRNA and mRNA) and proteomic biomarkers contained in tumor exh-EVs and to enable sensitive detection of human primary and secondary lung cancers during their early development.