From motion to molecules: exercise-triggered large EVs suppress oncogenic signaling in lung adenocarcinoma cells - an...

Although regular physical activity is known to reduce cancer risk, the circulating molecular mechanisms responsible for these effects remain incompletely defined. Exercise alters the release and composition of extracellular vesicles (EVs), suggesting a role in systemic anti-tumor signaling. However, the contribution of large EVs (L-EVs) ─ a distinct and understudied EV subtype ─ to the regulation of lung adenocarcinoma cell behaviour has not been examined to date.

L-EVs were isolated from platelet-free plasma of healthy, regularly exercising older adults with over 25 years of training history (aged 61 ± 2 years), collected at rest (Pre) and after (Post) an acute endurance exercise bout. EV fractions were characterized via transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and antibody-based profiling. PC9 lung adenocarcinoma cells were incubated with plasma-derived L-EV-enriched fractions for 24 h. Cell viability and metabolic activity were evaluated using ATP-based luminescence and XTT assays following L-EV treatment. miRNA and gene expression changes were assessed using RT-qPCR with TaqMan arrays. Differentially expressed miRNAs (DEmiRs) and genes (DEGs) were analysed using Ingenuity Pathway Analysis (IPA) to evaluate regulatory networks and predict functional outcomes.

Exercise significantly increased plasma nanoparticle concentration (mean increase of 5.05 × 10⁷ ± 9.99 × 10⁶ particles/mL; p < 0.001, Cohen's d = 1.685). TEM and NTA confirmed successful isolation of L-EVs, with mode sizes of 136.1 ± 23.6 nm pre-exercise and 129.5 ± 17.7 nm post-exercise. In PC9 cells, both Pre- and Post-L-EVs reduced metabolic activity compared to control conditions, as demonstrated by ATP-based and XTT assays, indicating a functional impact on cell viability (p < 0.001). Transcriptomic analysis revealed distinct expression changes between conditions. Post-L-EVs downregulated oncogenic and immune evasion-associated genes (MAX, FASLG, and ELK1) while upregulating pro-apoptotic and cell cycle inhibitory genes (CASP9, FADD, and CDKN2B). Several DEmiRs, including miR-21-5p, miR-301b-3p, and miR-193a-3p, shifted in directions opposite to their known oncogenic patterns in NSCLC, suggesting a tumor-suppressive influence. IPA predicted enhanced apoptotic signaling and reduced metastatic potential following Post-L-EV treatment.

Exercise-conditioned plasma-derived enriched L-EVs modulate gene and miRNA expression in lung adenocarcinoma cells toward a tumor-suppressive profile. By attenuating oncogenic and immune-evasion signals while enhancing apoptotic and cell cycle-inhibitory pathways, Post-L-EVs offer preliminary mechanistic insight into the anti-cancer potential of physical activity. Their origin from older adults engaged in long-term exercise highlights the systemic benefits of sustained activity and the promise of L-EVs as biomarkers of exercise responsiveness.