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  • br aspirin treatment Fig E Western blot


    aspirin treatment (Fig. 3E). Western blot coupled with ELISA (Fig. 3F) indicated that the hypoxia-mediated activation of HIF-1α/COX-2/PGE2 signaling could be attenuated by aspirin; the second and third genera-tions of ALDH+ Biotin-HPDP showed similar results (Supplementary Fig. 1). These findings suggest that aspirin inhibits the hypoxia-mediated maintenance of stemness in ALDH+ cells.
    3.4. Aspirin affects the characteristics of exosomes secreted by lung cancer cells under hypoxic conditions
    Since cancer-derived exosomes contribute to TME recruitment and reprogramming, we isolated exosomes from the conditioned medium and characterized A549 exosomes by electron microscopy and Western blot. Transmission electron microscopic analysis revealed that the exosomes were smaller than 200 nm (Fig. 4A). CD63 and CD81, which commonly use membrane-bound exosomal markers, were detected in A549 exosomes. To validate the purity of the obtained exosomes, we examined the presence of the endoplasmic reticulum marker calnexin in A549 exosomes. A lack of calnexin expression indicated successful isolation of exosomes from the conditioned medium (Fig. 4B). Exosome quantification analysis showed that hypoxia increased the number of exosomes whereas aspirin reduced exosome numbers in aspirin-treated cells in hypoxia (Fig. 4C).
    HIF-1α may be involved in hypoxic enhancement of exosome re-lease in breast cancer cells [26]. We examined the expression of exo-somal HIF-1α and its downstream effector COX-2 after aspirin treat-ment under hypoxic conditions. In Fig. 4D, hypoxia resulted in a significant induction of exosomal HIF-1α and COX-2, and this hypoxia-mediated up-regulation was abrogated by aspirin. To study the effect of aspirin on exosomal miRNAs, miR-135b and miR-210 were selected as potential candidates by their ability to mediate the hypoxic effect in exosomes [24–26]; miR-16 was also chosen as a stable endogenous 
    reference miRNA [24,27]. While levels of exosomal miR-135b and miR-210 were elevated under hypoxic conditions, aspirin remarkably in-hibited the expression of these two exosomal miRNAs (Fig. 4E). These findings indicate that aspirin not only reduces hypoxia-enhanced exo-some numbers but also changes the content of exosomes from A549 cells.
    3.5. Aspirin inhibits the paracrine effect of hypoxic exosomes
    Since the cancer-promoting factors (HIF-1a, COX2, miR-135b, and miR-210) are abnormally elevated in hypoxic exosomes, we compared the exocrine effects of exosomes on other A549 cells under different conditions. As shown in Fig. 5A, compared with that in PBS, co-culture with hypoxic exosomes significantly increased cell proliferation after 48 or 72 h of treatment. A decrease in proliferation-promoting effect was observed in A549 cells co-cultured with exosomes secreted by aspirin-treated cells (Fig. 5A). Transwell migration assay showed that normoxic exosomes slightly promote the migration of A549 cells while the pre-sence of hypoxic exosomes significantly increases A549 cell migration. By contrast, the migration-promoting ability of exosomes from aspirin-treated cells was significantly down-regulated (Fig. 5B,C). In vitro an-giogenesis experiments showed similar changes in the angiogenesis capacity of HUVEC cells (Fig. 5D–F). Taken together, the results in-dicate that aspirin attenuates the paracrine-promoting effects of hy-poxic exosomes on the proliferation, migration, and angiogenesis of other cells.
    To further establish the relationship between proliferation and stemness of A549 cells and exosomes under hypoxic conditions, we used the exosome secretion inhibitor GW4869 for validation experi-ments. Currently, GW4869, a neutral sphingomyelinase inhibitor, is the most widely used pharmacological agent for blocking exosome gen-eration [28–30]. As shown in Fig. 6A, compared with that in DMSO, co-