Fig. 1
From: Tumor induces muscle wasting in mice through releasing extracellular Hsp70 and Hsp90
Cancer cachexia co-exists with high circulating levels of Hsp70 and Hsp90 associated with tumor cell-released EVs. a Cachexia-inducing tumor cells constitutively release high levels of Hsp70 and Hsp90. Hsp70 and Hsp90 content in conditioned media of cachexia-inducing tumor cells (LLC, C26, H1299, BxPC3 and AGS) are compared with that of non-cachexia-inducing tumor cell EL4 and non-tumorigenic cells (NL20, HPDC and C2C12 myotubes). All cells were incubated for 48 h in regular media (RPMI 160 or DMEM supplemented with 10% fetal bovine serum) and then analyzed for Hsp70 and Hsp90 content by ELISA. Data (n = 3) were analyzed by analysis of variance (ANOVA). b Cachexia co-exists with elevated circulating Hsp70 and Hsp90 in tumor-bearing mice. Hsp70 and Hsp90 levels in the sera of PBS-injected control, EL4 tumor- or LLC tumor-bearing C57BL/6 mice on day 21 of tumor implant, and Apcmin/+ mice that were pre-cachectic (12 weeks of age) or cachectic (20 weeks of age) were determined by ELISA. Data (n = 5) were analyzed by ANOVA (for C57BL/6 mice) or Student t-test (for Apcmin/+ mice). * denotes a difference among bracketed C57BL/6 mice or with pre-cachectic Apcmin/+ mice (P < 0.05). c Particle size of EVs isolated from LCM and the serum of cachectic LLC tumor-bearing mice were analyzed using ZetaView® Nanoparticle Tracking Analyzer. d Tumor-released Hsp70 and Hsp90 are associated with CD9/TSG101/AchE-positive EVs. EVs were isolated from LCM and subjected to immunoprecipitation using CD9 antibody with pre-immune IgG as control. EVs, precipitated pellet and supernatant were analyzed with western blotting. e Cachexia-inducing tumor cells constitutively release high levels of EVs. EVs were isolated from diverse tumor cell-conditioned media and quantified by AchE activity. Data (n = 3) were analyzed by ANOVA. f Cachexia is associated with elevated circulating EVs in tumor-bearing mice. EV levels in serum of indicated mice were determined. Data (n = 5) were analyzed by ANOVA (for C57BL/6 mice) or Student t-test (for Apcmin/+ mice). * denotes a difference among bracketed C57BL/6 mice or pre-cachectic Apcmin/+ mice (P < 0.05)
