Composition of EVs markers under normoxic and hypoxic conditions depends on the expression level of adaptor protein Ruk/CIN85 in mouse renal carcinoma Renca cells

Abstract Aim: To isolate and characterize extracellular vesicles (EVs) produced by mouse renal carcinoma Renca cells with different expression levels of the adaptor protein Ruk/CIN85 under normoxia and hypoxia conditions. Methods: The density gradient centrifugation was used to isolate EVs from the conditioned medium of Renca cells cultured under normoxia and hypoxia conditions. Further characterization of EVs was performed by using nanoparticle tracking analysis (NTA), electron microscopy and Western Blot analysis. Results: Significant differences in average particle size between EVs produced by sublines studied under experimental conditions were not found. At the same time, concentration of particles produced by Ruk/CIN85 overexpressing cells turned out to be an order of magnitude higher in hypoxia in comparison to normoxia conditions. It was shown that under normoxia conditions the content of both Ruk/CIN85 and EVs’ markers Alix and CD81 was increased in vesicles produced by Renca cells with Ruk/CIN85 overexpression in comparison with those from control mock-transfected cells. Under hypoxia conditions, the content of studied proteins decreased by more than two orders of magnitude in EVs secreted by Renca cells with up-regulation of adaptor protein whereas the content of Ruk/CIN85 and CD81 increased in EVs from mock-transfected cells. Conclusions: It has been demonstrated that the adaptor protein Ruk/CIN85 is a novel component of EVs produced by tumor cells that may play a role in the control of EV composition under normoxia and hypoxia

Extracellular vesicles produced by mouse breast adenocarcinoma 4T1 cells with up- or down-regulation of adaptor protein Ruk/CIN85 differentially modulate the biological properties of 4T1 WT cells

Abstract Extracellular vesicles (EVs) are secreted by most cell types under both physiological and pathological conditions and were proposed to be actively involved in intercellular communication. The mode of EVs action is dependent on their cargos composition. EVs play an important role in tumor initiation, recurrence, metastasis and therapeutic resistance. EVs marker proteins Alix and Tsg101 and cortactin are the binding partners of adaptor protein Ruk/CIN85. The present study aims to analyze the regulatory effects of EVs produced by 4T1 cells with overexpression (RukUp) or down-regulation (RukDown) of adaptor protein Ruk/CIN85 on proliferation rate, migration and invasion activity of parental 4T1 WT cells. EVs from conditioned medium of 4T1 RukUp or RukDown cells were isolated by differential centrifugation followed by further purification using Exo-spin™ kit (Cell Guidance Systems). The number and size of EVs were characterized by NTA (Malvern Panalytical NanoSight NM300) instrument. The content of marker proteins and Ruk/CIN85 in isolated EVs was analyzed by Western-blotting. The viability, migration and invasion activity of 4T1 WT cells were studied using MTT-test, scratch-test and Boyden chamber assay, respectively. It was demonstrated for the first time that adaptor protein Ruk/CIN85 is a constitutive component of EVs produced by 4T1 cells. It was also shown that EVs produced by 4T1 cells with different levels of Ruk/CIN85 expression are characterized by a specific profile of the content of its multiple molecular forms. It turned out that the ability of EVs to modulate the proliferative activity, motility and invasiveness of 4T1 WT cells was tightly correlated with the biological properties of 4T1 cells that produce EVs (highly aggressive 4T1 RukUp cells or weakly invasive 4T1 RukDown cells). Our data suggest that adaptor protein Ruk/CIN85 is not only a constitutive component of cargos composition of EVs produced by tumor cells but, depending on its content in EVs, plays an active role in the control of carcinogenesis