title: Characterization of uptake and internalization of exosomes by bladder cancer cells.
abstract: Bladder tumors represent a special therapeutic challenge as they have a high recurrence rate requiring repeated interventions and may progress to invasive or metastatic disease. Exosomes carry proteins implicated in bladder cancer progression and have been implicated in bladder cancer cell survival. Here, we characterized exosome uptake and internalization by human bladder cancer cells using Amnis ImageStreamX, an image cytometer. Exosomes were isolated by ultracentrifugation from bladder cancer culture conditioned supernatant, labeled with PKH-26, and analyzed on the ImageStreamX with an internal standard added to determine concentration. Exosomes were cocultured with bladder cancer cells and analyzed for internalization. Using the IDEAS software, we determined exosome uptake based on the number of PKH-26+ spots and overall PKH-26 fluorescence intensity. Using unlabeled beads of a known concentration and size, we were able to determine concentrations of exosomes isolated from bladder cancer cells. We measured exosome uptake by recipient bladder cancer cells, and we demonstrated that uptake is dose and time dependent. Finally, we found that uptake is active and specific, which can be partially blocked by heparin treatment. The characterization of cellular uptake and internalization by bladder cancer cells may shed light on the role of exosomes on bladder cancer recurrence and progression.
authors.affiliation: Department of Urology, Loyola University Chicago, Maywood, IL 60153, USA.
authors.forename: Carrie A
authors.initials: CA
authors.lastname: Franzen
authors.affiliation: FACS Core Facility, Research Services, Loyola University Chicago, Maywood, IL 60153, USA.
authors.forename: Patricia E
authors.initials: PE
authors.lastname: Simms
authors.affiliation: Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA.
authors.forename: Adam F
authors.initials: AF
authors.lastname: Van Huis
authors.affiliation: Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA ; Oncology Institute, Loyola University Chicago, Maywood, IL 60153, USA ; Department of Pathology, Loyola University Chicago, Maywood, IL 60153, USA.
authors.forename: Kimberly E
authors.initials: KE
authors.lastname: Foreman
authors.affiliation: Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA ; Oncology Institute, Loyola University Chicago, Maywood, IL 60153, USA ; Department of Surgery, Loyola University Chicago, Maywood, IL 60153, USA.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Kuo
authors.affiliation: Department of Urology, Loyola University Chicago, Maywood, IL 60153, USA ; Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA ; Oncology Institute, Loyola University Chicago, Maywood, IL 60153, USA ; Department of Surgery, Loyola University Chicago, Maywood, IL 60153, USA.
authors.forename: Gopal N
authors.initials: GN
authors.lastname: Gupta
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival; D004705:Endocytosis / Q000235:genetics*; D055354:Exosomes; D006801:Humans; D009364:Neoplasm Recurrence, Local / Q000378:metabolism* / Q000473:pathology; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology