Functional roles of tissue factor and EMMPRIN/CD147 present on bladder urothelial carcinoma-derived extracellular vesicles.
Authors: Darabi Masoud , Boddu Vijay Kumar , Zamzow Piet , Siggel Constanze , Gorantla Sivahari Prasad , Kramer Mario W. , Merseburger Axel S. , Cramer Lena , von Bubnoff Nikolas , Jafari Mehrdad , Gieseler Frank 
Published: June 2023

e16557 Background: Extracellular vesicles are involved in the homeostatic functions and extracellular matrix remodeling in the body. We previously found that the antithrombotic drug Tinzaparin, at clinical concentrations, induces the sufficient release of the tissue factor pathway inhibitor TFPI to functionally inhibit the prothrombotic effector tissue factor expressed on the vesicles released by malignant tumor cells. Treatment of in vitro cancer cells with Tinzaparin results in the inhibition of the tissue factor/ERK pathway. Our hypothesis is that vesicles derived from metastatic urinary tumors are enriched in tissue factor; therefore, their elusive functioning is affected by the suppressed tissue factor caused by TFPI. Here, we show that this observation may have major clinical relevance. Methods: To test the contribution of extracellular vesicles to the mechanism and extent of cell invasion, we assessed two human adherent bladder urothelial carcinoma cell lines, RT4 and J82, which represent non-muscle and muscle-invasive diseases, respectively. Urine samples were obtained from a cohort of 75 outpatients who underwent transurethral resection subdivided by the presence of cancer or muscle invasion. Vesicles were isolated using sequential centrifugation and further validated for their size, nanoparticle tracking analysis, and surface markers using flow cytometry. The tissue factor activity index levels of the vesicles and the potential for in vitro induction of matrix metalloproteinases (MMPs) were quantified using colorimetric enzymatic methods. The levels of phosphorylated and total Erk1/2 were measured using an in vitro kinase assay and western blot. Results: Vesicles derived from the invasive cells J82 displayed higher levels of the MMP inducer EMMPRIN/CD147. The distinct pattern of surface markers in muscle-invasive-derived vesicles, featuring enhanced potential for inducing Erk1/2 phosphorylation and in vitro MMP activation as key processes of extracellular matrix degradation, was drastically suppressed by a potent, specific small molecule CD147 inhibitor. There was higher tissue factor activity, a key initiator of coagulation, among the patients with bladder urothelial carcinoma than those without. In the subgroup analysis, the tissue factor activity index and CD147 were higher in patients with muscle-invasive disease than in patients with non-muscle-invasive disease, showing high accuracy in distinguishing patients regarding invasion potential. Conclusions: The tissue factor activity index of urine extracellular vesicles may be considered a marker of bladder urothelial carcinoma. In addition to tissue factor expression, CD147 is crucial in promoting the potential of extracellular vesicles to induce MMP-mediated extracellular matrix degradation and, consequently, cell invasion, thereby allowing metastatic disease progression.