Targeting cancer-derived extracellular vesicles by combining CD147 inhibition with tissue factor pathway inhibitor for the management of urothelial cancer cells

Abstract

Background: Extracellular vesicles (EVs), including microvesicles, hold promise for the management of bladder urothelial carcinoma (BLCA), particularly because of their utility in identifying therapeutic targets and their diagnostic potential using easily accessible urine samples. Among the transmembrane glycoproteins highly enriched in cancer-derived EVs, tissue factor (TF) and CD147 have been implicated in promoting tumor progression. In this in vitro study, we explored a novel approach to impede cancer cell migration and metastasis by simultaneously targeting these molecules on urothelial cancer-derived EVs.

Methods: Cell culture supernatants from invasive and non-invasive bladder cancer cell lines and urine samples from patients with BLCA were collected. Large, microvesicle-like EVs were isolated using sequential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and flow cytometry. The impact of urinary or cell supernatant-derived EVs on cellular phenotypes was evaluated using cell-based assays following combined treatment with a specific CD147 inhibitor alone or in combination with a tissue factor pathway inhibitor (TFPI), an endogenous anticoagulant protein that can be released by low-molecular-weight heparins.

Results: We observed that EVs obtained from the urine samples of patients with muscle-invasive BLCA and from the aggressive bladder cancer cell line J82 exhibited higher TF activity and CD147 expression levels than did their non-invasive counterparts. The shedding of GFP-tagged CD147 into isolated vesicles demonstrated that the vesicles originated from plasma cell membranes. EVs originating from invasive cancer cells were found to trigger migration, secretion of matrix metalloproteinases (MMPs), and invasion. The same induction of MMP activity was replicated using EVs obtained from urine samples of patients with invasive BLCA. EVs derived from cancer cell clones overexpressing TF and CD147 were produced in higher quantities and exhibited a higher invasive potential than those from control cancer cells. TFPI interfered with the effect when used in conjunction with the CD147 inhibitor, further suppressing homotypic EV-induced migration, MMP production, and invasion.

Conclusions: Our findings suggest that combining a CD147 inhibitor with low molecular weight heparins to induce TFPI release may be a promising therapeutic approach for urothelial cancer management. This combination can potentially suppress the tumor-promoting actions of cancer-derived microvesicle-like EVs, including collective matrix invasion.

Keywords: Bladder carcinoma; CD142; EMMPRIN; Microvesicles.

Fig. 1

Workflow of sample processing and experimental design. Conditioned media were collected over a 24 h incubation period from two human cell lines, RT4 and J82, grown in monolayer culture. Urine samples were collected from patients with bladder urothelial carcinoma. Extracellular vesicles (EVs) were isolated and characterized using scanning electron microscopy (SEM), nanoparticle tracking analysis (NTA), and high-resolution flow cytometry. The homotypic effects of EVs were evaluated via cell-based functional assays with different readouts of cellular phenotypes (A). Cell and EV morphology were visualized using light microscopy and SEM, respectively (B). Cells (C) and cell- and urine-derived Evs (D) were analyzed for surface markers. The particle number and size distribution of the EVs were analyzed in unfractionated conditioned medium, centrifugation supernatant containing small EVs (sEVs), and centrifugation pellet containing large, microvesicle-like EVs (lEVs) by NTA (E), and the presence of tetraspanin EV markers was analyzed by flow cytometry (F) of both the EV pellet and centrifuge supernatant derived from muscle-invasive J82 cells. The parameters of the urine-derived EVs are presented as medians with 10th and 90th percentiles and were compared using the non-parametric Mann–Whitney U test. MFI, mean fluorescence intensity

Fig. 2

Association of tissue factor (TF) and CD147 overexpression with extracellular vesicle (EV) production and invasion potential. The expression of green fluorescent protein (GFP)-fusion CD147 on the plasma membrane of J82 cells was observed using confocal microscopy (A). To assess the vesicular export of CD147, GFP-labeled vesicles were derived from J82 cells transfected with the CD147-GFP construct and analyzed using flow cytometry (B). The lysates of the EVs were prepared using 0.1% Triton™ X-100. EVs were derived from human bladder urothelial carcinoma J82 cells co-transfected with TF and CD147 plasmids. The EVs were quantified using NTA in the 48-h culture supernatant of J82 cells overexpressing CD147 and TF versus that of the control transfection condition (C). Invasion potency was measured by a Transwell assay, with 6–8 images of the cells that had passed through the coated microporous membrane after 24 h per experiment (D)

Fig. 3

Impact of the combined inhibitors treatment on extracellular vesicle (EV)-induced migration potential. J82 human bladder urothelial carcinoma cells were exposed to 150 µg/ml protein from J82 cell-derived EVs and treated with either 5 µM CD147 inhibitor AC-73 alone (A) or a combination of 2.5 µM AC-73 and tissue factor pathway inhibitor (TFPI, 300 ng/ml, B) for 16 h. Relative migration was quantified via a Transwell assay utilizing 6–8 images of cells that traversed the microporous membrane per experiment

Fig. 4

Impact of treatment with the combined inhibitors on extracellular vesicle (EV)-induced matrix metalloproteinases (MMPs) release. J82 human bladder urothelial carcinoma cells were treated with J82 cell-derived EVs or with a pool of isolated EVs from urine samples collected from patients with muscle-invasive bladder cancer (A), along with either 2.5 µM or 5 µM of the CD147 inhibitor AC-73, in the absence or presence of tissue factor pathway inhibitor (TFPI, 300 ng/ml, B) for 24 h. Recombinant CD147 (rCD147) was used as a positive control to induce MMP activity. The induction of MMPs was measured using colorimetric enzymatic methods

Fig. 5

Impact of treatment with the combined inhibitors on extracellular vesicle (EV)-induced invasion potential. J82 human bladder urothelial carcinoma cells were exposed to 150 µg/ml protein from J82 cell-derived EVs or with a pool of isolated EVs from urine samples collected from patients with muscle-invasive bladder cancer, and treated with either 5 µM of the CD147 inhibitor AC-73 alone (A) or a combination of 2.5 µM AC-73 and tissue factor pathway inhibitor (TFPI, 300 ng/ml, B) for 24 h. Invasion potency was measured via a Transwell assay utilizing 6–8 images of cells that traversed the microporous membrane per experiment

Fig. 6

Impact of treatment with the combined inhibitors on the extracellular vesicle (EV) binding capability. J82 human bladder urothelial carcinoma cells were exposed in suspension to carboxyfluorescein succinimidyl ester fluorescent dye (CFSE)-labeled J82 cell-derived EVs and treated with either 5 µM CD147 inhibitor AC-73 alone or a combination of 2.5 µM AC-73 and tissue factor pathway inhibitor (TFPI, 300 ng/ml). The cells were analyzed for green fluorescence in the FITC channel using flow cytometry