Extracellular vesicle-mediated transfer of CLIC1 protein is a novel mechanism for the regulation of glioblastoma growth

Abstract

Little progresses have been made in the treatment of glioblastoma (GBM), the most aggressive and lethal among brain tumors. Recently we have demonstrated that Chloride Intracellular Channel-1 (CLIC1) is overexpressed in GBM compared to normal tissues, with highest expression in patients with poor prognosis. Moreover, CLIC1-silencing in cancer stem cells (CSCs) isolated from human GBM patients negatively influences proliferative capacity and self-renewal properties in vitro and impairs the in vivo tumorigenic potential. Here we show that CLIC1 exists also as a circulating protein, secreted via extracellular vesicles (EVs) released by either cell lines or GBM-derived CSCs. Extracellular vesicles (EVs), comprising exosomes and microvesicles based on their composition and biophysical properties, have been shown to sustain tumor growth in a variety of model systems, including GBM. Interestingly, treatment of GBM cells with CLIC1-containing EVs stimulates cell growth both in vitro and in vivo in a CLIC1-dose dependent manner. EVs derived from CLIC1-overexpressing GBM cells are strong inducers of proliferation in vitro and tumor engraftment in vivo. These stimulations are significantly attenuated by treatment of GBM cells with EVs derived from CLIC1-silenced cells. However, CLIC1 modulation appears to have no direct role in EV structure, biogenesis and secretion. These findings reveal that, apart from the function of CLIC1 cellular reservoir, CLIC1 contained in EVs is a novel regulator of GBM growth.

Keywords: cancer stem cells; cell proliferation; extracellular vesicles; glioblastoma; tumor growth.

Figure 1. CLIC1 protein is secreted by glioblastoma cells

A. CLIC1 expression was analyzed by Western blot in cell lysates (left panel) and conditioned media (right panel) of several GBM cell lines. GAPDH was used as loading control. A representative immunoblot is shown. B. U87 MG cells were mock infected or infected with CLIC1 GFP fusion protein. Cells lysates and conditioned media were immunoblotted (Input), or immunoprecipitated against GFP-tag (IP anti-GFP) and then immunoblotted with CLIC1 antibody. A representative immunoblot is shown. C. U87 MG cells expressing Green Fluorescent Protein (GFP) were co-cultured with U87 MG cells expressing the FLAG-tagged isoform of CLIC1 for 24 hours. Cells were stained using anti-FLAG antibody (red). Arrowheads mark FLAG-tagged isoform of CLIC1 on U87 MG GFP cells. Scale bar, 20 μm.

Figure 2. CLIC1 protein is secreted by GBM cells via Evs

A. Size profile of EVs released by U87 MG cells measured by NTA. Shown the profile from a representative experiment. B. EVs were divided into four different dimensional classes, and their abundance was measured by NTA. Shown the profile from a representative experiment. C. Representative transmission electron micrograph of GBM cell-derived EVs (100 nm EVs, white arrow; 40 nm EVs, black arrows, Upper). Representative immuno-EM image of CD63 staining of GBM cell-derived EV (Lower). Scale bar, 100 nm. At least 200 EVs from 10 sections were examined in two independent experiments. D. Whole cell lysates (WCL) from different GBM cell lines and lysates from corresponding EVs were analysed for the indicated proteins by Western blotting. A representative immunoblot is shown. E. Representative immuno-EM image of GBM cell-derived EVs. Double-staining was conducted using anti-CD63 antibody (10 nm gold particles, indicated by arrowheads) and anti-CLIC1 antibody (15 nm gold particles, indicated by arrows) Scale bar, 110 nm. At least 300 EVs from 30 sections were examined in three independent experiments. F. CLIC1 and CD63 colocalization was assessed in U87 MG cells by in situ proximal ligation assay (PLA). Scale bar, 20 μm. At least 10 cells from 5 sections were examined in three independent experiments.

Figure 3. CLIC1-containing EVs regulate the proliferative response of GBM cells

A. Whole cell lysates (WCL) obtained from U87 MG control cells (NT), CLIC1 silenced cells (siCLIC1) and CLIC1 overexpressing cells (CLIC1 FLAG), as well as lysates of corresponding EVs, were analysed by Western blotting using antibodies against CLIC1, and the EV markers CD63 and tsg101. Vinculin was used as loading control. A representative immunoblot is shown. B. U87 MG cells were incubated with EVs (50 μg/ml) derived from U87 MG NT, U87 MG siCLIC1 and U87 MG CLIC1 FLAG cells, or PBS as control (veichle). Cell growth was measured 72, 96 and 120 hours after EV administration. Three independent experiments were performed; error bars represent standard error;

Figure 4. CLIC1-containing EVs enhance GBM growth in vivo

U87 MG cells were incubated with EVs (1 μg/ml) derived from U87 MG NT, U87 MG siCLIC1 and U87 MG CLIC1 FLAG cells and subcutaneously injected into one flank of nude mice (n = 30 animals per group). As controls, untreated U87 MG cells were injected as well. Average tumor volumes at the indicated time points were measured. B. Tumors that formed after treatments described in A were surgically resected and weighted. Three independent experiments were performed. Error bars in A represent standard errors; solid lines in B are median values while error bars stand for minimum and maximum values. GLM tests of between-subjects effects showed statistically significant difference for the relative tumor growth according to the time, the treatment, and the interaction between those two variables. *p < 0.05.

Figure 5. CLIC1 secreted protein resides in EVs released from GBM CSCs

A. Representative immunoblots showing the expression of CLIC1 in three different GBM CSCs and in their respective media. GAPDH was used as loading control. B. Size profile of GBM CSC-derived EVs measured by NTA. Shown is the profile from a representative experiment. C. hGBM#10 CSC whole cell lysates (WCL) and lysates from corresponding EVs were analysed for the indicated proteins by Western blotting. A representative immunoblot is shown. D. Whole cell lysates (WCL) obtained from control hGBM#10 CSCs (NT) and CLIC1 silenced hGBM#10 CSCs (siCLIC1), as well as lysates of corresponding EVs, were analysed by Western blotting using antibodies against CLIC1, and the EV markers CD63 and tsg101. A representative immunoblot is shown. E. hGBM#10 CSCs were cultured in presence of EVs (50 μg/ml) derived from either NT or siCLIC1 hGBM#10 CSCs for 120 hours and assessed for cell growth by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Three independent experiments were performed; error bars represent standard error; *p < 0.05.

Figure 6. EV size and yield are not affected by CLIC1 modulation in GBM cells

A. Size distribution of EVs shedded by U87 MG NT, U87 MG siCLIC1 and U87 MG CLIC1 FLAG cells, measured by NTA. B. The yield of EVs shedded by U87 MG NT, U87 MG siCLIC1 and U87 MG CLIC1 FLAG cells, measured by NTA. C. EVs were divided into three different dimensional classes, and their abundance was measured by NTA. A-C data are from a representative experiment. D. EVs derived from U87 MG NT, U87 MG siCLIC1 and U87 MG CLIC1 FLAG cells were labelled with PKH26 dye, and added to U87 MG recipient cells for 24 hours. EV uptake efficiency was expressed as percentage of PKH26 positive cells measured at different time points. Two-way ANOVA. Contribution of “treatment” and “time”: *p < 0.05. Results shown in B-D represent mean ± standard errors from three independent experiments.