Mesenchymal stem cells deliver synthetic microRNA mimics to glioma cells and glioma stem cells and inhibit their cell migration and self-renewal

Abstract

MicroRNAs (miRNAs) have emerged as potential cancer therapeutics; however, their clinical use is hindered by lack of effective delivery mechanisms to tumor sites. Mesenchymal stem cells (MSCs) have been shown to migrate to experimental glioma and to exert anti-tumor effects by delivering cytotoxic compounds. Here, we examined the ability of MSCs derived from bone marrow, adipose tissue, placenta and umbilical cord to deliver synthetic miRNA mimics to glioma cells and glioma stem cells (GSCs). We examined the delivery of miR-124 and miR-145 mimics as glioma cells and GSCs express very low levels of these miRNAs. Using fluorescently labeled miRNA mimics and in situ hybridization, we demonstrated that all the MSCs examined delivered miR-124 and miR-145 mimics to co-cultured glioma cells and GSCs via gap junction- dependent and independent processes. The delivered miR-124 and miR-145 mimics significantly decreased the luciferase activity of their respected reporter target genes, SCP-1 and Sox2, and decreased the migration of glioma cells and the self-renewal of GSCs. Moreover, MSCs delivered Cy3-miR-124 mimic to glioma xenografts when administered intracranially. These results suggest that MSCs can deliver synthetic exogenous miRNA mimics to glioma cells and GSCs and may provide an efficient route of therapeutic miRNA delivery in vivo.

Figure 1. MSCs deliver miRNAs to co-cultured glioma cells

The expression of miR-124 and miR-145 was examined in NSCs, normal human astrocytes (NHA), the glioma cells U87 and A172, the GSCs HF2414, HF2485 and HF2587 and in the different MSC preparations using real time PCR (A). BM-MSCs were transfected with Cy3 labeled miR-124 or miR-145 mimics. After 24 h, U87 or A172 cells labeled with Green CellTracker were added to the MSC cultures and the expression of the fluorescent miR-124 in the A172 cells or miR-145 in the U87 cells was analyzed 24 h later using a fluorescence microscope (B). The delivery of Cy3 miR-124 by BM-MSC to the A172 cells was also analyzed using FACS analysis; a - double negative co-cultured cells; b – co-cultured cells with green CellTracker-labeled A172 cells; c – co-cultured cells with MSCs transfected with Cy3-miR-124; d – co-cultured cells with double positive cells (C). BM-MSCs were transfected with a miR-145 mimic and were co-cultured with A172 cells labeled with CellTracker Red for an additional 24 h. In situ hybridization of miR-145 was then performed and the red labeled cells were visualized for the presence of green labeled miR-145. The cells were counted in each of ten random fields (D). The results are the means ± SE of three different experiments (A) or are representative of three different experiments that gave similar results (B-D). *p < 0.001.

Figure 2. MSC-delivered miR-124 mimic downregulates the expression of SCP-1 in glioma cells

U87 cells were transfected with either a control miR (Con-miR) or a miR-124 mimic and the expression of SCP-1 was examined using qRT-PCR (A) or Western blot analysis (B) after 3 days. U87 cells were transfected with a SCP-1 3’-UTR-luciferase plasmid followed by transfection with Con-miR or miR-124 mimic. In parallel, U87 cells expressing this plasmid were co-cultured with BM- MSCs or AD-MSCs that were transfected with either a con-miR or miR-124 mimic (C). The luciferase activity of the cells was determined after 72 h of transfection or co-culture (C). The results are the means ± SE of three different experiments. *p < 0.001.

Figure 3. Role of gap junction, exosomes and contact-independent miRNA delivery by MSCs

U87 cells transfected with a plasmid expressing SCP-1 3’-UTR-luciferase were co-cultured with BM-MSCs that were transfected with either a con-miR or miR-124 mimic. The cells were treated with carbenoxolone (150 μM) and the luciferase activity of the cells was determined after 48 h of co-culture (A). The contact independent miRNA delivery by MSCs was examined using a transwell chamber with a 0.4 μm filter. MSCs transfected with either a control miR or miR-124 were plated on the upper compartment, whereas U87 cells transfected with SCP-1 3’-UTR-luciferase were seeded on the bottom. Following 48 h, the level of miR-124 was examined in the U87 cells by real time PCR (B) and the luciferase activity was determined as described in Figure 2 (C). BM-MSCs were infected with a lentivirus vector expressing CD63-GFP and transfected with a Cy3 miR-124 mimic. The colocalization of the exosomes and miR-124 was analyzed by a fluorescence microscope and is demonstrated by the yellow dots in the merge image (D). Exosomes were isolated from BM-MSCs using the Exoquick kit or by ultracentrifugation as described in Methods. Both preparations expressed similar levels of CD81, CD9 and Alix (E, Figure S2). The levels of the exosome-delivered miR-124 in U87 cells were determined by real time PCR (F), and the effect of the exosomes on the expression of SCP-1 in these cells was demonstrated by Western blot analysis (G) and by the luciferase activity of U87 cells transfected with the SCP-1 3’-UTR reporter plasmid (H). BM-MSCs (5×10⁶ cells) were treated with GW4869 (5 μM), exosomes were isolated from control and treated cells using the Exoquick kit as described in the methods and the expression of CD81, CD8 and Alix was determined by Western blot analyses (I). BM-MSCs transfected with either control miR or miR-124 were co-cultured with U87 expressing the reporter plasmid SCP-1 3’-UTR. The co-cultures were treated with and without 5 μM GW4869 and the luciferase activity was measured and compared to activity in medium-treated cells (J). The results are the means ± SE of three different experiments (A,B,C,F,H) or are representative of three different experiments (D,E,G,I). *p < 0.001, **p<0.01.

Figure 4. MSC-delivered miR-124 decreases the migration of glioma cells

U87 cells were transfected with either control miR or miR-124 mimic and cell migration for 4 h was determined 48 h later using transwell migration (A). U87 cells (A,B) or U87 cells labeled with Red CellTracker (C,D) were cultured with BM-MSCs transfected with either a con-miR or miR-124 mimic. The migration of the co-cultured cells (A,B) and the Red CellTracker labeled U87 cells (C,D) was determined after 48 h using transwell migration assay. The results are representative of three different experiments that gave similar results. *p < 0.001.

Figure 5. MSCs deliver miRNA mimics to GSCs and decrease their self-renewal

BM-MSCs were transfected with Cy3 miR-124 or Cy3 miR-145 mimics (A) and AD-MSCs or MSCs derived from umbilical cord or placenta were transfected with Cy3 labeled miR-124 (B). After 24 h, HF2414 GSCs labeled with Green CellTracker were added to the cultured MSCs for an additional 48 h. The levels of the fluorescent miR-124 and miR-145 (A) and miR-124 (B) were analyzed using a fluorescence microscope. HF2414 or HF2485 GSCs transfected with a control miR or miR-124 mimic (-MSCs) or co-cultured with BM-MSCs transfected with either a control miR, miR-145 (C) or miR-124 (H) mimics were collected after 48 h and analyzed for self renewal for 14 days. HF2414 and HF2485 GSCs were transfected with a miR-145 mimic. After 48 h, the cells were analyzed for the expression of Sox2 mRNA using real time PCR (D) and Sox2 protein using Western blot analysis (E). BM-MSC or AD-MSCs were transfected with a control miR, miR-145 (F) or miR-124 (G) mimics. After 24 h, HF2414 GSCs transfected with either the Sox-2 3’-UTR-luciferase (F) or the SCP-1 3’-UTR-luciferase (G) plasmids were added to the cultured MSCs. The luciferase activity of Sox2-3’UTR (F) or the SCP-1-3’UTR (G) expressed in the GSCs was analyzed after 48 h. The results are representative of three different experiments that gave similar results. *p < 0.001.

Figure 6. In vivo transfer of Cy3-miR-124 mimic to glioma xenografts by BM-MSCs

Glioma xenografts were established from U87-GFP cells. Twenty-two days post tumor cell implantation, BM-MSCs transfected with a Con-miR or with Cy3-mir-124 for 24 h, were injected into the ipsilateral hemisphere (n = 8 for each group), and animals were sacrificed 3 days later. Frozen brain sections were viewed under fluorescence microscopy. H&E staining of frozen sections and fluorescent microscopy demonstrated the presence of MSCs transfected with Cy3-miR-124 in the tumor periphery and within the tumor (A). Merged images (right panels) of GFP-fluorescent tumor cells (left panels) with Cy3-labeled miR-124-transfected MSCs (middle panels) demonstrate Cy3-miR-124 within tumor cells (B). The results are representative of eight different fields that were viewed in each of five different mice (magnification X40). The expression of miR-124 was examined in sorted GFP- positive disaggregated tumor cells derived from 8 mice with U87 xenografts treated with MSCs transfected with a control miR or from 8 mice with U87 xenografts treated with MSCs transfected with Cy3 miR-124 using real-time PCR as described in the methods (C). *p<0.05.