The application of mRNA-based gene transfer in mesenchymal stem cell-mediated cytotoxicity of glioma cells

Abstract

Since the tumor-oriented homing capacity of mesenchymal stem cells (MSCs) was discovered, MSCs have attracted great interest in the research field of cancer therapy mainly focused on their use as carries for anticancer agents. Differing from DNA-based vectors, the use of mRNA-based antituor gene delivery benefits from readily transfection and mutagenesis-free. However, it is essential to verify if mRNA transfection interferes with MSCs' tropism and their antitumor properties. TRAIL- and PTEN-mRNAs were synthesized and studied in an in vitro model of MSC-mediated indirect co-culture with DBTRG human glioma cells. The expression of TRAIL and PTEN in transfected MSCs was verified by immunoblotting analysis, and the migration ability of MSCs after anticancer gene transfection was demonstrated using transwell co-cultures. The viability of DBTRG cells was determined with bioluminescence, live/dead staining and real time cell analyzer. An in vivo model of DBTRG cell-derived xenografted tumors was used to verify the antitumor effects of TRAIL- and PTEN-engineered MSCs. With regard to the effect of mRNA transfection on MSCs' migration toward glioma cells, an enhanced migration rate was observed with MSCs transfected with all tested mRNAs compared to non-transfected MSCs (p<0.05). TRAIL- and PTEN-mRNA-induced cytotoxicity of DBTRG glioma cells was proportionally correlated with the ratio of conditioned medium from transfected MSCs. A synergistic action of TRAIL and PTEN was demonstrated in the current co-culture model. The immunoblotting analysis revealed the apoptotic nature of the cells death in the present study. The growth of the xenografted tumors was significantly inhibited by the application of MSCPTEN or MSCTRAIL/PTEN on day 14 and MSCTRAIL on day 28 (p<0.05). The results suggested that anticancer gene-bearing mRNAs synthesized in vitro are capable of being applied for MSC-mediated anticancer modality. This study provides an experimental base for further clinical anticancer studies using synthesized mRNAs.

Exosomes derived from microRNA-199a-overexpressing mesenchymal stem cells inhibit glioma progression by down-regulating AGAP2

“…FITC-labeled mouse anti-human antibodies against CD34, CD44, CD73, CD90 and CD105 were used to identify surface antigens by flow cytometry. CD44, CD73, CD90 and CD105 were used as markers of hMSCs and CD34 was used as marker for hematopoietic stem cells [21]. We found that CD44, CD73, CD90 and CD105 were all positively expressed in hMSCs while CD34 was negatively expressed, which implies the successful isolation and culture of hMSCs (Figure 6A).…”

Intracranial Delivery of Synthetic mRNA Using Common Transfection Reagent

“…In vitro synthesis of Luc-mRNA and TRAIL-mRNA Luc-mRNA and TRAIL-mRNAs were synthesized in vitro as previously described [6]. Brie y, the human 5'UTR with Kozak sequence and 3'UTR sequence were commercially synthesized by Integrated DNA Technologies (Coralville, Iowa) and sub-cloned into pcDNA3.3.…”

“…The cell viability was detected by real-time assessment by the xCELLigence cell analyzer (RTCA, Roche, USA) as previously described [6]. A volume of 100 μl of DBTRG-Luc cell suspension (5 × 10 3 cells) was seeded in E-Plate 16.…”

Therapeutic Potential of Mesenchymal Stem Cells for Cancer Therapy

“…A recent study using lentiviral transductions showed that human umbilical cord MSCs expressing interleukin-18 inhibit the proliferation and metastasis of breast cancer in mice (Liu et al, 2018). Genetically engineered MSCs with TRAIL have also shown strong anti-tumor activity in different types of cancer (Ciavarella et al, 2012;Fakiruddin et al, 2014;Guo et al, 2016;Jiang et al, 2016). In a fascinating study, X. Jiang and colleagues developed a non-viral method using nanoparticles to produce human MSCs engineered to express the suicide protein TRAIL for targeting and eradicating intracranial gliomas in mice (Jiang et al, 2016).…”

“…For instance, Bone Morphogenetic Protein 4 (BMP4)expressing MSCs were found to efficiently suppress tumor growth and prolong survival of glioma-bearing mice (Li et al, 2014;Mangraviti et al, 2016). Similarly, MSCs modified to express the tumor-suppressor gene Phosphatidylinositol 3,4,5-Trisphosphate 3-Phosphatase (PTEN) induced cytotoxicity of glioma cells (Guo et al, 2016).…”