| More than of90%of all oral neoplasms are oral squamous cell carcinoma (OSCC),80%of which are tougue squamous cell carcinoma(TSCC). However, despite the advances in the standard treatment guidelines (i.e., surgery and radiotherapy), the mortality rate of OSCC has remained largely unchanged for decades, with a five-year survival rate of approximately50%. Recently, researchers turn to explore a new type of therapeutic strategy---targeted molecular therapies, which is a strategy of finding an ideal combination of bio-drug or cytokines killing tumor cells and being safe for normal cells and vehicle selectively migrating toward tumor sites. Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), a member of the TNF superfamily, is a type-2transmembrane death ligand that causes apoptosis of transformed cells, but not in most normal cells. Thus, TRAIL has the potential of becoming an extremely exciting molecule for tumor therapy. Several studies have shown the anti-tumor activity of recombinant human TRAIL (rhTRAIL). However, several problems are still encountered, including short pharmacokinetic half-life and the requirements of frequent and high doses to produce the desired effects by system delivery of recombinant TRAIL. Recent research has verified that MSCs derived from human bone marrow can migrate toward several tumors. Human G-MSCs have several advantages over BM-MSCs. G-MSCs are homogenous, easy to isolated and proliferate faster than BM-MSCs without any growth factor. Moreover, G-MSCs display stable morphology and maintain MSC characteristic at high passages. Importantly, G-MSCs maintain normal karyotype and telomerase activity in long-term culture, and are not tumorigenic. These advantages suggest that gingiva-derived MSCs (G-MSCs) maybe an effective or even better vehicle for delivering anti-cancer bio-drugs. In the present study, we used G-MSCs as a cell-based vehicle and tested their migration ability toward TSCC. We expressed TRAIL in G-MSCs by using lentivirus and investigated the anti-tumor effect of engineered G-MSCs through three delivery routes (concurrent injection with tumor cells, intra-tumor injection, and tail vein injection). The study included the following three parts: Part1The observation of G-MSC migration toward TSCC cell lines in vitroObjective To explored whether human gingival-derived MSCs (G-MSCs) can migrate toward TSCC cell lines. Methods G-MSCs were acquired by magnetic-activated cell sorting through the Stro-1antibody from human dental gingival cells. Transwell plates were used for the mingration assays in vitro. Results The G-MSCs isolated from human gingival were fibroblast-like cells (Fig.1A). The G-MSCs differentiated toward fat cells and bone cells, as detected by Oil-O Red dyeing and Alizarin Red S dyeing, respectively. The in vitro migration assay indicated that G-MSCs can migrate toward the TSCC cell lines (Ca127and Tca8113). The numbers of migrated G-MSCs toward Ca127and Tca8113cells (Cal27:466.8±30.4; Tca8113:416.8±19.4cells/field) were more than ones toward293T (115.6±7.6) and medium (65.8±14.8)(P<0.001, ANOVA). Conclusion Gingival-derived MSCs can migrate torward TSCC cell lines.Part2The construction TRAIL expressing G-MSCsObjective To construct TRAIL expressing G-MSCs. Methods The lentivirus plasmid pLL3.7was used as the backbone for the incorporation of TRAIL DNA. The Nhe I and Age I restriction sites were located between the CMV-promoter and the eGFP in the pLL3.7plasmid. FMDV2A sequence was synthesized to connect TRAIL and eGFP. The plasmid pLL3.7was excised using the Nhe I and Age I restriction sites. Subsequently, human TRAIL (amino acids1-281, from pCMV-SPORT6, GenBank accession:BC032722) was amplified. Restriction sites and2A sequence were amplified by PCR and inserted into the plasmid between the CMV-promoter and eGFP to produce plasmid pLLT through the Nhe I and Age I restriction sites.293FT were used to package the lentivirus (LV) through the transfection reagent Lipofectamin2000TM (Invitrogen, Carlsbad, CA). The LV packaged by pLLT, psPAX2and pMD2.G plasmids (Addgene Inc., Cambridge, MA) were named LV-pLLT and ones by pLL3.7. psPAX2and pMD2.G plasmids were named LV-pLL3.7. G-MSCs transfect LV-Pllt were named G-MSCFLT which expressed TRAIL and GFP, and ones transfect LV-pLL3.7 were named G-MSCFL which expressed GFP only. The infection efficiency was evaluated by flow cytometer. The infected cells were GFP-positive cells. The expression of TRAIL was detected by Western Blot and ELISA. Results The TRAIL gene can be integrated into G-MSCs gene group at high infection efficiency (88.66%±0.13%) by lentivirus vector system. Western blot verified G-MSCFLT can express TRAIL protein. Conclusion we can integrate full length of TRAIL gene into vehicle cell G-MSCs gene group succesfully and efficiently through lentivirus infection.Part3Anti-tumor effect of TRAIL-expressing G-MSCs in vitro and vivoObjective To study Anti-tumor effect of TRAIL-expressing G-MSCs in vitro and vivo. Methods Anti-tumor assay were performed by direct co-cultures of TSCC (Tca8113or Ca127) and engineered G-MSCs (G-MSCFL/G-MSCFLT) at a ratio1:1. The apoptosis of tumor cells induced by G-MSCFLT was detected by flow cytometer grating on GFP-negative cells and using Annexin V-PE/7-ADD antibody kits. Meanwhile, in vivo anti-tumor assays were performed by administering G-MSCFLT to nude mice locally (mixed-injection with tumor cells and intra-tumor injection) and systematically (tail vein injection). The anti-tumor effect was evaluated by tumor weight and volumn, and tumor cell apoptosis was detected by in situ cell death,, detection kits. Result The death and apoptosis of the Tca8113and Ca127cells co-cultured with G-MSCFLT significantly increased compared with those of the cells co-cultured with G-MSCFL (Tca8113:48.45%±3.56%vs.17.82%±3.03%, P<0.01, ANOVA)(Ca127:52.63±16.03vs.15.92±10.64, P<0.01, ANOVA). For the mixed-injection of Tca8113and G-MSCs/G-MSCFL/G-MSCFLT at a1:1ratio, no tumors were found in G-MSCFLT Group. For the mixed-injection of Tca8113and G-MSCFLT at a ratio of2:1, tumors were formed in all mice. However, G-MSCFLT still significantly inhibited the tumor growth compared with the injection of Tca8113cells alone (85±42.72mg vs.738±430.49mg; P<0.01, Kruskal-Wallis). For the intra-tumor injection, G-MSCFLT can reduce tumor weight compared to blank controls (124mg±60.66vs.738mg±430.49)(P<0.01, Kruskal-Wallis). While there was no significant difference of anti-tumor effect between G-MSCFL and G-MSCFLT group (weight:370mg±217.41vs.124mg±60.66, P>0.05, Kruskal-Wallis). For the systemic delivery of engineered G-MSCs, the tumors in Group G-MSCFLT were smaller than ones in blank control group and Group G-MSCFL (weight. Group G-MSCFLT vs. blank control group:45.00±23.80mg vs.360±239.69mg; P<0.05. Kruskal-Wallis; Group G-MSCFLT vs. Group G-MSCFL:45.00±23.80mg vs.125.00±51.96mg; P<0.05, Kruskal-Wallis). Direct frozen section analysis and in situ cell death detection showed that G-MSCFLT engrafted to the tumor tissues, and delivered TRAIL which induced apoptosis of tumor cells dramatically. Conclusion It verified that G-MSCFLT can migrate toward TSCC and reduce or even inhibit TSCC growth regardless of method of administration. Furthermore, these data emphasized the effectiveness of G-MSCs as a vehicle for cell-based gene therapy and the anti-tumor activity of TRAIL-expressing G-MSCs. |
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