Nucleofection optimization and in vitro anti-tumourigenic effect of TRAIL-expressing human adipose-derived mesenchymal stromal cells

Abstract

BackgroundTumour homing capacity of engineered human adipose-derived mesenchymal stromal cells (ADMSCs) expressing anti-tumour agents might be the key for a much safer and yet efficient targeted tumour therapy. However, ADMSCs exhibit resistant to most gene transfection techniques and the use of highly efficient viral vectors has several disadvantages primarily concerning safety risk. Here, we optimized the use of highly efficient and safe nucleofection-based transfection using plasmid encoded for TNF-Related Apoptosis Inducing Ligand (TRAIL) into ADMSCs and investigated the potential anti-tumourigenic of TRAIL-expressing ADMSCs (ADMSCs-TRAIL) on selected cancer models in vitro.MethodsDifferent concentration of TRAIL-encoded plasmid and ADMSCs were nucleofected and the percentage of fluorescence cells were analyzed to determine the optimal condition. TRAIL protein and mRNA were validated in nucloeofected ADMSCs using ELISA and RT-PCR respectively. Evaluation of TRAIL specific death receptors were performed on both tumours (A549/lung tumour, LN18/glioblastoma and HepG2/hepatocellular carcinoma) and haematological malignant lines (REH/acute lymphocytic leukaemia, K562/chronic myelogenous leukaemia and KMS-28BM/multiple myeloma) using flow cytometry. ADMSCs-TRAIL was subsequently assessed for anti-tumourigenic properties using both proliferation assay (MTS assay) and apoptosis assay (Annexin-V / Propidium Iodide staining).ResultsNucleofection showed increased total plasmid concentration (2 μg to 8 μg) resulted in significantly higher reporter expression (11.33% to 39.7%) with slight reduction on cells viability (~10%). ADMSCs-TRAIL significantly inhibited ~50% of cell proliferation in LN18, signifying sensitivity of the cell to ADMSCs-TRAIL mediated inhibition. Inhibition of both tumour and malignant lines proliferation by ADMSCs-TRAIL conditioned medium noticed in HepG2, A549 and REH respectively, whereas K562 and KMS-28BM malignant lines exhibit resistant to ADMSCs-TRAIL mediated inhibition. Moreover, we found that native ADMSCs alone were capable of inducing apoptosis in both LN18 and HepG2 tumour lines, despite substantial increased on the percentage of apoptosis by ADMSCs-TRAIL.ConclusionADMSCs-TRAIL selectively inhibit cancer model and markedly induces apoptosis. Through investigation of the specific TRAIL death receptors expression, we saw that the receptors expression did influence the sensitivity of some but not all cancer lines to TRAIL-mediated inhibition. This study provides further insight into the anti-tumourigenic potential of ADMSCs-TRAIL on different cancer models.

Comparison and immunobiological characterization of retinoic acid inducible gene-I-like receptor expression in mesenchymal stromal cells

“…Afterwards, we investigated the mechanism underlying this cell death by checking the expression of TRAIL and Noxa recognized to be involved in the extrinsic and intrinsic apoptotic pathways respectively. MSCs are not known to express TRAIL but are considered as promising tool in cancer therapy when transfected with TRAIL-encoded plasmid 39 , 40 . We observed that RLR activation in MSCs induced the expression of TRAIL mRNA and that expression was ligand dose and MSC-type dependent, whit the highest expression observed in WJ-MSCs.…”

Immunomodulatory effects of OX40Ig gene-modified adipose tissue-derived mesenchymal stem cells on rat kidney transplantation

“…However, the cytogenetic stability of MSCs following viral transduction needs to be established to the allay safety issues of malignant transformation. The nucleofection technology is a safe non-viral electroporation-based transfection system (24,25). In this study, we modified ADSCs with the plasmid pcDNA3.1 that can expressed the OX40Ig fusion protein in eukaryotic expressiion systems by nucleofection.…”

Combined treatment, based on lysomustine administration with mesenchymal stem cells expressing cytosine deaminase therapy, leads to pronounced murine Lewis lung carcinoma growth inhibition

“…Several studies have used an approach related to the enhancement of certain properties of MSCs; for example, the tumor tropism of MSCs by inducing the over expression of CXCR1 or pre‐exposure of MSCs to soluble factors . In addition, improving the MSC properties is achieved by increasing the transfection effectiveness of MSCs . An alternative approach is to use modified MSCs in combination with other anticancer agents, which allows more effective therapy.…”