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T1 MESENCHYMAL STEM CELLS REDUCE LUNG METASTASES BY CONTROLLED TARGETED DELIVERY OF TUMOUR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND
MR Loebinger, SM Janes. Centre for Respiratory Research, University College London, London, UK
Background: Recent studies suggest that bone marrow-derived mesenchymal stem cells (MSC) incorporate within tumour stroma. We hypothesised that MSC engineered to produce and deliver tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a transmembrane protein that causes selective apoptosis of tumour cells, would home to and kill cancer cells in a lung metastatic cancer model.
Methods: TRAIL and the IRES-eGFP reporter gene, under the control of a tetracycline promoter were generated and subcloned into an HIV-1-based self-inactivating lentiviral vector. Expression of GFP and TRAIL in vitro, in 293T cells and MSC (MOI 10) was confirmed by flow cytometry, reverse transcription PCR, Western blot and ELISA. Apoptotic function of MSC expressing TRAIL (GFP positive) was established in co-culture with cancer cell lines (Hela, MDAMB231 and A549) (DiI-labelled) using Annexin V flow cytometry and microscopy. MSC homing was examined using chemotaxis assays with or without chemokine blocking antibodies. In vivo subcutaneous and systemically delivered metastatic lung xenograft cancer models were performed in NOD/SCID mice (MDAB231 cells).
Results: MSC were transduced with greater than 80% efficiency (flow cytometry) and expressed TRAIL under the control of doxycycline (reverse transcription PCR, ELISA, Western blot). Transduced and activated MSC caused lung (A549), breast (MDAB231) and cervical (Hela) tumour cell death and apoptosis in co-culture experiments (all p<0.001). Tumour cell death was maintained at low (1 : 16) MSC/tumour cell ratios (p<0.001) and reduced by a TRAIL neutralising antibody (p<0.001). Tumour cells expressing a dominant negative Fas-associated death domain (dnFADD) showed that TRAIL induced death was via the extrinsic apoptotic pathway (p<0.001). Transwell migration studies demonstrated preferential homing of MSC to tumour cells compared with control cells (p<0.001), which was not significantly blocked by anti-CXCL12 antibody. Subcutaneous xenograft experiments demonstrated that directly delivered TRAIL-expressing MSC …
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