Potent in vivo anti-tumor activity of extracellular vesicles isolated from genetically engineered primary mesenchymal stromal cells expressing the trans-membrane TNF-Related Apoptosis-Inducing Ligand (TRAIL)

Introduction: TNF-related apoptosis-inducing ligand (TRAIL) is a a wide variety of tumor cells with minimal effects on
normal cells. Despite its in vitro activity, recombinant soluble TRAIL has so far shown limited efficacy in vivo. In contrast,
recent reports have shown that significant apoptosis can be observed both in vitro and in vivo when TRAIL is expressed on
the cell membrane (mTRAIL). A further innovation might be the delivery of bioactive proapoptotic TRAIL through its
expression by extracellular vescicles (EVs), the nanovesicular organelles secreted by cells. In fact, EVs are viewed as an
effective tool for intercellular cross-talk and receptor discharge. The trans-membrane expression of TRAIL ligand within the
double layer exosomal membrane may induce a more potent death signal when compared with the soluble molecule.
Material and Methods: Mesenchymal Stromal Cells (MSC) from bone marrow were cultured in vitro and used for EVs
production. Cultured MSC in 75 cm2 flasks, at 80% confluence were infected with a lentivector encoding TRAIL, maintained
in culture, and cell-supernatants repeatedly collected over several days, ultracentrifugated, with EVs-containing pellet
harvested in PBS. EVs were produced also from uninfected MSC as control (EVs-CTRL). EVs were characterized by flow
cytometry for expression of MSC markers and mTRAIL, EV size was evaluated by NanoSight technology. Total protein
concentration was used to quantify EVs, Western Blot analysis was performed to characterize membrane-bound TRAIL. In
vitro analysis was performed on SU-DHL-4 (human B cell lymphoma) and MEL-1300 (human melanoma) cell lines, exposed
for 24 hours to 20-100 μg/ml EVs-TRAIL or EVs-CTRL. Annexin/propidium iodide assay was used to quantify
apoptotic/necrotic cells. For the in vivo assessments, SU-DHL-4 and MEL-1300 cells were transduced with Luc-Lentiviral
particles to obtain Luciferase positive cell lines. These cells were used to engraft NOD scid gamma (NSG) mice (2x106
SU-DHL-4 and 3x105 MEL-1300 cells for each subcutaneous injection point). To visualize tumor cells, mice were injected
intraperitoneum with luciferin and analyzed with the Xenogen system. Mice bearing subcutaneous tumor nodules received
single intravenous injections of 100, 200, 300 μg or multiple (x 3) 200 μg injections of either EVs-TRAIL or EVs-CTRL.
Results: FACS analysis showed strong TRAIL expression on EVs from TRAIL-infected MSC compared to EVs-CTRL, with a
high proportion of positive particles (median 85%, range 78-93). In addition, EVs-TRAIL displayed MSC membrane markers,
i.e. CD 105, CD 90, CD73 and CXCR4. Western Blot analysis under non-reducing conditions showed the presence of TRAIL
ligand, with strong prevalence of dimeric TRAIL isoform (barely detectable the trimeric isoform, undetectable monomeric
isoforms). NanoSight analysis revealed that EVs had a variable size, up to approximately 400 nm in diameter, with a
predominant peak at 273 nm. A strong and dose-dependent cytotoxic effect was observed on SU-DHL-4 cells exposed to
EVs-TRAIL (annexin/PI+ve cells: up to 87% for 100 μg/ml EVs-TRAIL), compared to EVs-CTRL exposure (15%
Annexin/PI+ve cells for 100 μg/ml EVs-TRAIL). A similar, albeit less pronounced in vitro cytotoxic effect of EVs-TRAIL was
observed on the melanoma MEL-1300 cell line. The anti-tumor effect was remarkably strong when EVs-TRAIL were injected
in vivo in mice bearing either SU-DHL-4 or MEL-1300 nodules. A marked reduction of the tumor luminescence from
1.2x1010 photon/sec to <108 photon/sec was observed at seven days since a single EVs-TRAIL injection at 200 and 300 μg.
Multiple administrations of 200 μg EVs-TRAIL induced the strongest luminescence reduction, as observed in MEL-1300
bearing NSG mice.