Cytochalasin B-induced membrane vesicles from human mesenchymal stem cells overexpressing TRAIL, PTEN and IFN-ß1 can kill carcinoma cancer cells.

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are of interest as a new vector for the delivery of therapeutic agents into the tumor microenvironment. Cell-free EV-based therapy has a number of advantages over cell-based therapy, since the use of EVs allows avoiding potential undesirable transformation associated with MSCs. MSC-derived EVs can transfer natural proteins with immunomodulatory or antitumor properties. The aim of this study was to produce vesicles from mesenchymal stem cells with simultaneous overexpression of TRAIL, PTEN and IFN-ß1 and analyze its antitumor and immunomodulatory properties. In this work, a stable line of human adipose tissue-derived mesenchymal stem cells (hADSCs) with simultaneous overexpression of TRAIL, PTEN and IFN-ß1 was produced. To obtain this cell line hADSCs were genetically modified with a genetic multicistronic cassette encoding TRAIL, PTEN, and IFN-ß1 genes separated with a self-cleaving P2A peptide nucleotide sequence. Membrane vesicles (CIMVs) were obtained from genetically modified hADSCs using cytochalasin B treatment. Antitumor and immunomodulatory properties of the CIMVs were analyzed in vitro. It was shown that CIMVs isolated from genetically modified hADSCs overexpressing TRAIL, PTEN and IFN-ß1 genes are able to activate human immune cells and induce apoptosis in various types of carcinomas in vitro. Thus, the immunomodulatory and antitumor properties of CIMVs were shown. However, further studies on animal models in vivo are required.

Storage stability and delivery potential of cytochalasin B induced membrane vesicles.

Cell-free therapies based on extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are considered as a promising tool for stimulating regeneration and immunomodulation. The need to develop a practical approach for large-scale production of vesicles with homogenous content led to the implementation of cytochalasin B-induced to induce microvesicles (CIMVs) biogenesis. CIMVs mimic natural EVs in size and composition of the surrounding cytoplasmic membrane. Previously we observed that MSC derived CIMVs demonstrate the same therapeutic angiogenic and immunomodulatory activity as the parental MSCs, making them a potentially scalable cell-free therapeutic option. However, little is known about their storage stability and delivery potential. We determined that different storage conditions alter the protein concentration within the solution used to store CIMVs over time, this concided with a decrease in the amount of CIMVs due to gradual degradation. We established that freezing and storage CIMVs in saline at -20 °C reduces degredation and prolongs their shelf life. Additionally, we found that freeze-thawing preserved the CIMVs morphology better than freeze drying and subsequent rehydration which resulted in aggregation of CIMVs. Collectively our data demonstrates for the first time, that the most optimal method of CIMVs storage is freezing at -20 °C, to preserve the CIMVs in the maximum quantity and quality with retention of effective delivery. These findings will benefit the formation of standardized protocols for the use of CIMVs for both basic research and clinical application.

Cytochalasin B-Induced Membrane Vesicles from Human Mesenchymal Stem Cells Overexpressing IL2 Are Able to Stimulate CD8+ T-Killers to Kill Human Triple Negative Breast Cancer Cells.

Interleukin 2 (IL2) was one of the first cytokines used for cancer treatment due to its ability to stimulate anti-cancer immunity. However, recombinant IL2-based therapy is associated with high systemic toxicity and activation of regulatory T-cells, which are associated with the pro-tumor immune response. One of the current trends for the delivery of anticancer agents is the use of extracellular vesicles (EVs), which can carry and transfer biologically active cargos into cells. The use of EVs can increase the efficacy of IL2-based anti-tumor therapy whilst reducing systemic toxicity. In this study, human adipose tissue-derived mesenchymal stem cells (hADSCs) were transduced with lentivirus encoding IL2 (hADSCs-IL2). Membrane vesicles were isolated from hADSCs-IL2 using cytochalasin B (CIMVs-IL2). The effect of hADSCs-IL2 and CIMVs-IL2 on the activation and proliferation of human peripheral blood mononuclear cells (PBMCs) as well as the cytotoxicity of activated PBMCs against human triple negative cancer MDA-MB-231 and MDA-MB-436 cells were evaluated. The effect of CIMVs-IL2 on murine PBMCs was also evaluated in vivo. CIMVs-IL2 failed to suppress the proliferation of human PBMCs as opposed to hADSCs-IL2. However, CIMVs-IL2 were able to activate human CD8+ T-killers, which in turn, killed MDA-MB-231 cells more effectively than hADSCs-IL2-activated CD8+ T-killers. This immunomodulating effect of CIMVs-IL2 appears specific to human CD8+ T-killer cells, as the same effect was not observed on murine CD8+ T-cells. In conclusion, the use of CIMVs-IL2 has the potential to provide a more effective anti-cancer therapy. This compelling evidence supports further studies to evaluate CIMVs-IL2 effectiveness, using cancer mouse models with a reconstituted human immune system.

Mesenchymal Stem Cell Derived Biocompatible Membrane Vesicles Demonstrate Immunomodulatory Activity Inhibiting Activation and proliferation of Human Mononuclear Cells.

Immune-mediated diseases are characterized by abnormal activity of the immune system. The cytochalasin B-induced membrane vesicles (CIMVs) are innovative therapeutic instruments. However, the immunomodulating activity of human mesenchymal stem cell (MSC)-derived CIMVs (CIMVs-MSCs) remains unknown. Therefore, we sought to investigate the immunological properties of CIMVs-MSCs and evaluate their effect on human peripheral blood mononuclear cells (PBMCs). We found that CIMVs-MSCs are primarily uptaken by monocytes and B-cells. Additionally, we demonstrated that CIMVs-MSCs inhibit phytohemagglutinin (PHA)-induced proliferation of PBMCs, with more pronounced effect on T-lymphocytes expansion as compared to that of B-cells. In addition, activation of T-helpers (CD4+CD25+), B-cells (CD19+CD25+), and T-cytotoxic lymphocytes (CD8+CD25+) was also significantly suppressed by CIMVs-MSCs. Additionally, CIMVs-MSCs decreased secretion of epidermal growth factor (EGF) and pro-inflammatory Fractalkine in a population of PBMCs, while the releases of FGF-2, G-CSF, anti-inflammatory GM-CSF, MCP-3, anti-inflammatory MDC, anti-inflammatory IL-12p70, pro-inflammatory IL-1b, and MCP-1 were increased. We analyzed the effect of CIMVs-MSCs on an isolated population of CD4+ and CD8+ T-lymphocytes and demonstrated their different immune response and cytokine secretion. Finally, we observed that no xenogeneic nor allogeneic transplantation of CIMVs induced an immune response in mice. Our data suggest that CIMVs-MSCs have immunosuppressive properties, are potential agents for immunomodulating treatment, and are worthy of further investigation.

Angiogenic Activity of Cytochalasin B-Induced Membrane Vesicles of Human Mesenchymal Stem Cells.

: The cytochalasin B-induced membrane vesicles (CIMVs) are suggested to be used as a vehicle for the delivery of therapeutics. However, the angiogenic activity and therapeutic potential of human mesenchymal stem/stromal cells (MSCs) derived CIMVs (CIMVs-MSCs) remains unknown. OBJECTIVES: The objectives of this study were to analyze the morphology, size distribution, molecular composition, and angiogenic properties of CIMVs-MSCs. METHODS: The morphology of CIMVs-MSC was analyzed by scanning electron microscopy. The proteomic analysis, multiplex analysis, and immunostaining were used to characterize the molecular composition of the CIMVs-MSCs. The transfer of surface proteins from a donor to a recipient cell mediated by CIMVs-MSCs was demonstrated using immunostaining and confocal microscopy. The angiogenic potential of CIMVs-MSCs was evaluated using an in vivo approach of subcutaneous implantation of CIMVs-MSCs in mixture with Matrigel matrix. RESULTS: Human CIMVs-MSCs retain parental MSCs content, such as growth factors, cytokines, and chemokines: EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNα2, IFN-γ, GRO, IL-10, MCP-3, IL-12p40, MDC, IL-12p70, IL-15, sCD40L, IL-17A, IL-1RA, IL-1a, IL-9, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP_1a, MIP-1b, TNF-α, TNF-ß, VEGF. CIMVs-MSCs also have the expression of surface receptors similar to those in parental human MSCs (CD90+, CD29+, CD44+, CD73+). Additionally, CIMVs-MSCs could transfer membrane receptors to the surfaces of target cells in vitro. Finally, CIMVs-MSCs can induce angiogenesis in vivo after subcutaneous injection into adult rats. CONCLUSIONS: Human CIMVs-MSCs have similar content, immunophenotype, and angiogenic activity to those of the parental MSCs. Therefore, we believe that human CIMVs-MSCs could be used for cell free therapy of degenerative diseases.