Participation of suicide gene extracellular vesicles in metastasis prevention.

The incidence of distant metastases is associated with most cancer-related mortalities. Extracellular vesicles (EVs), secreted from tumors and cancer-associated fibroblasts, are involved in the metastatic process mediating their organotropism through their involvement in the pre-metastatic niche formation. We have been developing suicide gene therapy mediated by EVs secreted from mesenchymal stem/ stromal cells, tumor cells, and cancer-associated fibroblasts. Suicide gene EVs conjugated with prodrug are tumor tropic, penetrate tumor cells, and kill them by intracellular conversion of nontoxic prodrug to an efficient anti-cancer drug. Here, we discuss findings regarding the possibility of using suicide gene EVs as a novel therapeutic approach for metastases, via pre-metastatic niche modification. The suicide gene EVs provide a future perspective for metastasis prevention.

Suicide-Gene-Modified Extracellular Vesicles of Human Primary Uveal Melanoma in Future Therapies.

Extracellular vesicles secreted from uveal melanoma (UM) cells are involved in the establishment of the premetastatic niche and display transforming potential for the formation of metastases, preferentially in the liver. In this study, we cultivated human primary UM cells and uveal melanoma-associated fibroblasts in vitro to be transduced by infection with a retrovirus containing the suicide gene-fused yeast cytosine deaminase::uracil phospho-ribosyl transferase (yCD::UPRT). A homogenous population of yCD::UPRT-UM cells with the integrated provirus expressed the gene, and we found it to continuously secrete small extracellular vesicles (sEVs) possessing mRNA of the suicide gene. The yCD::UPRT-UM-sEVs were internalized by tumor cells to the intracellular conversion of the prodrug 5-fluorocytosine (5-FC) to the cytotoxic drug 5-fluorouracil (5-FU). The host range of the yCD::UPRT-UM-sEVs was not limited to UMs only. The yCD::UPRT-UM-sEVs inhibited the growth of the human cutaneous melanoma cell line A375 and uveal melanoma cell line MP38, as well as other primary UMs, to various extents in vitro. The yCD::UPRT-UM-sEVs hold the therapeutic and prophylactic potential to become a therapeutic drug for UM. However, the use of yCD::UPRT-UM-sEVs must first be tested in animal preclinical studies.

Tumor-targeted suicide gene-directed enzyme prodrug therapy mediated by extracellular vesicles.

In this article, we describe the gene-directed enzyme prodrug therapy, also known as the "Trojan Horse" therapy mediated by exosomes - small extracellular vesicles (sEVs) secreted from mesenchymal stem/stromal cells (MSCs) and cancer cells. MSC-EVs possess strong migrating tropism toward tumor sites. EVs derived from tumor cells mimic the parental cells in an invasive metastatic growth trait and the capability to reprogram the recipient cells. The behavior of these EVs when modified with the suicide gene predestinates them to be a drug with guided intracellular action. EVs with therapeutic suicide gene are prepared from cells with integrated retrovirus vector containing its genetic message. These EVs are internalized by tumor cells and the product of the gene converts the non-toxic prodrug into a cytotoxic drug inside the cell causing its suicide. The action of two suicide gene systems are described: the yCD::UPRT-MSC/5-FC system and the HSVTK-MSC-GCV system. Suicide gene EVs either MSCs or tumor cell origin due to their intrinsic targeting capabilities, high modification flexibility, as well as biological barrier permeability represent potential drugs for tumors untreatable with present standard cancer therapies.

Extracellular vesicles derived from dental mesenchymal stem/stromal cells with gemcitabine as a cargo have an inhibitory effect on the growth of pancreatic carcinoma cell lines in vitro.

Extracellular vesicles (EVs) are nowadays a target of interest in cancer therapy as a successful drug delivering tool. Based on their many beneficial biocompatible properties are designed to transport nucleic acids, proteins, various nanomaterials or chemotherapeutics. Extracellular vesicles derived from mesenchymal stem/stromal cells (MSCs) possess their tumor-homing abilities. This inspired us to engineer the MSC's EVs to be packed with chemotherapeutic agents and deliver it as a Trojan horse directly into tumor cells. In our study, human dental pulp MSCs (DP-MSCs) were cultivated with gemcitabine (GCB), which led to its absorption by the cells and subsequent secretion of the drug out into conditioned media in EVs. Concentrated conditioned media containing small EVs (potentially exosomes) significantly inhibited the cell growth of pancreatic carcinoma cell lines in vitro. DP-MSCs were simultaneously engineered to express a suicide gene fused yeast cytosinedeaminase:uracilphosphoribosyltransferase (yCD::UPRT). The product of the suicide gene converts non-toxic prodrug 5-fluorocytosine (5-FC) to highly cytotoxic chemotherapeutic drug 5-fluorouracil (5-FU) in the recipient cancer cells. Conversion of 5-FC to 5-FU had an additional effect on cancer cell's growth inhibition. Our results showed a therapeutic potential for DP-MSC-EVs to be designed for successful delivering of chemotherapeutic drugs, together with prodrug suicide gene therapy system.

Low-Dose-Rate Radiation-Induced Secretion of TGF-ß3 Together with an Activator in Small Extracellular Vesicles Modifies Low-Dose Hyper-Radiosensitivity through ALK1 Binding.

Hyper-radiosensitivity (HRS) is the increased sensitivity to low doses of ionizing radiation observed in most cell lines. We previously demonstrated that HRS is permanently abolished in cells irradiated at a low dose rate (LDR), in a mechanism dependent on transforming growth factor ß3 (TGF-ß3). In this study, we aimed to elucidate the activation and receptor binding of TGF-ß3 in this mechanism. T-47D cells were pretreated with inhibitors of potential receptors and activators of TGF-ß3, along with addition of small extracellular vesicles (sEVs) from LDR primed cells, before their radiosensitivity was assessed by the clonogenic assay. The protein content of sEVs from LDR primed cells was analyzed with mass spectrometry. Our results show that sEVs contain TGF-ß3 regardless of priming status, but only sEVs from LDR primed cells remove HRS in reporter cells. Inhibition of the matrix metalloproteinase (MMP) family prevents removal of HRS, suggesting an MMP-dependent activation of TGF-ß3 in the LDR primed cells. We demonstrate a functional interaction between TGF-ß3 and activin receptor like kinase 1 (ALK1) by showing that TGF-ß3 removes HRS through ALK1 binding, independent of ALK5 and TGF-ßRII. These results are an important contribution to a more comprehensive understanding of the mechanism behind TGF-ß3 mediated removal of HRS.

Gene-Directed Enzyme/Prodrug Therapy of Rat Brain Tumor Mediated by Human Mesenchymal Stem Cell Suicide Gene Extracellular Vesicles In Vitro and In Vivo.

MSC-driven, gene-directed enzyme prodrug therapy (GDEPT) mediated by extracellular vesicles (EV) represents a new paradigm-cell-free GDEPT tumor therapy. In this study, we tested the efficacy of yeast cytosine deaminase::uracilphosphoribosyl transferase (yCD::UPRT-MSC)-exosomes, in the form of conditioned medium (CM) to inhibit the growth of C6 glioblastoma cells both in vitro and in vivo. MSCs isolated from human adipose tissue, umbilical cord, or dental pulp engineered to express the yCD::UPRT gene secreted yCD::UPRT-MSC-exosomes that in the presence of the prodrug 5-fluorocytosine (5-FC), inhibited the growth of rat C6 glioblastoma cells and human primary glioblastoma cells in vitro in a dose-dependent manner. CM from these cells injected repeatedly either intraperitoneally (i.p.) or subcutaneously (s.c.), applied intranasally (i.n.), or infused continuously by an ALZET osmotic pump, inhibited the growth of cerebral C6 glioblastomas in rats. A significant number of rats were cured when CM containing yCD::UPRT-MSC-exosomes conjugated with 5-FC was repeatedly injected i.p. or applied i.n. Cured rats were subsequently resistant to challenges with higher doses of C6 cells. Our data have shown that cell-free GDEPT tumor therapy mediated by the yCD::UPRT-MSC suicide gene EVs for high-grade glioblastomas represents a safer and more practical approach that is worthy of further investigation.

Intracellular prodrug gene therapy for cancer mediated by tumor cell suicide gene exosomes.

Recently, we reported about exosomes possessing messenger RNA (mRNA) of suicide gene secreted from mesenchymal stem/stromal cells (MSCs) engineered to express the suicide gene-fused yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT). The yCD::UPRT-MSC exosomes are internalized by tumor cells and intracellularly convert prodrug 5-fluorocytosine (5-FC) to cytotoxic drug 5-fluorouracil (5-FU). Human tumor cells with the potential to metastasize release exosomes involved in the creation of a premetastatic niche at the predicted organs. We found that cancer cells stably transduced with yCD::UPRT gene by retrovirus infection released exosomes acting similarly like yCD::UPRT-MSC exosomes. Different types of tumor cells were transduced with the yCD::UPRT gene. The homogenous cell population of yCD::UPRT-transduced tumor cells expressed the yCD::UPRT suicide gene and secreted continuously exosomes with suicide gene mRNA in their cargo. All tumor cell suicide gene exosomes upon internalization into the recipient tumor cells induced the cell death by intracellular conversion of 5-FC to 5-FU and to 5-FUMP in a dose-dependent manner. Most of tumor cell-derived suicide gene exosomes were tumor tropic, in 5-FC presence they killed tumor cells but did not inhibit the growth of human skin fibroblast as well as DP-MSCs. Tumor cell-derived suicide gene exosomes home to their cells of origin and hold an exciting potential to become innovative specific therapy for tumors and potentially for metastases.

Suicide Gene Therapy Mediated with Exosomes Produced by Mesenchymal Stem/Stromal Cells Stably Transduced with HSV Thymidine Kinase.

Mesenchymal stem/stromal cells (MSCs) prepared from various human tissues were stably transduced with the suicide gene herpes simplex virus thymidine kinase (HSVTK) by means of retrovirus infection. HSVTK-transduced MSCs express the suicide gene and in prodrug ganciclovir (GCV) presence induced cell death by intracellular conversion of GCV to GCV-triphosphate. The homogenous population of HSVTK-MSCs were found to release exosomes having mRNA of the suicide gene in their cargo. The exosomes were easily internalized by the tumor cells and the presence of ganciclovir caused their death in a dose-dependent manner. Efficient tumor cell killing of glioma cell lines and primary human glioblastoma cells mediated by HSVTK-MSC exosomes is reported. Exosomes produced by suicide gene transduced MSCs represent a new class of highly selective tumor cell targeted drug acting intracellular with curative potential.

Mesenchymal Stem Cell Exosome-Mediated Prodrug Gene Therapy for Cancer.

Exosomes derived from human mesenchymal stem cells (MSCs) engineered to express the suicide gene yeast cytosine deaminase::uracil phosphoribosyl transferase (yCD::UPRT) represent a new therapeutic approach for tumor-targeted innovative therapy. The yCD::UPRT-MSC-exosomes carry mRNA of the suicide gene in their cargo. Upon internalization by tumor cells, the exosomes inhibit the growth of broad types of cancer cells in vitro, in the presence of a prodrug. Here we describe the method leading to the production and testing of these therapeutic exosomes. The described steps include the preparation of replication-deficient retrovirus possessing the yCD::UPRT suicide gene, and the preparation and selection of MSCs transduced with yCD::UPRT suicide gene. We present procedures to obtain exosomes possessing the ability to induce the death of tumor cells. In addition, we highlight methods for the evaluation of the suicide gene activity of yCD::UPRT-MSC-exosomes.

Prodrug suicide gene therapy for cancer targeted intracellular by mesenchymal stem cell exosomes.

The natural behavior of mesenchymal stem cells (MSCs) and their exosomes in targeting tumors is a promising approach for curative therapy. Human tumor tropic mesenchymal stem cells (MSCs) isolated from various tissues and MSCs engineered to express the yeast cytosine deaminase::uracil phosphoribosyl transferase suicide fusion gene (yCD::UPRT-MSCs) released exosomes in conditional medium (CM). Exosomes from all tissue specific yCD::UPRT-MSCs contained mRNA of the suicide gene in the exosome's cargo. When the CM was applied to tumor cells, the exosomes were internalized by recipient tumor cells and in the presence of the prodrug 5-fluorocytosine (5-FC) effectively triggered dose-dependent tumor cell death by endocytosed exosomes via an intracellular conversion of the prodrug 5-FC to 5-fluorouracil. Exosomes were found to be responsible for the tumor inhibitory activity. The presence of microRNAs in exosomes produced from naive MSCs and from suicide gene transduced MSCs did not differ significantly. MicroRNAs from yCD::UPRT-MSCs were not associated with therapeutic effect. MSC suicide gene exosomes represent a new class of tumor cell targeting drug acting intracellular with curative potential.

Dental Mesenchymal Stem/Stromal Cells and Their Exosomes.

Stem cells derived from human dental pulp tissue (DP-MSC) differ from the other mesenchymal stem cells prepared from bone marrow or adipose tissue due to their embryonic origin from the neural crest and are of special interest because of their neurotropic character. Furthermore, the therapeutic potential of DP-MSCs is realized through paracrine action of extracellularly released components, for which exosomes play an important role. In this review, we intend to explore the properties of these cells with an emphasis on exosomes. The therapeutic applicability of these cells and exosomes in dental practice, neurodegenerative diseases, and many other difficultly treatable diseases, like myocardial infarction, focal cerebral ischemia, acute lung or brain injury, acute respiratory distress syndrome, acute inflammation, and several others is concisely covered. The use of cellular exosomes as an important diagnostic marker and indicator of targeted cancer therapies is also discussed, while the importance of stem cells from human exfoliated deciduous teeth as a source of evolutionally young cells for future regenerative therapies is stressed. We conclude that exosomes derived from these cells are potent therapeutic tools for regenerative medicine in the near future as clinical administration of DP-MSC-conditioned medium and/or exosomes is safer and more practical than stem cells.