RESUMO
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.
Assuntos
Antineoplásicos/uso terapêutico , Genes Transgênicos Suicidas , Terapia Genética/métodos , Neoplasias/terapia , Pró-Fármacos/administração & dosagem , Animais , Antineoplásicos/farmacologia , Engenharia Celular/métodos , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Citosina Desaminase/genética , Exossomos/genética , Flucitosina/administração & dosagem , Flucitosina/metabolismo , Fluoruracila/metabolismo , Proteínas Fúngicas/genética , Vetores Genéticos/genética , Humanos , Camundongos , Pentosiltransferases/genética , Pró-Fármacos/metabolismo , Proteínas Recombinantes de Fusão/genética , Retroviridae/genética , Transdução Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Exossomos , Genes Transgênicos Suicidas , Terapia Genética/métodos , Neoplasias/terapia , Retroviridae/genética , Animais , Linhagem Celular , Citosina Desaminase/genética , Portadores de Fármacos , Proteínas Fúngicas/genética , Vetores Genéticos , Humanos , Células-Tronco Mesenquimais , Camundongos , Pentosiltransferases/genética , Leveduras/enzimologiaRESUMO
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.