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.     

Prodrug cancer gene therapy

There is no effective treatment for late stage and metastatic cancers of colorectal, prostate, pancreatic, breast, glioblastoma and melanoma cancers. Novel treatment modalities are needed for these late stage patients because cytotoxic chemotherapy offers only palliation, usually accompanied with systemic toxicities and poor quality of life. Gene directed enzyme prodrug therapy (GDEPT), which concentrates the cytotoxic effect in the tumor site may be one alternative. This review provides an explanation of the GDEPT principle, focusing on the development, application and potential of various GDEPTs. Current gene therapy limitations are in efficient expression of the therapeutic gene and in tumor-specific targeting. Therefore, the current status of research related to the enhancement of in situ GDEPT delivery and tumor-specific targeting of vectors is assessed. Finally, GDEPT versions of stem cell based gene therapy as another potential treatment modality for progressed tumors and metastases are discussed. Combinations of traditional, targeted, and stem cell directed gene therapy could significantly advance the treatment of cancer.     

Appropriate subcellular localisation of prodrug-activating enzymes has important consequences for suicide gene therapy

Escherichia coli B nitroreductase (NR) has been expressed stably in MDA-MB-361 human breast adenocarcinoma cells either as the wild-type protein (wtNR), which is distributed evenly between the cytoplasmic and nuclear compartments, or targeted to the mitochondrion (mtNR). Whereas bacterial NR is active as a dimer, a proportion of wtNR is monomeric. In contrast, mtNR is mostly dimeric, suggesting that it adopts a more stable, native conformation. Despite this, when tested in gene-directed enzyme prodrug therapy cell cytotoxicity studies, cells expressing wtNR or mtNR had similar sensitivity to the prodrug CB1954 and mounted similar bystander killing effects. Furthermore, when short prodrug exposures were given, wtNR was more efficient at killing cells than mtNR. These data demonstrate that the site of enzyme expression and prodrug activation is an important variable that requires consideration in suicide gene therapy approaches.     

Biology and therapeutic potential of mesenchymal stem cell‐derived exosomes

Mesenchymal stem cells (MSC) are multipotent stromal cells with the potential to differentiate into several cell types. MSC‐based therapy has emerged as a promising strategy for various diseases. Accumulating evidence suggests that the paracrine effects of MSC are partially exerted by the secretion of soluble factors, in particular exosomes. MSC‐derived exosomes are involved in intercellular communication through transfer of proteins, RNA, DNA and bioactive lipids, which might constitute a novel intercellular communication mode. This review illustrates the current knowledge on the composition and biological functions as well as the therapeutic potential of MSC‐derived exosomes in cancer, with a focus on clinical translation opportunities.     

Role of stem cell derived exosomes in tumor biology

Exosomes are nano‐scale messengers loaded with bio‐molecular cargo of RNA, DNA, and Proteins. As a master regulator of cellular signaling, stem cell (both normal, and cancer stem cells) secreted exosome orchestrate various autocrine and paracrine functions which alter tumor micro‐environment, growth and progression. Exosomes secreted by one of the two important stem cell phenotypes in cancers a) Mesenchymal stem cells, and b) Cancer stem cells not only promote cancerous growth but also impart therapy resistance in cancer cells. In tumors, normal or mesenchymal stem cell (MSCs) derived exosomes (MSC‐exo) modulate tumor hallmarks by delivering unique miRNA species to neighboring cells and help in tumor progression. Apart from regulating tumor cell fate, MSC‐exo are also capable of inducing physiological processes, for example, angiogenesis, metastasis and so forth. Similarly, cancer stem cells (CSCs) derived exosomes (CSC‐exo) contain stemness‐specific proteins, self‐renewal promoting regulatory miRNAs, and survival factors. CSC‐exo specific cargo maintains tumor heterogeneity and alters tumor progression. In this review we critically discuss the importance of stem cell specific exosomes in tumor cell signaling pathways with their role in tumor biology.     

Cancer exosomes trigger mesenchymal stem cell differentiation into pro-angiogenic and pro-invasive myofibroblasts

Stromal fibroblasts become altered in response to solid cancers, to exhibit myofibroblastic characteristics, with disease promoting influence. Infiltrating mesenchymal stem cells (MSC) may contribute towards these changes, but the factors secreted by cancer cells that impact MSC differentiation are poorly understood.We investigated the role of nano-metre sized vesicles (exosomes), secreted by prostate cancer cells, on the differentiation of bone-marrow MSC (BM-MSC), and the subsequent functional consequences of such changes. Purified exosomes impaired classical adipogenic differentiation, skewing differentiation towards alpha-smooth muscle actin (αSMA) positive myofibroblastic cells. A single exosomes treatment generated myofibroblasts secreting high levels of VEGF-A, HGF and matrix regulating factors (MMP-1, −3 and −13). Differentiated MSC had pro-angiogenic functions and enhanced tumour proliferation and invasivity assessed in a 3D co-culture model. Differentiation was dependent on exosomal-TGFβ, but soluble TGFβ at matched dose could not generate the same phenotype. Exosomes present in the cancer cell secretome were the principal factors driving this phenotype.Prostate cancer exosomes dominantly dictate a programme of MSC differentiation generating myofibroblasts with functional properties consistent with disease promotion.     

间充质干细胞源性外泌体及其在肿瘤治疗中的研究进展

外泌体（exosomes）是细胞内多囊泡体（multivesicular bodies,MVBs）与细胞膜融合后释放到细胞外直径为40~100nm的囊泡样小体。作为一种重要的细胞间信息传递分子及遗传物质传递载体,外泌体内含有蛋白质、RNA等多种活性物质,广泛分布于血液、尿液等体液中。目前发现多种类型细胞均可产生外泌体,尤其是间充质干细胞（MSCs）被认为是产生外泌体能力最强的细胞,并且MSCs源性外泌体（MSC-exosomes）与MSCs同样具有向炎症组织及肿瘤组织迁移的特性,为肿瘤治疗提供了一种新思路。由此,本文将从MSC-exosomes生物学特性、分离鉴定方法、肿瘤治疗潜能三方面进行综述。     

间充质干细胞来源的外泌体治疗放射性肠炎的疗效与安全性

目的：探讨间充质干细胞来源的外泌体（MSC-Exo）对人结肠癌CT26细胞体外增殖、迁移的影响及对放射性肠炎（RE）荷瘤小鼠模型的治疗效果及其安全性。方法：利用商品化含MSC-Exo的液体敷料产品（MSC-Exo产品）,通过纳米颗粒追踪分析、透射电镜、WB法对其中的MSC-Exo进行鉴定。采用CCK-8法和Transwell小室法检测MSC-Exo产品对结肠癌CT26细胞增殖、迁移的影响。构建CT26细胞荷瘤小鼠模型,连续7 d分别给予400μL MSC-Exo产品、MSC培养基或生理盐水灌胃,评估MSC-Exo产品在体内对肿瘤生长和小鼠生存的影响。构建RE荷瘤小鼠模型,连续7 d分别给予400μL MSC-Exo产品、MSC培养基、生理盐水灌胃治疗,通过小肠组织H-E染色法评估MSC-Exo产品治疗RE的有效性与安全性。结果：纳米颗粒追踪分析、透射电镜、WB法的鉴定结果确证了商品化的液体敷料中含有MSC-Exo有效成分。体外研究表明,MSC-Exo产品成分不会促进结肠癌CT26细胞的增殖和迁移,具有安全性。体内研究结果发现,MSC-Exo产品的灌胃给药并不影响荷瘤小鼠的肿瘤生长和生存...     

Complete regression of glioblastoma by mesenchymal stem cells mediated prodrug gene therapy simulating clinical therapeutic scenario

Suicide gene therapy mediated by mesenchymal stem cells with their ability to engraft into tumors makes these therapeutic stem cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we evaluated the therapeutic efficacy of human mesenchymal stem cells derived from bone marrow and from adipose tissue, engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase to treat intracerebral rat C6 glioblastoma in a simulated clinical therapeutic scenario. Intracerebrally grown glioblastoma was treated by resection and subsequently with single or repeated intracerebral inoculations of therapeutic stem cells followed by a continuous intracerebroventricular delivery of 5‐fluorocytosine using an osmotic pump. Kaplan–Meier survival curves revealed that surgical resection of the tumor increased the survival time of the resected animals depending on the extent of surgical intervention. However, direct injections of therapeutic stem cells into the brain tissue surrounding the postoperative resection cavity led to a curative outcome in a significant number of treated animals. Moreover, the continuous supply of therapeutic stem cells into the brain with growing glioblastoma by osmotic pumps together with continuous prodrug delivery also proved to be therapeutically efficient. We assume that observed curative therapy of glioblastoma by stem cell‐mediated prodrug gene therapy might be caused by the destruction of both tumor cells and the niche where glioblastoma initiating cells reside.     

Human adipose tissue-derived mesenchymal stem cells: characteristics and therapeutic potential as cellular vehicles for prodrug gene therapy against brainstem gliomas

Human mesenchymal stem cells (hMSCs) have emerged as attractive cellular vehicles for gene therapy against brain malignancy because of their targeted tropism for cancer and the intrinsic attribute of autologous transplantation. We evaluated the characteristics and therapeutic potential of human adipose tissue-derived MSCs (hAT-MSCs) and prodrug gene therapy against diffuse pontine gliomas.The hAT-MSCs were isolated from human adipose tissue and characterised for morphology, surface markers and potential to differentiate into mesenchymal and neuronal lineages. We genetically modified hAT-MSCs to express rabbit carboxylesterase (rCE) enzyme, which can efficiently convert the prodrug CPT-11 (irinotecan-7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin), into the active drug SN-38 (7-ethyl-10-hydroxycamptothecin). The migratory capacity of hAT-MSCs expressing rCE (hAT-MSC.rCE), their ability to convert CPT-11 to SN-38 and cytotoxic effect on F98 cells were evaluated in vitro. The therapeutic potential of hAT-MSC.rCE was confirmed using a rat brainstem glioma model.The hAT-MSCs showed fibroblast-like morphology and expressed hMSC-specific markers including CD73, CD90 and CD105. The hAT-MSCs could differentiate into a mesenchymal lineage and transdifferentiate into a neuronal lineage under optimum culture conditions. The hAT-MSC.rCE converted CPT-11 to SN-38 and preserved the tumour tropism of hAT-MSCs. Brainstem glioma-bearing rats treated with hAT-MSC.rCE and CPT-11 survived 5 d more than rats treated with CPT-11 only (p = 0.0018).Our study demonstrates that hAT-MSCs can be easily prepared and genetically modified as cellular vehicles for prodrug gene therapy and that they have therapeutic potential against brainstem gliomas.     

间充质干细胞基因治疗肿瘤新进展

骨髓间充质干细胞(MSC)是骨髓内除造血干细胞和多功能前祖细胞外另一种成体干细胞,具有很强的自我更新能力、多向分化潜能.该细胞具有明显的趋向肿瘤移动并与肿瘤组织楔合的生长特性.虽然骨髓间充质干细胞有向肿瘤干细胞转化的风险,但其作为一种克隆载体可以转导治疗基因在体内获得长期稳定表达,对肿瘤的靶向治疗有着广阔的前景.     

肿瘤自杀基因靶向治疗

自杀基因靶向疗法在恶性肿瘤的治疗方法中占有重要的位置。众多体内体外试验已经证明了自杀基因的肿瘤杀伤效应。目前,相关的临床试验也证实HSV-tk/GCV,CD/5-FC等自杀基因系统具有良好的抗肿瘤效应。然而在临床推广应用上还有很多问题需要解决,如基因靶向转移困难、基因转染效率较低、基因转移后的表达缺乏有效的调控手段和基因杀伤效应较低且难以控制等,这些问题均有待于我们在今后的研究中进一步解决。本文将从自杀基因的种类、载体、作用机制、旁观者效应和临床发展前景,尤其是纳米粒载体和肿瘤干细胞理论的应用等方面进行综述。     

乳腺癌干细胞信号转导通路及以其为靶点的干细胞治疗

20世纪以来,乳腺癌的发病率在世界范围内呈逐年上升趋势,且发病年龄日趋年轻化.据估计,2009年美国有192 370名女性被诊断患有乳腺癌,40 170名女性因乳腺癌而死亡.在我国,乳腺癌亦位居女性恶性肿瘤发病率首位.     

To grab the stroma by the horns: From biology to cancer therapy with mesenchymal stem cells

Mesenchymal stem or stromal cells (MSCs) are precursor cells that play important roles in tumorigenesis. MSCs are recruited to tumors from local and distant sources to form part of the tumor microenvironment. MSCs influence tumor progression by interacting with cancer cells, endothelial cells, immune cells, and cancer stem cells, in a context-dependent network. This review aims to synthesize this emerging yet controversial field to identify key questions regarding the mechanisms of MSC mobilization and survival in blood; homing to tumors, metastases, and premetastatic sites; spatiotemporal organization and differentiation; and interaction with immune cells and cancer stem cells. Understanding the fundamental biology underlying mesenchymal stem cell and tumor interactions has the potential to inform our knowledge of cancer initiation and progression as well as lead to novel therapeutics for cancer. Furthermore, knowledge of endogenous mechanisms can be used to “program” exogenous MSCs for targeted chemotherapeutic delivery to tumors and metastases. Emerging studies will provide crucial insight into the mechanisms of tumor interactions with the whole organism including MSCs.     

Novel treatments for hepatocellular cancer

Hepatocellular cancer (HCC) has always been considered a therapeutic challenge, given the cytoxic drug resistant nature of the cancer and associated disorder in liver function, reducing the safety of many conventional chemotherapy agents. The Multikinase inhibitor sorafenib has been found to prolong survival in patients with advanced HCC, by around 3 months compared to placebo, but novel treatments need to be explored. Current experimental therapeutic approaches encompass a broad range of science, ranging from intrahepatic irradiation to virus directed immunotherapy. This chapter presents a horizon scan of novel treatments which are currently at early stages of trial development.     

自杀基因治疗恶性肿瘤的研究现状及展望

肿瘤的自杀基因疗法是近年来肿瘤基因治疗的研究热点,是一种具有潜在临床应用前景的新的肿瘤基因治疗策略。本文就近年来自杀基因疗法在肿瘤治疗中取得的研究进展,分别从作用机制、自杀基因系统、旁观者效应、自杀基因的转导以及联合基因治疗等几个方面进行综述。     

肿瘤HSV—tk／GCV自杀基因治疗系统的增效策略

HSV—tk／GCV系统是目前应用最广泛的自杀基因治疗系统之一,其重要性日益上升。然而,许多缺陷严重限制了其应用广度和治疗效率。为满足更多领域的研究需要并实现临床治疗,科研人员投入了大量精力改善HSV—tk／GCV系统,增强其治疗效率。增效策略主要包括加强亲和性、提高表达水平、双自杀基因系统联合、诱发免疫系统协同作用、激活细胞周期、与其他肿瘤治疗方法相结合等。本文在阐明HSV—tk／GCV系统的自杀机理的基础上,分析了多种增效策略及效果,列举了应用领域并对未来发展作探讨。     

羊水间充质干细胞在肿瘤治疗中的研究进展

羊水间充质干细胞是多能的非造血祖细胞,具有自我修复及多向分化潜能,如成骨细胞、成软骨细胞及成脂肪细胞。尽管其具有高度增殖能力,但是其在体内并没有成瘤的报道,甚至多次传代后其核型也正常。 AF-MSCs对肿瘤的趋向性及其作为治疗多种肿瘤的细胞载体已经被很多研究证实。本文总结近期国内外的相关文献,就羊水间充质干细胞在肿瘤治疗中的研究进展进行综述。     

肝癌干细胞表面标记物及靶向治疗的研究进展

肝癌是一种常常致命的恶性肿瘤,具有较高的复发率和化疗耐药性。 癌症的主要恶性表现包括复发、转移和化疗耐药性,且都与肿瘤干细胞（cancer stem cells,CSCs）的存在有关。 在过去的几十年中,CSCs已经在包括肝癌在内的许多肿瘤中被鉴定和确定。 越来越多的证据揭示了许多肝癌干细胞（liver cancer stem cells,LCSCs）的生物学行为及其调控的机制。 基于这些研究结果,LCSCs的根除是靶向治疗的目标之一。 本综述重点介绍LCSCs表面标记物最新进展及其靶向治疗策略的发展。