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.     

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.     

Exosome‐mediated regulation of tumor immunology

Exosomes are representative extracellular vesicles (EV) derived from multivesicular endosomes (MVE) and have been described as new particles in the communication of neighborhood and/or distant cells by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and nucleotides including micro (mi) RNAs. Exosomes from immune cells and tumor cells act in part as a regulator in tumor immunology. CD8⁺ T cells that show potent cytotoxic activity against tumor cells reside as an inactive naïve form in the T‐cell zone of secondary lymphoid organs. Once receiving tumor‐specific antigenic stimulation by dendritic cells (DC), CD8⁺ T cells are activated and differentiated into effector CTL. Subsequently, CTL circulate systemically, infiltrate into tumor lesions through the stromal neovasculature where mesenchymal stromal cells, for example, mesenchymal stem cells (MSC) and cancer‐associated fibroblasts (CAF), abundantly exist, destroy mesenchymal tumor stroma in an exosome‐mediated way, go into tumor parenchyma, and attack tumor cells by specific interaction. DC‐derived and regulatory T (Treg) cell‐derived exosomes, respectively, promote and inhibit CTL generation in this setting. In this review, we describe the roles of exosomes from immune cells and tumor cells on the regulation of tumor progression.     

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.     

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.     

Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth

Exosomes are 30–150 nm vesicles secreted by a wide range of mammalian cells that can contain microRNA (miRNA). To test if marrow stromal cell (MSC) exosomes could be used as a vehicle for delivery of anti-tumor miRNAs, we transfected MSCs with a miR-146b expression plasmid, and harvested exosomes released by the MSCs. Intra-tumor injection of exosomes derived from miR-146-expressing MSCs significantly reduced glioma xenograft growth in a rat model of primary brain tumor.     

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。     

Mesenchymal stem cell‐derived CCN2 promotes the proliferation,migration and invasion of human tongue squamous cell carcinoma cells

Recent studies have demonstrated that mesenchymal stem cells (MSC) exhibit a tropism to tumors and form the tumor stroma. In addition, we found that MSC can secrete different types of factors. However, the involvement of MSC‐derived factors in human tongue squamous cell carcinoma (TSCC) growth has not been clearly addressed. The CCN family includes multifunctional signaling molecules that affect the initiation and development events of various tumors. In our study, we report that CCN2/connective tissue growth factor (CTGF) was the most highly induced among the CCN family members in MSC that were co‐cultured with TSCC cells. To evaluate the relationship between CCN2 and TSCC growth, we downregulated MSC‐derived CCN2 expression with shRNA targeting CCN2 and found that MSC‐secreted CCN2 promotes TSCC cell proliferation, migration and invasion. We also confirmed that MSC‐derived CCN2 partially accelerated tumor growth in vitro. Taken together, these results suggest that MSC‐derived CCN2 contributes to the promotion of proliferation, migration and invasion of TSCC cells and may be a possible therapy target in the future.     

PGE2/EP4 antagonism enhances tumor chemosensitivity by inducing extracellular vesicle‐mediated clearance of cancer stem cells

Cells expressing mesenchymal/basal phenotypes in tumors have been associated with stem cell properties. Cancer stem cells (CSCs) are often resistant to conventional chemotherapy. We explored overcoming mesenchymal CSC resistance to chemotherapeutic agents. Our goal was to reduce CSC numbers in vivo, in conjunction with chemotherapy, to reduce tumor burden. Analysis of clinical samples demonstrated that COX‐2/PGE₂/EP₄ signaling is elevated in basal‐like and chemoresistant breast carcinoma and is correlated with survival and relapse of breast cancer. EP₄ antagonism elicts a striking shift of breast cancer cells from a mesenchymal/CSC state to a more epithelial non‐CSC state. The transition was mediated by EP₄ antagonist‐induced extracellular vesicles [(EVs)/exosomes] which removed CSC markers, mesenchymal markers, integrins, and drug efflux transporters from the CSCs. In addition, EP₄ antagonism‐induced CSC EVs/exosomes can convert tumor epithelial/non‐CSCs to mesenchymal/CSCs able to give rise to tumors and to promote tumor cell dissemination. Because of its ability to induce a CSC‐to‐non‐CSC transition, EP₄ antagonist treatment in vivo reduced the numbers of CSCs within tumors and increased tumor chemosensitivity. EP₄ antagonist treatment enhances tumor response to chemotherapy by reducing the numbers of chemotherapy‐resistant CSCs available to repopulate the tumor. EP₄ antagonism can collaborate with conventional chemotherapy to reduce tumor burden.     

Mesenchymal stem cell signaling in cancer progression

Mesenchymal (multipotent) stem/stromal cells (MSCs) may affect cancer progression through a number of secreted factors triggering activation of various cell signaling pathways. Depending on receptor status, phosphatase and tensin homolog (PTEN) status, or Wnt activation in the cancer cells, the signals may either result in increased growth and metastasis or lead to inhibition of growth with increased cell death. Thus, MSCs can play a dual role in cancer progression depending on the cellular context wherein they reside. The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway has a central role in regulating tumor growth, and several MSC secreted factors stimulate activation of this pathway. A comprehensive understanding of the signals regulating MSC–tumor cross-talk is highly important for the development of MSCs as potential therapeutic vehicles. Thus, the presented review focuses on factors released by MSCs and on the dual role they may have on various stages of tumorigenesis.     

外泌体传输microRNA在肿瘤研究中的进展*

外泌体是一类可以在细胞间传递直径为30～100 nm的内吞衍生囊泡,可包含与其来源和功能相关的蛋白质和RNA 等物质。外泌体作为一种天然的载体,已被视为一种新型的药物传输系统用于肿瘤的治疗。microRNA（miRNA ）是一类新型的RNA调控基因,不仅仅存在于细胞内,亦存在于细胞外,这些细胞外miRNA 可作为分泌型信号分子影响受体细胞表型,并一定程度上反映出供体细胞内的分子改变,具有一定的诊断及潜在的治疗用途。肿瘤患者血液中外泌体高于正常人,并能够包裹肿瘤相关miRNA 行使生物学功能,因此机体通过外泌体传输特异性miRNA 可能在肿瘤的发病过程中扮演着重要的角色。本文将对外泌体作为miRNA 的传输载体在肿瘤发生发展以及肿瘤治疗等方面的研究进行综述。      

Accelerated growth of B16BL6 tumor in mice through efficient uptake of their own exosomes by B16BL6 cells

Exosomes are extracellular vesicles released by various cell types and play roles in cell–cell communication. Several studies indicate that cancer cell‐derived exosomes play important pathophysiological roles in tumor progression. Biodistribution of cancer cell‐derived exosomes in tumor tissue is an important factor for determining their role in tumor proliferation; however, limited studies have assessed the biodistribution of exosomes in tumor tissues. In the present study, we examined the effect of cancer‐cell derived exosomes on tumor growth by analyzing their biodistribution. Murine melanoma B16BL6‐derived exosomes increased the proliferation and inhibited the apoptosis of B16BL6 cells, which was associated with an increase and decrease in the levels of proliferation‐ and apoptosis‐related proteins, respectively. GW4869‐induced inhibition of exosome secretion decreased the proliferation of B16BL6 cells, and treatment of GW4869‐treated cells with B16BL6‐derived exosomes restored their proliferation. Next, we treated B16BL6 tumors in mice with B16BL6‐derived exosomes and examined the biodistribution and cellular uptake of these exosomes. After the intratumoral injection of radiolabeled B16BL6‐derived exosomes, most radioactivity was detected within the tumor tissues of mice. Fractionation of cells present in the tumor tissue showed that fluorescently labeled exosomes were mainly taken up by B16BL6 cells. Moreover, intratumoral injection of B16BL6‐derived exosomes promoted tumor growth, whereas intratumoral injection of GW4869 suppressed tumor growth. These results indicate that B16BL6 cells secrete and take up their own exosomes to induce their proliferation and inhibit their apoptosis, which promotes tumor progression.     

Regulation of exosome release from mammary epithelial and breast cancer cells – A new regulatory pathway

PurposeExosomes are small 50–100 nm sized extracellular vesicles released from normal and tumour cells and are a source of a new intercellular communication pathway. Tumour exosomes promote tumour growth and progression. What regulates the release and homoeostatic levels of exosomes, in cancer, in body fluids remains undefined.MethodsWe utilised a human mammary epithelial cell line (HMEC B42) and a breast cancer cell line derived from it (B42 clone 16) to investigate exosome production and regulation. Exosome numbers were quantified using a Nanosight LM10 and measured in culture supernatants in the absence and presence of exosomes in the medium. Concentrated suspensions of exosomes from the normal mammary epithelial cells, the breast cancer cells and bladder cancer cells were used. The interaction of exosomes with tumour cells was also investigated using fluorescently labelled exosomes.ResultsExosome release from normal human mammary epithelial cells and breast cancer cells is regulated by the presence of exosomes, derived from their own cells, in the extracellular environment of the cells. Exosomes from normal mammary epithelial cells also inhibit exosome secretion by breast cancer cells, which occurs in a tissue specific manner. Labelled exosomes from mammary epithelial cells are internalised into the tumour cells implicating a dynamic equilibrium and suggesting a mechanism for feedback control.ConclusionsThese data suggest a previously unknown novel feedback regulatory mechanism for controlling exosome release, which may highlight a new therapeutic approach to controlling the deleterious effects of tumour exosomes. This regulatory mechanism is likely to be generic to other tumours.     

Exosome‐transmitted p120‐catenin suppresses hepatocellular carcinoma progression via STAT3 pathways

Hepatocellular carcinoma (HCC) is a fatal disease with increasing morbidity and poor prognosis due to surgical recurrence and metastasis. Moreover, the molecular mechanism of HCC progression remains unclear. Although the role of p120‐catenin (p120ctn) in liver cancer is well studied, the effects of secreted p120ctn transported by exosomes are less understood. Here, we show that p120ctn in exosomes secreted from liver cancer cells suppresses HCC cell proliferation and metastasis and expansion of liver cancer stem cells (CSCs). Mechanically, exosome p120ctn inhibits HCC cell progression via the STAT3 pathway, and the STAT3 inhibitor S3I‐201 abolishes the observed effects on growth, metastasis, and self‐renewal ability between exosome p120ctn‐treated HCC cells and control cells. Taken together, we propose that p120ctn‐containing exosomes derived from cancer cells inhibit the progression of liver cancer and may offer a new therapeutic strategy.     

MicroRNA control of epithelial‐mesenchymal transition in cancer stem cells

Cancer stem cells (CSCs) represent a small subset of cancer cell populations that possess characteristics associated with normal stem cells. They have the ability to self‐renew, and are able to generate diverse tumor cells and account for metastases. Therefore, CSCs are widely accepted as potential mediators of therapeutic resistance and novel targets for anti‐cancer treatments. Recent progress has highlighted the significance of epithelial–mesenchymal transition (EMT) process in CSC formation, as well as the crucial role of microRNAs in controlling EMT and cancer metastasis. MicroRNAs are also reported to take part in the control of CSC functions and the regulation of cancer progression by affecting EMT process. Thus, it is highly crucial to develop deeper understanding of the mechanisms that how microRNAs control EMT processes and regulate CSC functions for better therapeutics of cancer disease. Herein we make this review to summarize the current understanding of the regulatory mechanisms of EMT in CSC initiation, with a special focus on the role of microRNAs in EMT control, and discuss the implications of targeting CSCs for cancer therapeutics.     

Mesenchymal stem cell‐derived CXCL16 promotes progression of gastric cancer cells by STAT3‐mediated expression of Ror1

Bone marrow‐derived mesenchymal stem or stromal cells (MSC) have been shown to be recruited to various types of tumor tissues, where they interact with tumor cells to promote their proliferation, survival, invasion and metastasis, depending on the type of the tumor. We have previously shown that Ror2 receptor tyrosine kinase and its ligand, Wnt5a, are expressed in MSC, and Wnt5a‐Ror2 signaling in MSC induces expression of CXCL16, which, in turn, promotes proliferation of co–cultured MKN45 gastric cancer cells via the CXCL16‐CXCR6 axis. However, it remains unclear how CXCL16 regulates proliferation of MKN45 cells. Here, we show that knockdown of CXCL16 in MSC by siRNA suppresses not only proliferation but also migration of co–cultured MKN45 cells. We also show that MSC‐derived CXCL16 or recombinant CXCL16 upregulates expression of Ror1 through activation of STAT3 in MKN45 cells, leading to promotion of proliferation and migration of MKN45 cells in vitro. Furthermore, co–injection of MSC with MKN45 cells in nude mice promoted tumor formation in a manner dependent on expression of Ror1 in MKN45 cells, and anti–CXCL16 neutralizing antibody suppressed tumor formation of MKN45 cells co–injected with MSC. These results suggest that CXCL16 produced through Ror2‐mediated signaling in MSC within the tumor microenvironment acts on MKN45 cells in a paracrine manner to activate the CXCR6‐STAT3 pathway, which, in turn, induces expression of Ror1 in MKN45 cells, thereby promoting tumor progression.     

Recent advances on the role of tumor exosomes in immunosuppression and disease progression

Exosomes are endosomal-derived nanovesicles released by most cells types, including tumor cells, and principally involved in intercellular communication in physiology and disease. Tumor exosomes are gaining increasing interest in medicine and oncology as efficient tools for the delivery of defined signals. Representing the acellular replicas of tumor cells, they contain a great variety of bioactive molecules, such as proteins, RNA, miRNA and DNA. Their great ability to recirculate in body fluids and their structure allow them to transport their cargo to distant targets. Major studies have shown that tumor exosomes convey information not only between tumor cells but also to other cell types, including different immune cell components. There is increasing evidence that these nanovesicles may contribute to cancer progression by influencing different immune cell types, likely blunting specific T cell immunity and skewing innate immune cells toward a pro-tumorigenic phenotype. Because of this function and the additional property to deliver molecular signals modulating neoangiogenesis and stroma remodeling, tumor exosomes are believed to play a role in tumor progression by favoring metastatic niche onset. This review outlines the recent knowledge on immune suppressive mechanisms mediated by tumor exosomes. We will discuss our view on the role of these nanovesicular structures in cancer progression and how their presence could interfere with cancer therapy.     

小细胞肺癌干细胞信号通路的研究进展

小细胞肺癌是具有高度侵袭性的肺肿瘤,其主要临床特征是化疗有效率高但易在短时间内复发转移,这一特点可能与肿瘤干细胞的存在有关。肿瘤干细胞被认为是恶性肿瘤发生发展、耐药、复发及转移的根源。目前多认为肿瘤干细胞与正常干细胞有着相同的信号通路,如Hedgehog、Notch、Wnt等通路。本文就这几条信号通路在小细胞肺癌干细胞中所起的作用以及针对这几条信号通路治疗药物的研究进展和可能的信号通路交互作用等方面进行综述。     

Human breast and melanoma cancer stem cells biomarkers

Cancer progression in humans is difficult to infer because we do not routinely sample patients at multiple stages of their disease. The identification cancer stem cell (CSC) subpopulations inside tumor opens a new view of cancer development, since it implies that tumors can only be eradicated by targeting CSCs. Several markers have been proposed in the literature to identify CSCs both in breast and melanoma but no consensus has been reached, leading to the hypothesis that the CSC phenotype might be dynamically switched. Herein we provide a critical discussion of the biological markers described in the literature for breast cancer and melanoma. Due to its complexity the field would benefit from an interdisciplinary approach to investigate tumor heterogeneity and its progression. Similar considerations could also be relevant for normal tissue stem cells.