Therapeutic effect of genetically engineered mesenchymal stem cells in rat experimental leptomeningeal glioma model

Disseminating disease of high grade gliomas is difficult to treat. We examined the therapeutic effect of intrathecal administration of mesenchymal stem cells transduced with herpes simplex virus-thymidine kinase gene (MSCtk) followed by systemic ganciclovir (GCV) administration in rat experimental leptomeningeal glioma model. First, to examine in vivo bystander effect, rats were intrathecally co-injected with a mixture of MSCtk and C6 cells and then, intraperitoneally administered with GCV or saline for 10 days (co-injection model). Next, to examine the therapeutic effect of MSCtk/GCV therapy, MSCtk cells were intrathecally administered 1 day after C6 injection and then, GCV or saline was administered (treatment model). GCV administration significantly reduced tumor size on day 14 both in the co-injection model (0.41 ± 0.22 vs. 3.10 ± 0.97 mm², p < 0.01) and in the treatment model (0.73 ± .29 vs. 2.84 ± 0.82 mm², p < 0.01). Survival was also significantly prolonged in GCV group both in the co-injection model (29.2 ± 3.3 vs. 18.8 ± 0.8 days, p < 0.001) and in the treatment model (21.5 ± 1.5 vs. 17.2 ± 0.5 days, p < 0.001). This study provided a novel treatment strategy for leptomeningeal glioma dissemination using intrathecal MSCtk injection followed by systemic GCV administration.     

Therapeutic efficacy and safety of TRAIL-producing human adipose tissue-derived mesenchymal stem cells against experimental brainstem glioma

Mesenchymal stem cells (MSCs) have an extensive migratory capacity for gliomas, which is comparable to that of neural stem cells. Among the various types of MSCs, human adipose tissue-derived MSCs (hAT-MSC) emerge as one of the most attractive vehicles for gene therapy because of their high throughput, lack of ethical concerns, and availability and ease of isolation. We evaluated the therapeutic potential and safety of genetically engineered hAT-MSCs encoding the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against brainstem gliomas. Human AT-MSCs were isolated from human fat tissue, characterized, and transfected with TRAIL using nucleofector. The therapeutic potential of TRAIL-producing hAT-MSCs (hAT-MSC.TRAIL) was confirmed using in vitro and in vivo studies. The final fate of injected hAT-MSCs was traced in long-survival animals. The characterization of hAT-MSCs revealed the expression of MSC-specific cell-type markers and their differentiation potential into mesenchymal lineage. Short-term outcomes included a 56.3% reduction of tumor volume (P < .001) with increased apoptosis (3.03-fold, P < .05) in animals treated with hAT-MSC.TRAIL compared with the control groups. Long-term outcomes included a significant survival benefit in the hAT-MSC.TRAIL-treated group (26 days of median survival in the control group vs 84 days in the hAT-MSC.TRAIL-treated group, P < .0001), without any evidence of mesenchymal differentiation in vivo. Our study demonstrated the therapeutic efficacy and safety of nonvirally engineered hAT-MSCs against brainstem gliomas and showed the possibility of stem-cell-based targeted gene therapy for clinical application.     

Therapeutic effect of neural stem cells expressing TRAIL and bortezomib in mice with glioma xenografts

Treatment of glioblastoma remains a challenge in neuro-oncology. We investigated if treatment with neural stem cells engineered to express membrane-bound TRAIL (NSCs-mTRAIL) alone or in combination with proteasome inhibitors is a feasible therapeutic approach for experimental glioma. Glioma cells showed resistance to soluble TRAIL and proteasome inhibitors alone, but responded well to their combined treatment. In co-culture with NSCs-mTRAIL, glioma cells appeared to be more prone to apoptosis than to treatment with soluble TRAIL, which was enhanced by proteasome inhibitor bortezomib. In vivo, the survival of animals bearing intracranial glial xenografts was significantly improved by NSCs-mTRAIL. The addition of bortezomib further enhanced the efficacy of NSCs-TRAIL treated group in one of examined tumor models. These data demonstrate that therapy with NSCs-mTRAIL is a potent cell based approach for treatment of glioma. Such an approach warrants further search for therapeutics capable of increasing sensitivity of glioma cells to mTRAIL in vivo.     

9L脑胶质瘤干细胞模型建立及MRI成像表现

目的:建立9L脑胶质瘤干细胞F344大鼠载瘤模型,采用3.0T磁共振成像评价肿瘤生长。方法:采用流式细胞仪分选CD133表达阳性细胞群,免疫荧光检测分选后细胞群的干细胞标记CD133、Nestin的表达情况。利用三维立体定向技术建立脑胶质瘤干细胞大鼠载瘤模型,定期行MR检查,观察颅内肿瘤生长情况。HE染色和免疫组织化学染色法观察肿瘤组织细胞形态和GFAP、S-100蛋白表达情况。结果:分选后细胞群CD133、Nestin蛋白表达阳性;干细胞荷瘤大鼠中位生存时间为（21.00±0.33）天;接种后1~3周MR扫描示肿瘤持续生长,增强扫描瘤体显像清晰,第3周末瘤周水肿显著;病理结果示肿瘤浸润生长,局部见出血、坏死,免疫组化示肿瘤组织胶质纤维GFAP阳性, S-100蛋白弱阳性。结论:流式技术分选9L脑胶质瘤干细胞建立的F344大鼠脑胶质瘤模型稳定可靠,符合恶性胶质瘤生物学特点;3.0T MR扫描仪能够动态观察脑胶质瘤生长状态。     

Therapeutic potential of human mesenchymal stem cells producing IL-12 in a mouse xenograft model of renal cell carcinoma

Mesenchymal stem cells (MSCs) represent a new tool for delivery of therapeutic agents to cancer. The cytokine interleukin-12 (IL-12) has demonstrated a potent anti-tumor activity in a variety of mouse tumor models. In this study, human MSCs were isolated from human bone marrow and identified by phenotype analysis and differentiation assays. The anti-tumor activity of human MSCs stably transduced with a recombinant adenoviral vector expressing the murine IL-12 (MSC/IL-12) were evaluated in a mouse xenograft model of renal cell carcinoma (RCC). Expression and bioactivity of the transgenic protein IL-12 from adenoviral vector were confirmed prior to in vivo studies. A nude mouse model of RCC was developed by subcutaneously injection of 786-0 cells into nude mice. MSC/IL-12 was injected into the lateral tail vein with single dose. Results indicated that systemic administration of MSC/IL-12 reduced the growth of 786-0 RCC and significantly prolonged mouse survival. These transfected cells could home to tumors after intravenous injection and largely produce local IL-12 protein. In contrast, systemic level of IL-12 was modestly elevated. Further studies showed that the anti-tumor activity of the MSC/IL-12 was dependent on the presence of natural killer (NK) cells and IFN-γ in this experimental setting. These data demonstrate the potential of adult MSC constitutively producing IL-12 to reduce the growth of RCC and enhance the tumor-bearing mouse survival.     

Efficacy of suicide gene therapy in hypoxic rat 9L glioma cells

Viral vector mediated suicide gene therapy (SGT) involving thymidine kinase (TK) or cytosine deaminase (CD) have considerable promise in the treatment of malignant brain tumors. An unresolved issue is to what extent tumor hypoxia influences the outcome of SGT since brain tumors characterized by regions of hypoxia have potentially reduced cellular metabolism and SGT's cytotoxicity is manifest through cellular metabolism. We studied in vitro and in vivo, the effect of hypoxia on the cytotoxicity of SGT in rat 9L glioma cells. Neither acute nor chronic hypoxia affected the cell killing of SGT by TK or CD. In vivo confirmation that SGT efficacy was not adversely affected by tumor hypoxia using the hypoxic cell marker pimonidazole was shown by the absence of a change in tumor hypoxia by SGT. These studies support the use of SGT utilizing either TK or CD gene strategies even when tumors are characterized by a hypoxic microenvironment.     

Cytotoxic effect and uptake of estramustine in a rat glioma model

The cytotoxic effect of estramustine-phosphate (EMP) and the uptake in tumor tissue were investigated in a rat glioma model in vitro and in vivo. EMP, a combination of nornitrogen mustard and 17beta-estradiol, is a cytotoxic drug which main target is assumed to be the microtubule system. EMP and its metabolite estramustine (EaM) have a demonstrated anti-tumorous effect on human glioma cells in vitro. The drug uptake in tumor tissue and subsequently also the cytotoxic effect, is believed to depend, at least partially, on a specific estramustine-binding protein (EMBP) which is present in human glioma tissue. In this study we have examined the effects and pharmacokinetics of EaM in the nitrosourea induced BT4C rat glioma model. The tumor was characterized by infiltrative growth with a histopathological picture resembling gliosarcoma. The presence of EMBP was demonstrated by immunohistology. In vitro EMP caused a dose-related inhibition of BT4C-cell growth. In vivo, in the rat model, a significant inhibition of tumor growth was obtained after administration of EaM 20 mg/kg/d i.p. The pharmacokinetics of EaM resembled that found in the human clinical situation with EaM as the main metabolite accumulating in tumor tissue. The mean concentration ratio of EaM was 15.6 in tumor versus serum, and 1.8 in tumor versus normal brain of 1.8. The cytotoxic effect demonstrated in the rat glioma model justifies further evaluation of EMP/EaM in the treatment of malignant gliomas.     

Three-dimensional assessment of bystander effects of mesenchymal stem cells carrying a cytosine deaminase gene on glioma cells

Stem cells carrying a suicide gene have emerged as therapeutic candidates for their cytotoxic bystander effects on neighboring cancers, while being non-toxic to other parts of the body. However, traditional cytotoxicity assays are unable to adequately assess the therapeutic effects of bystander cells. Here, we report a method to assess bystander effects of therapeutic stem cells against 3-dimensionally grown glioma cells in real time. U87 glioma cells were stably transduced to express a green fluorescence protein and co-cultivated with mesenchymal stem cells engineered to carry a bacterial cytosine deaminase gene (MSC/CD). Following addition of a 5-fluorocytine (5-FC) prodrug to the co-culture, fluorescence from U87 cells was obtained and analyzed in real time. Notably, the IC₅₀ of 5-FC was higher when U87 cells were grown 3-dimensionally in soft agar medium for 3 weeks, as compared to those grown for one week in two-dimensional monolayer cultures. Additionally, more MSC/CD cells were required to maintain a similar level of efficacy. Since three-dimensional growth of glioma cells under our co-culture condition mimics the long-term expansion of cancer cells in vivo, our method can extend to an in vitro assay system to assess stem cell-mediated anti-cancer effects before advancing into preclinical animal studies.     

Tumoricidal bystander effect in the suicide gene therapy using mesenchymal stem cells does not injure normal brain tissues

In our previous rat study, an established intracranial C6 glioma was successfully treated using intratumoral injection of mesenchymal stem cells transduced with the herpes simplex virus-thymidine kinase gene (MSCtk) and systemic administration of ganciclovir (GCV). In the present study, effect of the "bystander effect" associated with the MSCtk/GCV strategy on the background normal brain tissues was examined in both in vitro and in vivo conditions. Rat MSCtk and C6 glioma cells were mixed and seeded on the rat primary neuron and glia co-culture in the medium containing GCV to generate the bystander effect and the numbers of background cells were counted on day 0, 2 and 7. Though the number of MSCtk and C6 cells decreased rapidly due to the bystander effect, most of the neurons and glias survived on day 7. Next, rats were intracranially injected with the MSCtk and C6 cells and then intraperitoneally administered with GCV for 7days. No remarkable histological abnormality including apoptosis was observed in the background brain tissues near the injection site. The present study has demonstrated that the tumoricidal bystander effect does not injure the background normal brain tissue significantly and that the suicide gene therapies are sufficiently safe.     

Pathological features of highly invasive glioma stem cells in a mouse xenograft model

Glioma stem cells (GSCs) may be a source of tumor progression and recurrence after multimodal therapy, because of their high invasive potential. The purpose of this study was to compare the invasive and migratory properties of GSCs and non-GSCs and examine the distribution of these cells in a mouse xenograft model. Three GSC lines, G144, Y02, and Y10, cultured from human glioblastoma, were used in the study. Matrigel-invasion assays of infiltration and time-lapse studies of migration were performed for comparison of the GSCs with the corresponding differentiated non-GSC lines. Cells were also transplanted into mouse brain and the different distribution of GSCs and non-GSCs was examined in the tumor xenograft model. All 3 GSC lines had greater invasion and migration ability than the corresponding non-GSCs. In vivo, GSCs infiltrated more widely than non-GSCs and reached the contralateral hemisphere via the corpus callosum in the early stage of tumorigenesis. GSCs also primarily penetrated the subventricular zone (SVZ). GSCs have high invasive potential and tend to be present in the outer tumor bulk and infiltrate the contralateral hemisphere via the corpus callosum, in addition to penetrating the SVZ.     

骨髓间充质干细胞向脑胶质瘤定向迁移及其DAPI标记的研究

目的:探讨骨髓间充质干细胞(BSMCs)向C6胶质瘤定向迁移的能力以及DAPI用于BMSCs向胶质瘤体内迁移示踪的价值。方法:直接贴壁法分离培养纯化BMSCs。利用Transwell小室建立体外迁移模型检测BMSCs向C6胶质瘤细胞定向迁移的能力。立体定向法建立大鼠C6胶质瘤模型,利用DAPI体外标记培养、纯化的BMSCs,经荷瘤侧颈内动脉灌注,观察BMSCs向C6胶质瘤组织的定向迁移能力,并评价这一过程中DAPI用于BMSCs标记的价值。结果:通过直接贴壁法分离、培养、传代,获得了纯化的BMSCs。体外迁移实验证实,BMSCs具有向C6胶质瘤细胞定向迁移的能力,迁移细胞数量为400倍视野下(32.1±10.5)/HP。DAPI标记后BMSCs细胞核呈蓝色荧光,阳性率达100%。经荷瘤大鼠颈内动脉灌注后BMSCs可以存活,并表现出向脑胶质瘤趋化迁移的特性,分布区域主要位于肿瘤内部血管周边。结论:DAPI可以用于BMSCs的体内示踪,BMSCs具有通过血肿瘤屏障向C6胶质瘤定向迁移的能力,经颈内动脉灌注是其有效的移植途径。     

Therapeutic efficacy of antiglioma mesenchymal extracellular matrix 131I-radiolabeled murine monoclonal antibody in a human glioma xenograft model

The development of Mabs, particularly those reactive with primary brain tumors but not with normal brain, provides a potential means of delivering therapeutic agents selectively to human malignant gliomas. Mab 81C6, an IgG2b immunoglobulin, which defines an epitope of the glioma-associated extracellular matrix protein tenascin, has been shown to bind to human glioma cell lines, glioma xenografts in nude mice, and primary human gliomas, but not to normal adult or fetal brain. To test the therapeutic potential of this Mab for targeted delivery of isotopes, nude mice bearing progressively growing s.c. xenografts of D-54 MG, a human glioma cell line, were given injections via the tail vein of either buffer, unlabeled 81C6, 131I-labeled 81C6, or 131I-labeled 45.6, a nonspecific control Mab of the same isotype. Specific activities of the Mab range from 6.0 to 15.5 mCi/mg with protein doses from 7.6 to 167 micrograms. The doses given by injection per animal for labeled 81C6 were 50, 250, 500, and 1000 mu Ci and 500 and 1000 mu Ci for 45.6. Tumor response was measured by growth delay in reaching 1000 or 5000 mm3 tumor volumes using the Wilcoxon rank sum test, and by comparing the proportion of tumors that had regression in volume after treatment using the Fisher exact test. Statistically significant growth delays at 1000 mm3 were noted in 1 of 3 experiments with 500 mu Ci 81C6 (P less than 0.001) and 2 of 3 for 1000 mu Ci 81C6 (P = 0.001 and less than 0.001). At 5000 mm3, statistically significant growth delays were seen with radiolabeled 81C6 in 2 of 2 experiments at 250 mu Ci (P = 0.01 and 0.02), 4 of 4 at 500 mu Ci (P = 0.03-less than 0.001), and 2 of 2 at 1000 mu Ci (P = less than or equal to 0.001) and with radiolabeled 45.6 in 1 of 1 at 1000 mu Ci (P = 0.01). The percentage of animals with tumor regression progressively increased with increasing doses of isotope. For radiolabeled 45.6, there were 0 of 10 regressors at 500 and 1 of 10 at 1000 mu Ci. For radiolabeled 81C6, there were 0 of 6 regressors at 50 mu Ci, 1 of 16 (6%) at 250 mu Ci, 7 of 38 (18%) at 500, and 15 of 28 (54%) at 1000 mu Ci. Statistically significant tumor regression was seen only at doses of 500 and 1000 mu Ci of 131I-81C6.(ABSTRACT TRUNCATED AT 400 WORDS)     

骨髓间充质干细胞表达外源性IL-12对胶质瘤C6细胞增殖的影响

目的:探讨以骨髓间充质干细胞（mesenchymal stem cells,MSCs） 为基因治疗载体表达外源性IL12对胶质瘤C6细胞增殖的影响。方法：分离培养大鼠MSCs, 腺病毒介导IL12基因转染大鼠MSCs(AdIL12MSCs),RTPCR 及Western Blotting检测AdIL12MSCs中IL12基因mRNA及蛋白表达。MTT法检测AdIL12MSCs分泌的外源性IL12对C6胶质瘤细胞增殖活性 的影响,光镜下观察外源性IL12对C6细胞形态的影响。结果：腺病毒介导IL12基因成功转染MSCs形成AdIL12MSC,其IL12基因在mRNA及蛋白水平均有明显表达。AdIL12MSC分泌的外源性IL12显著抑制胶质瘤C6细胞的增殖（P<0.05）。结论：转染IL12的MSCs(AdIL12MSC)能够在mRNA及蛋白水平表达外源性IL12基因,显著抑制胶质瘤C6细胞的增殖。     

骨髓间充质干细胞表达外源性IL-12对胶质瘤C6细胞增殖的影响

目的: 探讨以骨髓间充质干细胞(mesenchymal stem cells,MSCs) 为基因治疗载体表达外源性IL-12对胶质瘤C6细胞增殖的影响.方法:分离培养大鼠MSCs, 腺病毒介导IL-12基因转染大鼠MSCs(AdIL-12-MSCs),RT-PCR 及Western Blotting检测AdIL-12-MSCs中IL-12基因mRNA及蛋白表达.MTT法检测AdIL-12-MSCs分泌的外源性IL-12对C6胶质瘤细胞增殖活性的影响,光镜下观察外源性IL-12对C6细胞形态的影响.结果:腺病毒介导IL-12基因成功转染MSCs形成AdIL-12-MSC,其IL-12基因在mRNA及蛋白水平均有明显表达.AdIL-12-MSC分泌的外源性IL-12显著抑制胶质瘤C6细胞的增殖(P<0.05).结论:转染IL-12的MSCs(AdIL-12-MSC)能够在mRNA及蛋白水平表达外源性IL-12基因,显著抑制胶质瘤C6细胞的增殖.     

Application of mesenchymal stem cells as a vehicle to deliver replication-competent adenovirus for treating malignant glioma

Although gene therapy was regarded as a promising approach for glioma treatment,its therapeutic efficacy was often disappointing because of the lack of efficient drug delivery systems.Mesenchymal stem cells(MSCs) have been reported to have a tropism for brain tumors and thus could be used as delivery vehicles for glioma therapy.Therefore,in this study,we attempted to treat glioma by using MSCs as a vehicle for delivering replication-competent adenovirus.We firstly compared the infectivity of type 3,type 5,and type 35 fiber-modified adenoviruses in MSCs.We also determined suitable adenovirus titer in vitro and then used this titer to analyze the ability of MSCs to deliver replication-competent adenovirus into glioma in vivo.Our results indicated that type 35 fiber-modified adenovirus showed higher infectivity than did naked type 3 or type 5 fiber-modified adenovirus.MSCs carrying replication-competent adenovirus significantly inhibited tumor growth in vivo compared with other control groups.In conclusion,MSCs are an effective vehicle that can successfully transport replication-competent adenovirus into glioma,making it a potential therapeutic strategy for treating malignant glioma.     

Inhibitory effect and mechanism of mesenchymal stem cells on melanoma cells

Purpose

To explore the inhibitory effect and mechanism of MSCs on melanoma proliferation.

Methods

The inhibitory effect of MSCs on melanoma A375 cells was detected by co-culture and conditioned medium (CM) experiments using MTT method. The cell cycle was analyzed by flow cytometry. Then, Western Blot experiment detected the expression of proteins related to NF-κB signaling in A375 cells. The expression of IL-1Ra in MSCs was proved by RT-PCR. The over-expression and silencing vector pcDNA3.1-EGFP-IL-1Ra and pGPH1-IL-1R were constructed and transfected into MSCs cells. After that, the changes of inhibitory effect and cell cycle from MSCs-S and MSCs-O CM on A375 cells were explored. The expression of proteins related to NF-κB signaling in A375 cells after MSCs-S or MSCs-O CM treatment was detected by Western Blot. MSCs, MSCs-S, or MSCs-O and A375 cells were co-injected into nude mice under the arms, the growth of tumor was observed, the frozen sections were made, and H&E staining of tumor tissue was performed.

Results

The proliferation of A375 cells was inhibited and the cell cycle of A375 was arrested by MSCs. The expressions of cytokines related to NF-κB signaling were down-regulated. Over-expression and silence of Interleukin 1 receptor antagonist (IL-1Ra), specifically blocking activation of NF-κB signaling, indicated that inhibitory effect from MSCs was enhanced or weakened respectively, which suggested that IL-1Ra was involved in the inhibitory effect. In vivo, tumor initiation and growth were significantly inhibited when A375 cells were co-injected with MSCs into nude mice, which were related to the expression level of IL-1Ra.

Conclusion

MSCs could inhibit the proliferation and tumor initiation of melanoma A375 cells through NF-κB signaling. MSCs could secret IL-1Ra and inhibit expressions of NF-κB signaling-related factors of tumor cells, and cause cell cycle arrest in G1 phase.

     

PEX-producing human neural stem cells inhibit tumor growth in a mouse glioma model.

A unique characteristic of neural stem cells is their capacity to track glioma cells that have migrated away from the main tumor mass into the normal brain parenchyma. PEX, a naturally occurring fragment of human metalloproteinase-2, acts as an inhibitor of glioma and endothelial cell proliferation, migration, and angiogenesis. In the present study, we evaluated the antitumor activity of PEX-producing human neural stem cells against malignant glioma. The HB1.F3 cell line (immortalized human neural stem cell) was transfected by a pTracer vector with PEX. The retention of the antiproliferative activity and migratory ability of PEX-producing HB1.F3 cells (HB1.F3-PEX) was confirmed in vitro. For the in vivo studies, DiI-labeled HB1.F3-PEX cells were stereotactically injected into established glioma tumor in nude mice. Tumor size was subsequently measured by magnetic resonance imaging and at the termination of the studies by histologic analysis including tumor volume, microvessel density, proliferation, and apoptosis rate. Histologic analysis showed that DiI-labeled HB1.F3-PEX cells migrate at the tumor boundary and cause a 90% reduction of tumor volume (P < 0.03). This reduction in tumor volume in animals treated with HB1.F3-PEX was associated with a significant decrease in angiogenesis (44.8%, P < 0.03) and proliferation (23.6%, P < 0.03). These results support the use of neural stem cells as delivery vehicle for targeting therapeutic genes against human glioma.     

Characteristics of glioma stem cells

The cancer stem cell theory postulates that tumors are sustained by a select cell population with specific features, such as self-renewal ability and the capacity to give rise to a heterogeneous mass of tumor cells. The existence of such cells has been demonstrated for glioblastoma, with these cells being referred to as glioma stem cells (GSCs). Glioblastomas are notoriously heterogeneous tumors, however, and the isolation and characterization of their stem cells will require further investigations. Furthermore, the lack of unequivocal markers for GSCs and a partial overlap in characteristics with other cells often lead to confusion. Here, we review the characteristics necessary for a glioma cell to be considered a stem cell, and we adopt our murine glioblastoma model based on genetically modified neural stem cells to illustrate and discuss the GSC concept.     

Frontiers in targeting glioma stem cells

Patients with glioblastoma multiforme (GBM - WHO grade IV) seldom recover. This is due to the infiltrative nature of these tumours and the presence of cellular populations with ability to escape therapies and drive tumour recurrence and progression. In some cases, these resistant cells exhibit stem properties [glioma stem cells (GSC)]. This article aims at discussing relevant issues on GSC resistance to current therapies and outlines possible and promising avenues in regard to novel therapeutic strategies, such as pharmacological, immunological and viral interventions.