Effects of human placenta-derived mesenchymal stem cells with NK4 gene expression on glioblastoma multiforme cell lines

Poor prognosis and low survival are commonly seen in patients with glioblastoma multiforme (GBM). Due to the specific nature of solid tumors such as GBM, delivery of therapeutic agents to the tumor sites is difficult. So, one of the major challenges in the treatment of these tumors is a selection of appropriate method for drug delivery. Mesenchymal stem cells (MSCs) have a unique characteristic in migration toward the tumor tissue. In this regard, the present study examined the antitumor effects of manipulating human placenta-derived mesenchymal stem cells (PDMSCs) with NK4 expression (PDMSC-NK4) on GBM cells. After separation and characterization of PDMSCs, these cells were transduced with NK4 which was known as the antagonist of hepatocyte growth factor (HGF). The results indicated that engineered PDMSCs preferably migrate into GBM cells by transwell coculture system. In addition, the proliferation of the GBM cells significantly reduced after coculture with these cells. In fact, manipulated PDMSCs inhibited growth of tumor cells by induction of apoptosis. Our findings suggested that besides having antitumor effects, PDMSCs can also be applied as an ideal cellular vehicle to target the glioblastoma multiforme.     

Mesenchymal stem cells: a promising candidate in regenerative medicine

Mesenchymal stem cells were initially characterized as plastic adherent, fibroblastoid cells. In recent years, there has been an increasing focus on mesenchymal stem cells since they have great plasticity and are potential for therapeutic applications. Mesenchymal stem cells or mesenchymal stem cell-like cells have been shown to reside within the connective tissues of most organs. These cells can differentiate into osteogenic, adipogenic and chondrogenic lineages under appropriate conditions. A number of reports have also indicated that these cells possess the capacity to trans-differentiate into epithelial cells and lineages derived from the neuro-ectoderm, and in addition, mesenchymal stem cells can migrate to the sites of injury, inflammation, and to tumors. These properties of mesenchymal stem cells make them promising candidates for use in regenerative medicine and may also serve as efficient delivery vehicles in site-specific therapy.     

GDNF慢病毒载体感染食蟹猴骨髓间充质干细胞

骨髓间充质干细胞(mesenchymal stem cells,MSCs)是基因工程和细胞治疗的种子细胞之一,本研究利用含胶质源性神经营养因子(glial cell derived neurotrophic factor,GDNF)基因的慢病毒载体感染成年食蟹猴MSCs,探讨转染后GDNF在MSCs中的体外表达水平及其影响因素。首先,通过密度梯度离心法分离食蟹猴骨髓单核细胞(marrow mononuclear cells,MNCs),体外培养食蟹猴MSCs。同时构建表达GDNF的慢病毒载体,并感染食蟹猴MSCs,分别利用酶联免疫吸附(ELISA)方法和Real-time PCR方法,测定感染不同拷贝数病毒和不同转染组细胞GDNF的蛋白分泌水平和基因表达水平。实验结果显示,表达GDNF基因的慢病毒载体成功转染成年食蟹MSCs,体外培养的MSCs持续表达分泌GDNF。感染慢病毒的拷贝数可以影响GDNF分泌水平,相同条件下感染拷贝数越高,GDNF分泌量越多,其基因表达水平越高。     

IFN-γ-primed human bone marrow mesenchymal stem cells induce tumor cell apoptosis in vitro via tumor necrosis factor-related apoptosis-inducing ligand

Human mesenchymal stem cells hold promise as gene therapy vectors for delivery of various genes to solid tumors for either therapeutic or tumor-tracing purposes. However, whether Mesenchymal stem cells support or inhibit tumor growth remains unknown. Herein, we first observed that mesenchymal stem cells primed with IFN-γ selectively induced the death of tumor cell lines, but not normal cells. We further identified that IFN-γ-primed mesenchymal stem cells expressed tumor necrosis factor-related apoptosis-inducing ligand. Tumor-suppressive effect of IFN-γ-primed mesenchymal stem cells could be blocked by activity neutralization or expression reduction of tumor necrosis factor-related apoptosis-inducing ligand. Moreover, mesenchymal stem cells mediated apoptosis of tumor cells by activating caspase-3 in such cells, via a mechanism involving tumor necrosis factor-related apoptosis-inducing ligand. However, when IFN-γ-primed or non-primed mesenchymal stem cells were co-injected into nude mice along with H460 cells, tumor growth was much faster than that of the group receiving only tumor cells (p<0.01) because of the promoting vascularization effect of mesenchymal stem cells, although IFN-γ-primed mesenchymal stem cells also exerted a certain degree of tumor-suppressive effect compared with non-primed cells (2.79±0.9 g versus 2.03±0.6 g). Collectively, our findings show that IFN-γ-primed human mesenchymal stem cells could induce cancer cell apoptosis via TRAIL-mediated pathway. In addition, our data afford a novel explanation of the opposing effects of hMSCs presence on tumor growth in vitro and in vivo. Thus, more attention needs to be paid when seeking to exploit mesenchymal stem cells as a therapeutic option under the condition of malignant tumor.     

Evaluation of the efficiency of modified PAMAM dendrimer with low molecular weight protamine peptide to deliver IL-12 plasmid into stem cells as cancer therapy vehicles

Interleukin 12 (IL-12) is considered as an important molecule for cancer immunotherapy with significant roles in hindering tumor activity, mostly mediated by tumor-associated macrophages and anti-angiogenic factors. Mesenchymal stem cells (MSCs) have been come out as promising carriers to increase the accumulation of drug/gene in tumor sites. As a vehicle, MSCs have various advantages, including tumor-specific propensity and migratory ability; however, they have limited transfection efficiency, compared to other cells. In this study, we introduced a novel delivery system based on poly-(amidoamine) (PAMAM) (G5) to deliver a plasmid encoding IL-12 to MSCs. Initially, 30% of the amine surface of PAMAM was substituted by 10-bromodecanoic acid. Then, the low molecular weight of protamine peptide was conjugated to PAMAM and PAMAM-alkyl with N-succinimidyl 3-(2-pyridyldithio) propionate as a linker. Physicochemical properties of this modified PAMAM were evaluated, including size and surface charge, toxicity, transfection efficiency to deliver reporter and IL-12 genes into MSCs and finally the migration potential of the engineered stem cells into cancer and normal cell lines (HepG2 and NIH/3 T3). The results showed that alkyl-peptide modified PAMAM with low toxicity had a higher potential to deliver green fluorescent protein and IL-12 genes to stem cells, than PMAMAM, PAMAM-alkyl and PAMAM-peptide. These engineered stem cells had a greater ability to migrate to cancer cells than normal cells. It can be concluded that engineered stem cells containing the IL-12 gene can be considered as an efficient cell carrier for cancer immunotherapy. Further clinical studies are needed to confirm these results.     

Antitumor and antimetastatic activity of IL-23

The structure and T cell stimulatory effects of the recently discovered cytokine IL-23 are similar to, but distinct from, those of IL-12. Although the antitumor activities of IL-12 are well characterized, the effect of IL-23 on tumor growth is not known. In this study, murine CT26 colon adenocarcinoma and B16F1 melanoma cells were engineered using retroviral vectors to release single-chain IL-23 (scIL-23) to evaluate its antitumor activity. In BALB/c mice, scIL-23-transduced CT26 cells grew progressively until day 26 to an average size of 521 +/- 333 mm(3), then the tumors started to regress in most animals, resulting in a final 70% rate of complete tumor rejection. scIL-23 transduction also significantly suppressed lung metastases of CT26 and B16F1 tumor cells. In addition, mice that rejected scIL-23-transduced tumors developed a memory response against subsequent wild-type tumor challenge. Compared with scIL-12-expressing CT26 cells, scIL-23-transduced tumors lacked the early response, but achieved comparable antitumor and antimetastatic activity. These results demonstrated that IL-23, like IL-12, provided effective protection against malignant diseases, but it probably acted by different antitumor mechanisms. As a first step in identifying these antitumor mechanisms, tumor challenge studies were performed in immunocompromised hosts and in animals selectively depleted of various lymphocyte populations. The results showed that CD8(+) T cells, but not CD4(+) T cells or NK cells, were crucial for the antitumor activity of IL-23.     

Gene expression profile of cytokine and growth factor during differentiation of bone marrow-derived mesenchymal stem cell

Mesenchymal stem cells (MSCs), which are adherent stromal cells of a nonhematopoietic origin, have the ability to give rise to various differentiated cell types. MSCs regulate localization, self-renewal and differentiation of hematopoietic stem cells (HSCs) due to MSCs' secretion of cytokines and growth factors, the cell-to-cell interactions and the influence of the extracellular matrix proteins. Using RT-PCR analysis, we examined the expression levels of cytokines and growth factors from MSCs and their differentiated cell types, including osteoblasts, adipocytes and endothelial cells. Cytokine and growth factor genes, including IL-6, IL-8, IL-11, IL-12, IL-14, IL-15, LIF, G-CSF, GM-CSF, M-SCF, FL and SCF, were found to be expressed in the MSCs. In contrast, there was no IL-1alpha, IL-1beta, or IL-7 expression observed. The IL-12, IL-14, G-CSF, and GM-CSF mRNA expression levels either disappeared or decreased after the MSCs differentiated into osteoblasts, adipocytes, and endothelial cells. Among the differentiated cells derived from MSCs, osteoblasts, adipocytes, and endothelial cells expressed the osteopontin, aP2, and the VEGFR-2 gene, respectively. These profiles could help determine future clinical applications of MSCs and their derivatives for cell therapy.     

过表达白细胞介素10的间充质干细胞对炎症性肠病的治疗作用

炎症性肠病(inflammatory bowel disease,IBD)是一种以T细胞浸润至结肠为特征的难治性炎性自身免疫疾病。间充质干细胞 (mesenchymal stem cells,MSCs) 具有免疫抑制能力,在IBD的治疗中具有一定的潜力。但是由于MSCs在体内的免疫调节能力不稳定,所以其治疗效果会受到影响。本研究构建了过表达白细胞介素10(interleukin 10,IL-10)的工程化MSCs,并对其在IBD小鼠模型中的治疗潜力进行评估。MSCs经编码IL-10的慢病毒(lentivirus,LV)转染后,其表型和细胞增殖率均不发生变化。免疫细胞和 MSCs体外共培养的结果表明,与未修饰的MSCs相比,同过表达IL-10的MSCs共培养的免疫细胞中辅助T细胞1(T helper 1 cells,Th1)和辅助T细胞17(T helper 17 cells,Th17)数量显著性降低(P<0.05),同过表达IL-10的MSCs共培养的巨噬细胞细胞培养上清液,TNF-α含量显著性降低(P<0.0001)。右旋糖酐硫酸钠(dextran sodium sulfate,DSS)诱导IBD小鼠模型中,尾静脉注射过表达IL-10的MSCs与注射未修饰MSCs相比,过表达IL-10的MSCs具有更好的治疗效果,结肠长度、疾病活动指数(disease activity index,DAI)和结肠炎性细胞因子表达共同证明这一差异。实验结果均具有统计学差异(P<0.05)。总体而言,经LV转染过表达IL-10的MSCs可能是IBD的一种有希望的替代治疗选择。     

Local and systemic inhibition of lung tumor growth after nanoparticle-mediated mda-7/IL-24 gene delivery

The human melanoma differentiation associated gene-7 (mda-7), also known as interleukin-24 (IL-24), is a novel gene with tumor suppressor, antiangiogenic, and cytokine properties. In vitro adenovirus-mediated gene transfer of the human mda-7/IL-24 gene (Ad-mda-7) results in ubiquitous growth suppression of human cancer cells with minimal toxicity to normal cells. Intratumoral administration of Ad-mda-7 to lung tumor xenografts results in growth suppression via induction of apoptosis and antiangiogenic mechanisms. Although these results are encouraging, one limitation of this approach is that its locoregional clinical application-systemic delivery of adenoviruses for treatment of disseminated cancer is not feasible at the present time. An alternative approach that is suitable for systemic application is non-viral gene delivery. We recently demonstrated that DOTAP:cholesterol (DOTAP:Chol) nanoparticles effectively deliver tumor suppressor genes to primary and disseminated lung tumors. In the present study, therefore, we evaluated nanoparticle-mediated delivery of the human mda-7/IL-24 gene to primary and disseminated lung tumors in vivo. We demonstrate that DOTAP:Chol efficiently delivers the mda-7/IL-24 gene to human lung tumor xenografts, resulting in suppression of tumor growth. Growth-inhibitory effects were observed in both primary (P=0.001) and metastatic lung tumors (P=0.02). Furthermore, tumor vascularization was reduced in mda-7/IL-24-treated tumors. Finally, growth was also inhibited in murine syngenic tumors treated with DOTAP:Chol-mda-7 nanoparticles (P=0.01). This is the first report demonstrating (1) systemic therapeutic effects of mda-7/IL-24 in lung cancer, and (2) antitumor effects of human mda-7 in syngeneic cancer models. Our findings are important for the development of mda-7/IL-24 treatments for primary and disseminated cancers.     

EGFR Inhibition Abrogates Leiomyosarcoma Cell Chemoresistance through Inactivation of Survival Pathways and Impairment of CSC Potential

Background

Tumor cells with stem-like phenotype and properties, known as cancer stem cells (CSC), have been identified in most solid tumors and are presumed to be responsible for driving tumor initiation, chemoresistance, relapse, or metastasis. A subpopulation of cells with increased stem-like potential has also been identified within sarcomas. These cells are endowed with increased tumorigenic potential, chemoresistance, expression of embryonic markers, and side population(SP) phenotype. Leiomyosarcomas (LMS) are soft tissue sarcomas presumably arising from undifferentiated cells of mesenchymal origin, the Mesenchymal Stem Cells (MSC). Frequent recurrence of LMS and chemoresistance of relapsed patients may likely result from the failure to target CSC. Therefore, therapeutic cues coming from the cancer stem cell (CSC) field may drastically improve patient outcome.

Methodology/Principal Findings

We expanded LMS stem-like cells from patient samples in vitro and examined the possibility to counteract LMS malignancy through a stem-like cell effective approach. LMS stem-like cells were in vitro expanded both as “tumor spheres” and as “monolayers” in Mesenchymal Stem Cell (MSC) conditions. LMS stem-like cells displayed MSC phenotype, higher SP fraction, and increased drug-extrusion, extended proliferation potential, self-renewal, and multiple differentiation ability. They were chemoresistant, highly tumorigenic, and faithfully reproduced the patient tumor in mice. Such cells displayed activation of EGFR/AKT/MAPK pathways, suggesting a possibility in overcoming their chemoresistance through EGFR blockade. IRESSA plus Vincristine treatment determined pathway inactivation, impairment of SP phenotype, high cytotoxicity in vitro and strong antitumor activity in stem-like cell-generated patient-like xenografts, targeting both stem-like and differentiated cells.

Conclusions/Significance

EGFR blockade combined with vincristine determines stem-like cell effective antitumor activity in vitro and in vivo against LMS, thus providing a potential therapy for LMS patients.     

Delivery of sTRAIL variants by MSCs in combination with cytotoxic drug treatment leads to p53-independent enhanced antitumor effects

Mesenchymal stem cells (MSCs) are able to infiltrate tumor tissues and thereby effectively deliver gene therapeutic payloads. Here, we engineered murine MSCs (mMSCs) to express a secreted form of the TNF-related apoptosis-inducing ligand (TRAIL), which is a potent inducer of apoptosis in tumor cells, and tested these MSCs, termed MSC.sTRAIL, in combination with conventional chemotherapeutic drug treatment in colon cancer models. When we pretreated human colorectal cancer HCT116 cells with low doses of 5-fluorouracil (5-FU) and added MSC.sTRAIL, we found significantly increased apoptosis as compared with single-agent treatment. Moreover, HCT116 xenografts, which were cotreated with 5-FU and systemically delivered MSC.sTRAIL, went into remission. Noteworthy, this effect was protein 53 (p53) independent and was mediated by TRAIL-receptor 2 (TRAIL-R2) upregulation, demonstrating the applicability of this approach in p53-defective tumors. Consequently, when we generated MSCs that secreted TRAIL-R2-specific variants of soluble TRAIL (sTRAIL), we found that such engineered MSCs, labeled MSC.sTRAIL^DR5, had enhanced antitumor activity in combination with 5-FU when compared with MSC.sTRAIL. In contrast, TRAIL-resistant pancreatic carcinoma PancTu1 cells responded better to MSC.sTRAIL^DR4 when the antiapoptotic protein XIAP (X-linked inhibitor of apoptosis protein) was silenced concomitantly. Taken together, our results demonstrate that TRAIL-receptor selective variants can potentially enhance the therapeutic efficacy of MSC-delivered TRAIL as part of individualized and tumor-specific combination treatments.     

IL-27 mediates complete regression of orthotopic primary and metastatic murine neuroblastoma tumors: role for CD8+ T cells

We have shown previously that IFN-gamma-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8(+) T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-gamma. In addition, marked enhancement of local IFN-gamma gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8(+), but not CD4(+) T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.     

Oncolytic Adenovirus Expressing IL-23 and p35 Elicits IFN-γ- and TNF-α-Co-Producing T Cell-Mediated Antitumor Immunity

Cytokine immunogene therapy is a promising strategy for cancer treatment. Interleukin (IL)-12 boosts potent antitumor immunity by inducing T helper 1 cell differentiation and stimulating cytotoxic T lymphocyte and natural killer cell cytotoxicity. IL-23 has been proposed to have similar but not overlapping functions with IL-12 in inducing Th1 cell differentiation and antitumor immunity. However, the therapeutic effects of intratumoral co-expression of IL-12 and IL-23 in a cancer model have yet to be investigated. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral inoculation of oncolytic adenovirus co-expressing IL-23 and p35, RdB/IL23/p35. Intratumoral administration of RdB/IL23/p35 elicited strong antitumor effects and increased survival in a murine B16-F10 syngeneic tumor model. The levels of IL-12, IL-23, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were elevated in RdB/IL23/p35-treated tumors. Moreover, the proportion of regulatory T cells was markedly decreased in mice treated with RdB/IL23/p35. Consistent with these data, mice injected with RdB/IL23/p35 showed massive infiltration of CD4⁺ and CD8⁺ T cells into the tumor as well as enhanced induction of tumor-specific immunity. Importantly, therapeutic mechanism of antitumor immunity mediated by RdB/IL23/p35 is associated with the generation and recruitment of IFN-γ- and TNF-α-co-producing T cells in tumor microenvironment. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-23 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.     

Pre-existing tumor-sensitized T cells are essential for eradication of established tumors by IL-12 and cyclophosphamide plus IL-12.

The antitumor immune response activated by IL-12, especially by a combination of cyclophosphamide and IL-12 (Cy+IL-12), is clinically significant in certain experimental tumor models, in that a number of well-established (10-20 mm in diameter) s.c. tumors are completely eradicated. Furthermore, Cy+IL-12 treatment is also able to eradicate well-established grossly detectable experimental lung metastases and advanced ascites tumors. Despite the dramatic antitumor effects seen in some tumor models, Cy+IL-12 fails to induce regression of other established tumors. Characterization of tumor immunogenicity shows that all tumors responding to IL-12 and Cy+IL-12 treatments are immunogenic tumors, in that an antitumor immune response is detectable in tumor-bearing hosts upon tumor establishment. In contrast, none of the nonimmunogenic tumor responds to IL-12 and Cy+IL-12 treatments. Analysis of cellular requirements for successful tumor rejection through an adoptive cell transfer approach reveals that the presence of tumor-sensitized, but not naive, T cells is essential for tumor rejection by IL-12 and Cy+IL-12. Transfer of these tumor-sensitized T cells must be conducted before, but not after, IL-12 treatment in order for tumor rejection to occur. The requirement of sensitized T cells is also tumor specific. In mice bearing immunogenic tumors, the presence of pre-existing tumor-sensitized T cells is demonstrated by adoptive cell transfer experiments using purified spleen T cells from these mice. Results from our study show that Cy+IL-12-based immunotherapy of cancer may be highly effective and that pre-existing tumor-sensitized T cells are essential for the success of the therapy.     

食蟹猴胚胎干细胞向间充质前体细胞诱导分化及鉴定

间充质干细胞(mesenchymal stem cells,MSCs)可以诱导分化成脂肪、软骨、骨骼和骨骼肌细胞,并可作为骨骼、软骨或肌肉移植中的再生干细胞,广泛应用于细胞治疗和组织工程。胚胎干细胞(embryonic stem cells,ESCs)具有体外培养无限增殖和多向分化的特性,能被诱导分化为机体几乎所有的细胞类型。该研究通过无血清条件下诱导食蟹猴ESCs形成类胚体(embryoid bodies,EBs),然后在血清条件下贴壁分化EBs成间充质前体细胞(mesenchymal precursor cells,MPCs),再经过长期体外培养,纯化和扩增MPCs。结果显示,纯化后的MPCs具有MSCs生物学特征,并能在体外诱导分化成脂肪细胞和骨细胞。将这些细胞皮下注射给SCID小鼠,并未发现形成肿瘤,提示食蟹猴ESCs来源的MPCs具有一定的安全性。