Pigment epithelium-derived factor inhibits glioma cell growth in vitro and in vivo

Glioblastoma multiforme is the most common malignant brain tumor in adults, and it is among the most lethal of all cancers. Recent studies have shown that pigment epithelium-derived factor (PEDF) can induce differentiation and inhibit angiogenesis of several tumors. This study was designed to determine whether gliomas angiogenesis and tumor growth could be inhibited by PEDF. We found that PEDF down-regulated expression levels of vascular endothelial growth factor and up-regulated the expression of thrombospondin-2 and augmented apoptosis in a dose-dependent manner in both A172 and U87 glioma cells lines after 48 h of treatment. Analysis of the cell cycle showed arrest in the G₁ phase and block in S phase of the cell cycle. Meanwhile PEDF induced apoptosis was associated with increases of p53 and Bax and inhibition of Bcl-2. Conditioned medium with PEDF showed a significantly reductive effect on migration in vitro accompanied with a significant reduction of matrix metalloproteinase-9 expression. PEDF suppressed glioma cell migration in vitro and tumor burden in athymic nude mice. These results demonstrate for the first time inhibitory effects of PEDF on the growth and migration of human gliomas via induction of apoptosis and blocking of migratory-related factors. PEDF activation can be a novel approach for future therapeutic purposes against gliomas.     

The homing of human cord blood stem cells to sites of inflammation: Unfolding mysteries of a novel therapeutic paradigm for glioblastoma multiforme

Efficient homing of human umbilical cord blood mesenchymal stem cells (hUCBSC) to inflammation sites is crucial for therapeutic use. In glioblastoma multiforme, soluble factors released by the tumor facilitate the migratory capacity of mesenchymal stem cells toward glioma cells. These factors include chemokines and growth inducers. Nonetheless, the mechanistic details of these factors involved in hUCBSC homing have not been clearly delineated. The present study is aimed to deduce specific factors involved in hUCBSC homing by utilizing a glioma stem cell-induced inflammatory lesion model in the mouse brain. Our results show that hUCBSC do not form tumors in athymic nude mice brains and do not elicit immune responses in immunocompetent SKH1 mice. Further, hUCBSC spheroids migrate and invade glioma spheroids, while no effect was observed on rat fetal brain aggregates. Several cytokines, including GRO, MCP-1, IL-8, IL-3, IL-10, Osteopontin and TGF-β2, were constitutively secreted in the naive hUCBSC-conditioned medium, while significant increases of IL-8, GRO, GRO-α, MCP-1 and MCP-2 were observed in glioma stem cell-challenged hUCBSC culture filtrates. Furthermore, hUCBSC showed a stronger migration capacity toward glioma stem cells in vitro and exhibited enhanced migration to glioma stem cells in an intracranial human malignant glioma xenograft model. Our results indicate that multiple cytokines are involved in recruitment of hUCBSC toward glioma stem cells, and that hUCBSC are a potential candidate for glioma therapy.     

MicroRNA-145 Is Downregulated in Glial Tumors and Regulates Glioma Cell Migration by Targeting Connective Tissue Growth Factor

Glioblastomas (GBM), the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs) compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF) as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3′-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3′-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.     

Glioma Cell Death: Cell–Cell Interactions and Signalling Networks

The prognosis for patients with malignant gliomas is poor, but improvements may emerge from a better understanding of the pathophysiology of glioma signalling. Recent therapeutic developments have implicated lipid signalling in glioma cell death. Stress signalling in glioma cell death involves mitochondria and endoplasmic reticulum. Lipid mediators also signal via extrinsic pathways in glioma cell proliferation, migration and interaction with endothelial and microglial cells. Glioma cell death and tumour regression have been reported using polyunsaturated fatty acids in animal models, human ex vivo explants, glioma cell preparations and in clinical case reports involving intratumoral infusion. Cell death signalling was associated with generation of reactive oxygen intermediates and mitochondrial and other signalling pathways. In this review, evidence for mitochondrial responses to stress signals, including polyunsaturated fatty acids, peroxidising agents and calcium is presented. Additionally, evidence for interaction of glioma cells with primary brain endothelial cells is described, modulating human glioma peroxidative signalling. Glioma responses to potential therapeutic agents should be analysed in systems reflecting tumour connectivity and CNS structural and functional integrity. Future insights may also be derived from studies of signalling in glioma-derived tumour stem cells.     

IL-6 Secreted from Senescent Mesenchymal Stem Cells Promotes Proliferation and Migration of Breast Cancer Cells

Human mesenchymal stem cells (hMSCs) are currently investigated for a variety of therapeutic applications. However, MSCs isolated from primary tissue cannot meet clinical grade needs and should be expanded in vitro for several passages. Although hMSCs show low possibility for undergoing oncogenic transformation, they do, similar to other somatic cells, undergo cellular senescence and their therapeutic potential is diminished when cultured in vitro. However, the role of senescent MSCs in tumor progression remains largely elusive. In the current study, by establishing senescent human umbilical cord mesenchymal stem cells (s-UCMSCs) through the replicative senescence model and genotoxic stress induced premature senescence model, we show that s-UCMSCs significantly stimulate proliferation and migration of breast cancer cells in vitro and tumor progression in a co-transplant xenograft mouse model compared with ‘young’ counterparts (defined as MSCs at passage 5, in contrast to senescent MSCs at passage 45). In addition, we identified IL-6, a known pleiotropic cytokine, as a principal mediator for the tumor-promoting activity of s-UCMSCs by induction of STAT3 phosphorylation. Depletion of IL-6 from s-UCMSCs conditioned medium partially abrogated the stimulatory effect of s-UCMSCs on the proliferation and migration of breast tumor cells.     

Overexpression of HIF-1α in mesenchymal stem cells contributes to repairing hypoxic-ischemic brain damage in rats

Preclinical researches on mesenchymal stem cells (MSCs) transplantation, which is used to treat hypoxic-ischemic (HI) brain damage, have received inspiring achievements. However, the insufficient migration of active cells to damaged tissues has limited their potential therapeutic effects. There are some evidences that hypoxia inducible factor-1 alpha (HIF-1α) promotes the viability and migration of the cells. Here, we aim to investigate whether overexpression of HIF-1α in MSCs could improve the viability and migration capacity of cells, and its therapeutic efficiency on HI brain damage. In the study, MSCs with HIF-1α overexpression was achieved by recombinant lentiviral vector and transplanted to the rats subsequent to HI. Our data indicated that overexpression of HIF-1α promoted the viability and migration of MSCs, HIF-1α overexpressed MSCs also had a stronger therapeutic efficiency on HI brain damaged treatment by mitigating the injury on behavioral and histological changes evoked by HI insults, accompanied with more MSCs migrating to cerebral damaged area. This study demonstrated that HIF-1α overexpression could increase the MSCs’ therapeutic efficiency in HI and the promotion of the cells’ directional migration to cerebral HI area by overexpression may be responsible for it, which showed that transplantation of MSCs with HIF-1α overexpression is an attractive therapeutic option to treat HI-induced brain injury in the future.     

The neural stem cell fate determinant TLX promotes tumorigenesis and genesis of cells resembling glioma stem cells

A growing body of evidence indicates that deregulation of stem cell fate determinants is a hallmark of many types of malignancies. The neural stem cell fate determinant TLX plays a pivotal role in neurogenesis in the adult brain by maintaining neural stem cells. Here, we report a tumorigenic role of TLX in brain tumor initiation and progression. Increased TLX expression was observed in a number of glioma cells and glioma stem cells, and correlated with poor survival of patients with gliomas. Ectopic expression of TLX in the U87MG glioma cell line and Ink4a/Arf-deficient mouse astrocytes (Ink4a/Arf^-/- astrocytes) induced cell proliferation with a concomitant increase in cyclin D expression, and accelerated foci formation in soft agar and tumor formation in in vivo transplantation assays. Furthermore, overexpression of TLX in Ink4a/Arf^-/- astrocytes inhibited cell migration and invasion and promoted neurosphere formation and Nestin expression, which are hallmark characteristics of glioma stem cells, under stem cell culture conditions. Our results indicate that TLX is involved in glioma stem cell genesis and represents a potential therapeutic target for this type of malignancy.     

Mesenchymal Stem Cells Derived from Adipose Tissue vs Bone Marrow: In Vitro Comparison of Their Tropism towards Gliomas

Introduction

Glioblastoma is the most common primary malignant brain tumor, and is refractory to surgical resection, radiation, and chemotherapy. Human mesenchymal stem cells (hMSC) may be harvested from bone marrow (BMSC) and adipose (AMSC) tissue. These cells are a promising avenue of investigation for the delivery of adjuvant therapies. Despite extensive research into putative mechanisms for the tumor tropism of MSCs, there remains no direct comparison of the efficacy and specificity of AMSC and BMSC tropism towards glioma.

Methods

Under an IRB-approved protocol, intraoperative human Adipose MSCs (hAMSCs) were established and characterized for cell surface markers of mesenchymal stem cell origin in conjunction with the potential for tri-lineage differentiation (adipogenic, chondrogenic, and osteogenic). Validated experimental hAMSCs were compared to commercially derived hBMSCs (Lonza) and hAMSCs (Invitrogen) for growth responsiveness and glioma tropism in response to glioma conditioned media obtained from primary glioma neurosphere cultures.

Results

Commercial and primary culture AMSCs and commercial BMSCs demonstrated no statistically significant difference in their migration towards glioma conditioned media in vitro. There was statistically significant difference in the proliferation rate of both commercial AMSCs and BMSCs as compared to primary culture AMSCs, suggesting primary cultures have a slower growth rate than commercially available cell lines.

Conclusions

Adipose- and bone marrow-derived mesenchymal stem cells have similar in vitro glioma tropism. Given the well-documented ability to harvest larger numbers of AMSCs under local anesthesia, adipose tissue may provide a more efficient source of MSCs for research and clinical applications, while minimizing patient morbidity during cell harvesting.     

Mesenchymal stem cells promote cell invasion and migration and autophagy‐induced epithelial‐mesenchymal transition in A549 lung adenocarcinoma cells

Mesenchymal stem cells (MSCs) are recruited into the tumour microenvironment and promote tumour growth and metastasis. Tumour microenvironment‐induced autophagy is considered to suppress primary tumour formation by impairing migration and invasion. Whether these recruited MSCs regulate tumour autophagy and whether autophagy affects tumour growth are controversial. Our data showed that MSCs promote autophagy activation, reactive oxygen species production, and epithelial‐mesenchymal transition (EMT) as well as increased migration and invasion in A549 cells. Decreased expression of E‐cadherin and increased expression of vimentin and Snail were observed in A549 cells cocultured with MSCs. Conversely, MSC coculture‐mediated autophagy positively promoted tumour EMT. Autophagy inhibition suppressed MSC coculture‐mediated EMT and reduced A549 cell migration and invasion slightly. Furthermore, the migratory and invasive abilities of A549 cells were additional increased when autophagy was further enhanced by rapamycin treatment. Taken together, this work suggests that microenvironments containing MSCs can promote autophagy activation for enhancing EMT; MSCs also increase the migratory and invasive abilities of A549 lung adenocarcinoma cells. Mesenchymal stem cell‐containing microenvironments and MSC‐induced autophagy signalling may be potential targets for blocking lung cancer cell migration and invasion.     

The Involvement of CXCL11 in Bone Marrow-Derived Mesenchymal Stem Cell Migration Through Human Brain Microvascular Endothelial Cells

Bone marrow-derived mesenchymal stem cells (MSCs) transplant into the brain, where they play a potential therapeutic role in neurological diseases. However, the blood–brain barrier (BBB) is a native obstacle for MSCs entry into the brain. Little is known about the mechanism behind MSCs migration across the BBB. In the present study, we modeled the interactions between human MSCs (hMSCs) and human brain microvascular endothelial cells (HBMECs) to mimic the BBB microenvironment. Real-time PCR analysis indicated that the chemokine CXCL11 is produced by hMSCs and the chemokine receptor CXCR3 is expressed on HBMECs. Further results indicate that CXCL11 secreted by hMSCs may interact with CXCR3 on HBMECs to induce the disassembly of tight junctions through the activation of ERK1/2 signaling in the endothelium, which promotes MSCs transendothelial migration. These findings are relevant for understanding the biological responses of MSCs in BBB environments and helpful for the application of MSCs in neurological diseases.     

Mesenchymal Stem Cells Modified with a Single-Chain Antibody against EGFRvIII Successfully Inhibit the Growth of Human Xenograft Malignant Glioma

Background

Glioblastoma multiforme is the most lethal brain tumor with limited therapeutic options. Antigens expressed on the surface of malignant cells are potential targets for antibody-mediated gene/drug delivery.

Principal Findings

In this study, we investigated the ability of genetically modified human mesenchymal stem cells (hMSCs) expressing a single-chain antibody (scFv) on their surface against a tumor specific antigen, EGFRvIII, to enhance the therapy of EGFRvIII expressing glioma cells in vivo. The growth of U87-EGFRvIII was specifically delayed in co-culture with hMSC-scFvEGFRvIII. A significant down-regulation was observed in the expression of pAkt in EGFRvIII expressing glioma cells upon culture with hMSC-scFvEGFRvIII vs. controls as well as in EGFRvIII expressing glioma cells from brain tumors co-injected with hMSC-scFvEGFRvIII in vivo. hMSC expressing scFvEGFRvIII also demonstrated several fold enhanced retention in EGFRvIII expressing flank and intracranial glioma xenografts vs. control hMSCs. The growth of U87-EGFRvIII flank xenografts was inhibited by 50% in the presence of hMSC-scFvEGFRvIII (p<0.05). Moreover, animals co-injected with U87-EGFRvIII and hMSC-scFvEGFRvIII intracranially showed significantly improved survival compared to animals injected with U87-EGFRvIII glioma cells alone or with control hMSCs. This survival was further improved when the same animals received an additional dosage of hMSC-scFvEGFRvIII two weeks after initial tumor implantation. Of note, EGFRvIII expressing brain tumors co-injected with hMSCs had a lower density of CD31 expressing blood vessels in comparison with control tumors, suggesting a possible role in tumor angiogenesis.

Conclusions/Significance

The results presented in this study illustrate that genetically modified MSCs may function as a novel therapeutic vehicle for malignant brain tumors.     

The homing of human cord blood stem cells to sites of inflammation

Efficient homing of human umbilical cord blood mesenchymal stem cells (hUCBSC) to inflammation sites is crucial for therapeutic use. In glioblastoma multiforme, soluble factors released by the tumor facilitate the migratory capacity of mesenchymal stem cells toward glioma cells. These factors include chemokines and growth inducers. Nonetheless, the mechanistic details of these factors involved in hUCBSC homing have not been clearly delineated. The present study is aimed to deduce specific factors involved in hUCBSC homing by utilizing a glioma stem cell-induced inflammatory lesion model in the mouse brain. Our results show that hUCBSC do not form tumors in athymic nude mice brains and do not elicit immune responses in immunocompetent SKH1 mice. Further, hUCBSC spheroids migrate and invade glioma spheroids, while no effect was observed on rat fetal brain aggregates. Several cytokines, including GRO, MCP-1, IL-8, IL-3, IL-10, Osteopontin and TGF-β2, were constitutively secreted in the naive hUCBSC-conditioned medium, while significant increases of IL-8, GRO, GRO-α, MCP-1 and MCP-2 were observed in glioma stem cell-challenged hUCBSC culture filtrates. Furthermore, hUCBSC showed a stronger migration capacity toward glioma stem cells in vitro and exhibited enhanced migration to glioma stem cells in an intracranial human malignant glioma xenograft model. Our results indicate that multiple cytokines are involved in recruitment of hUCBSC toward glioma stem cells, and that hUCBSC are a potential candidate for glioma therapy.     

The NF-κB RelB Protein Is an Oncogenic Driver of Mesenchymal Glioma

High-grade gliomas, such as glioblastomas (GBMs), are very aggressive, invasive brain tumors with low patient survival rates. The recent identification of distinct glioma tumor subtypes offers the potential for understanding disease pathogenesis, responses to treatment and identification of molecular targets for personalized cancer therapies. However, the key alterations that drive tumorigenesis within each subtype are still poorly understood. Although aberrant NF-κB activity has been implicated in glioma, the roles of specific members of this protein family in tumorigenesis and pathogenesis have not been elucidated. In this study, we show that the NF-κB protein RelB is expressed in a particularly aggressive mesenchymal subtype of glioma, and loss of RelB significantly attenuated glioma cell survival, motility and invasion. We find that RelB promotes the expression of mesenchymal genes including YKL-40, a marker of the MES glioma subtype. Additionally, RelB regulates expression of Olig2, a regulator of cancer stem cell proliferation and a candidate marker for the cell of origin in glioma. Furthermore, loss of RelB in glioma cells significantly diminished tumor growth in orthotopic mouse xenografts. The relevance of our studies for human disease was confirmed by analysis of a human GBM genome database, which revealed that high RelB expression strongly correlates with rapid tumor progression and poor patient survival rates. Thus, our findings demonstrate that RelB is an oncogenic driver of mesenchymal glioma tumor growth and invasion, highlighting the therapeutic potential of inhibiting the noncanonical NF-κB (RelB-mediated) pathway to treat these deadly tumors.     

Proportional Upregulation of CD97 Isoforms in Glioblastoma and Glioblastoma-Derived Brain Tumor Initiating Cells

CD97 is a novel glioma antigen that confers an invasive phenotype and poor survival in patients with glioblastoma (GBM), the most aggressive primary malignant brain tumor. The short isoform of CD97, known as EGF(1,2,5), has been shown to promote invasion and metastasis, but its role in gliomas and GBM-derived brain tumor initiating cells (BTICs) has not been studied. We sought to characterize CD97 expression among gliomas and identify the specific isoforms expressed. The short isoform of CD97 was identified in GBM and GBM-derived BTICs, but not low grade or anaplastic astrocytomas. All samples expressing the EGF(1,2,5) isoform were also found to express the EGF(1,2,3,5) isoform. These isoforms are believed to possess similar ligand binding patterns and interact with chondroitin sulfate, a component of the extracellular matrix, and the integrin α5β1. Using data acquired from the Cancer Genome Atlas (TCGA), we show that CD97 is upregulated among the classical and mesenchymal subtypes of GBM and significantly decreased among IDH1 mutant GBMs. Given its proven roles in tumor invasion, expression among aggressive genetic subtypes of GBM, and association with overall survival, CD97 is an attractive therapeutic target for patients with GBM.     

Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

BackgroundEwing''s sarcoma (ES) is an aggressive cancer affecting children and young adults. We pre-clinically demonstrated that mesenchymal stromal/stem cells (MSCs) can deliver tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) against primary ES after local injection. However, ES is often metastatic calling for approaches able to support MSC targeting to the ES multiple remote sites. Considering that the disialoganglioside GD2 is expressed by ES and to optimise MSC tumour affinity, bi-functional (BF) MSCs expressing both TRAIL and a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR) were generated and challenged against ES.MethodsThe anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were tested in several in vitro ES models. Tumour targeting and killing by BF MSCs was further investigated by a novel immunodeficient ES metastatic model characterized by different metastatic sites, including lungs, liver and bone, mimicking the deadly clinical scenario.FindingsIn vitro data revealed both tumour affinity and killing of BF MSCs. In vivo, GD2 tCAR molecule ameliorated the tumour targeting and persistence of BF MSCs counteracting ES in lungs but not in liver.InterpretationWe here generated data on the potential effects of BF MSCs within a complex ES metastatic in vivo model, exploring also the biodistribution of MSCs. Our BF MSC-based strategy promises to pave the way for potential improvements in the therapeutic delivery of TRAIL for the treatment of metastatic ES and other deadly GD2-positive malignancies.     

Enhanced Antitumor Efficacy of an Oncolytic Herpes Simplex Virus Expressing an Endostatin–Angiostatin Fusion Gene in Human Glioblastoma Stem Cell Xenografts

Viruses have demonstrated strong potential for the therapeutic targeting of glioblastoma stem cells (GSCs). In this study, the use of a herpes simplex virus carrying endostatin–angiostatin (VAE) as a novel therapeutic targeting strategy for glioblastoma-derived cancer stem cells was investigated. We isolated six stable GSC-enriched cultures from 36 human glioblastoma specimens and selected one of the stable GSCs lines for establishing GSC-carrying orthotopic nude mouse models. The following results were obtained: (a) VAE rapidly proliferated in GSCs and expressed endo–angio in vitro and in vivo 48 h and 10 d after infection, respectively; (b) compared with the control gliomas treated with rHSV or Endostar, the subcutaneous gliomas derived from the GSCs showed a significant reduction in microvessel density after VAE treatment; (c) compared with the control, a significant improvement was observed in the length of the survival of mice with intracranial and subcutaneous gliomas treated with VAE; (d) MRI analysis showed that the tumor volumes of the intracranial gliomas generated by GSCs remarkably decreased after 10 d of VAE treatment compared with the controls. In conclusion, VAE demonstrated oncolytic therapeutic efficacy in animal models of human GSCs and expressed an endostatin–angiostatin fusion gene, which enhanced antitumor efficacy most likely by restricting tumor microvasculature development.     

CXC chemokine receptor 1 enhances the ability of human umbilical cord blood-derived mesenchymal stem cells to migrate toward gliomas

In this study, we showed that knocking-down interleukin-8 (IL-8) in glioma cells, or its receptor, CXC chemokine receptor 1 (CXCR1) in hUCB-MSCs reduced hUCB-MSC migration toward glioma cells in a Transwell chamber. In contrast, CXCR1-transfected hUCB-MSCs (CXCR1-MSCs) showed a superior capacity to migrate toward glioma cells in a Transwell chamber compared to primary hUCB-MSCs. Furthermore, these transfected cells also demonstrated the same ability to migrate toward tumors in mice bearing intracranial human gliomas as shown by histological and in vivo imaging analysis. Our findings indicate that overexpression of CXCR1 could be a useful tool for MSC-based gene therapy to achieve a sufficient quantity of therapeutic MSCs that are localized within tumors.