Paula Y.P. Lam

Human Bone Marrow‐Derived Mesenchymal Stem Cells Suppress Human Glioma Growth Through Inhibition of Angiogenesis

Tumor tropism of human bone marrow‐derived mesenchymal stem cells (MSC) has been exploited for the delivery of therapeutic genes for anticancer therapy. However, the exact contribution of these cells in the tumor microenvironment remains unknown. In this study, we examined the biological effect of MSC on tumor cells. The results showed that MSC inhibited the growth of human glioma cell lines and patient‐derived primary glioma cells in vitro. Coadministration of MSC and glioma cells resulted in significant reduction in tumor volume and vascular density, which was not observed when glioma was injected with immortalized normal human astrocytes. Using endothelial progenitor cells (EPC) from healthy donors and HUVEC endothelial cells, the extent of EPC recruitment and capacity to form endothelial tubes was significantly impaired in conditioned media derived from MSC/glioma coculture, suggesting that MSC suppressed tumor angiogenesis through the release of antiangiogenic factors. Further studies using antibody array showed reduced expression of platelet‐derived growth factor (PDGF)‐BB and interleukin (IL)‐1β in MSC/glioma coculture when compared with controls. In MSC/glioma coculture, PDGF‐BB mRNA and the corresponding proteins (soluble and membrane bound forms) as well as the receptors were found to be significantly downregulated when compared with that of glioma cocultured with normal human astrocytes or glioma monoculture. Furthermore, IL‐1β, phosphorylated Akt, and cathepsin B proteins were also reduced in MSC/glioma. Taken together, these data indicated that the antitumor effect of MSC may be mediated through downregulation of PDGF/PDGFR axis, which is known to play a key role in glioma angiogenesis. STEM Cells2013;31:146–155

Matrix metalloproteinase 1 is necessary for the migration of human bone marrow-derived mesenchymal stem cells toward human glioma.

Human mesenchymal stem cells (MSCs) have increasingly been used as cellular vectors for the delivery of therapeutic genes to tumors. However, the precise mechanism of mobilization remains poorly defined. In this study, MSCs that expressed similar cell surface markers and exhibited multilineage differentiation potentials were isolated from various donors. Interestingly, different MSC isolates displayed differential migration ability toward human glioma cells. We hypothesized that distinct molecular signals may be involved in the varied tumor tropisms exhibited by different MSC isolates. To test this hypothesis, gene expression profiles of tumor‐trophic MSCs were compared with those of non–tumor‐trophic MSCs. Among the various differentially regulated genes, matrix metalloproteinase one (MMP1) gene expression and its protein activities were enhanced by 27‐fold and 21‐fold, respectively, in highly migrating MSCs compared with poorly migrating MSCs. By contrast, there was no change in the transcriptional levels of other MMPs. Functional inactivation of MMP1 abrogated the migratory potential of MSCs toward glioma‐conditioned medium. Conversely, the nonmigratory phenotype of poorly migrating MSC could be rescued in the presence of either recombinant MMP1 or conditioned medium from the highly migrating MSCs. Ectopic expression of MMP1 in these poorly migrating cells also rendered the cells responsive to the signaling cues from the glioma cells in vivo. However, blocking the interaction of MMP1 and its cognate receptor PAR1 effectively diminished the migratory ability of MSCs. Taken together, this study provides, for the first time, supporting evidence that MMP1 is critically involved in the migration capacity of MSCs, acting through the MMP1/PAR1 axis. STEM CELLS 2009;27:1366–1375

Nanosized bioceramic particles could function as efficient gene delivery vehicles with target specificity for the spleen.

We have compared the ability of several nanosized bioceramic particles including negatively charged silica (SiO2), neutrally charged hydroxyapatite (HA) and positively charged zirconia (ZrO2) nanoparticles as non-viral vectors for efficient in vivo gene delivery. A mixture of highly monodispersed aqueous suspension of HA or SiO2 nanoparticles, coated with protamine sulfate (PS), complexed efficiently with plasmid DNA and significantly enhanced transgene expression in vitro. In comparison, ZrO2 nanoparticles gave poor transfection efficiency under similar conditions tested. It was also determined that, under the same conditions, PS-SiO2-DNA, but not PS-HA-DNA-nanoplexes, were able to mediate efficient transgene expression in vitro in the presence of 50% serum. Intraperitoneal injections of PS-SiO2-luciferase DNA nanoplexes targeted the highest level of transgene expression in the spleen of recipient mice that lasted for more than 48 h. Injection of PS-SiO2-pNGVL-hFLex-MUC-1 nanoplexes was able to mediate the production of Flt-3L in the sera of recipient mice. Simultaneously, the production of Flt-3L was accompanied by the stimulation of IL-2 and interferon-γ (IFN-γ). Most importantly, the injection of PS-SiO2-pNGVL-hFLex-MUC-1 nanoplexes could mount potent anti-tumour specific immune responses that led to the subsequent regression of parental tumor cells containing the muc-1 determinant.

Identification and characterization of novel human glioma-specific peptides to potentiate tumor-specific gene delivery.

Glioblastomas account for approximately 20% of all primary brain tumors in adults. Glioblastoma multiforme (GBM) is a highly malignant tumor. In spite of advances in surgery, chemotherapy, and radiotherapy, the life expectancy of the patient with glioblastoma is approximately 11 months. To enhance glioma-specific gene delivery, we employed a 12-mer phage display peptide library to isolate phages that bind specifically to human glioma cell lines. Here, we report the isolation and functional characterization of novel glioma-specific peptides that target transgenes specifically to a wide array of human glioblastomas in vitro and in vivo. One of the isolated peptides, tentatively denoted as MG11, is demonstrated to be glioma specific and gives an in vitro-binding enrichment of more than 5-fold for glioma cells when compared with nonglioma cells. Intravenous injection of phages bearing the MG11 peptide-binding motif enables the phages to home specifically to glioma xenografts. Most significantly, when Lissamine rhodamine-labeled MG11 peptide is injected intratumorally, it targets specifically to glioma xenografts instead of non-glioma-derived xenografts. In summary, our results suggest that the MG11 peptide is able to target specifically to tumors of glial origin, which would allow the design of applications related to the diagnosis and treatment of human gliomas.

Glioma-specific and cell cycle-regulated herpes simplex virus type 1 amplicon viral vector.

We have engineered a novel herpes simplex virus type 1 (HSV-1)-based amplicon viral vector, whereby gene expression is controlled by cell cycle events. In nondividing cells, trans-activation of the cyclin A promoter via interaction of the Gal4/NF-YA fusion protein with the Gal4-binding sites is prevented by the presence of a repressor protein, cell cycle-dependent factor 1 (CDF-1). CDF-1 is specifically expressed during the G(0)/G(1) phase of the cell cycle and its binding site is located within the cyclin A promoter. In actively proliferating cells, trans-activation could take place because of the absence of CDF-1. Our results showed that when all these cell cycle-specific regulatory elements are incorporated in cis into a single HSV-1 amplicon plasmid vector backbone (pC8-36), reporter luciferase activity is greatly enhanced. Transgene expression mediated by this series of HSV-1 amplicon plasmid vectors and amplicon viral vectors could be regulated in a cell cycle-dependent manner in a variety of cell lines. In a further attempt to target transgene expression to a selected group of actively proliferating cells such as glial cells, we have replaced the cytomegalovirus promoter of the pC8-36 amplicon plasmid with the glial cell-specific GFAP enhancer element. With this latter viral construct, cell type-specific and cell cycle-dependent transgene expression could subsequently be demonstrated specifically in glioma-bearing animals. Taken together, our results suggest that this series of cell cycle-regulatable HSV-1 amplicon viral vectors could potentially be adapted as useful tools for the treatment of human cancers.

Carbenoxolone enhances TRAIL-induced apoptosis through the upregulation of death receptor 5 and inhibition of gap junction intercellular communication in human glioma.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been used extensively in cancer therapy. However, more than half of glioblastoma multiforme are insensitive to the apoptotic effect of TRAIL. Improvement in therapeutic modalities that enhances the efficacy of TRAIL in glioma is much sought after. In this study, we combined the tumor selectivity of TRAIL and tumor-homing properties of mesenchymal stem cells (MSC) with gap junction (GJ) inhibitory effect of carbenoxolone (CBX) to target orthotopic glioma. MSC were engineered to express TRAIL (MSC-TRAIL) by incorporating the secretable trimeric form of TRAIL into a Herpes Simplex Virus (HSV) type I amplicon vector. Our results showed that combined treatment of MSC-TRAIL and CBX enhanced glioma cell death, especially in three primary human glioma isolates, of which two of those are marginally sensitive to TRAIL. CBX enhanced TRAIL-induced apoptosis through upregulation of death receptor 5, blockade of GJ intercellular communication, and downregulation of connexin 43. Dual arm therapy using TRAIL and CBX prolonged the survival of treated mice by ~27% when compared with the controls in an intracranial glioma model. The enhanced efficacy of TRAIL in combination with CBX coupled with the minimal cytotoxic nature of CBX suggested a favorable clinical usage of this treatment regimen.

Isolation of peptide ligands that interact specifically with human glioma cells

Poor prognosis of high grade gliomas coupled with the difficulty of widespread delivery of therapeutic agents prompted the search into new molecular targets. Our aim is to isolate glioma-specific peptide sequences that can be used for targeted delivery of therapeutic drugs and imaging tracer to accurately demarcate tumor volume as a response to therapy. Herein, we describe the isolation and characterization of a glioma-specific peptide sequence, GL1, that interact exclusively with human glioma cells lines and primary glioma cells derived from human biopsy in vitro. Further analysis showed that the receptors for GL1 were located on the external side of the plasma membrane, where the GL1 peptides could bind stably up to a period of 180 min. More importantly, GL1 phages home specifically to human glioma xenograft when administered through tail vein, a phenomenon that was not observed when non-specific phages were used as control. Taken together, our results confirmed that GL1 could represent a novel peptide that target to tumor of glial origins, and could potentially be used as a targeting moiety for the conjugation of therapeutic drugs or diagnostic imaging radiolabels.

Paracrine Factors of Human Fetal MSCs Inhibit Liver Cancer Growth Through Reduced Activation of IGF-1R/PI3K/Akt Signaling

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death in the world. The multikinase inhibitor sorafenib only demonstrated marginal improvement in overall survival for advanced disease prompted the search for alternative treatment options. Human mesenchymal stem cells (MSCs) have the ability to home to tumor cells. However, its functional roles on the tumor microenvironment remain controversial. Herein, we showed that conditioned media derived from human fetal MSC (CM-hfMSCs) expressed high level of the insulin growth factor binding proteins IGFBPs and can sequester free insulin-like growth factors (IGFs) to inhibit HCC cell proliferation. The inhibitory effect of IGFBPs on IGF signaling was further evident from the reduction of activated IGF-1R and PI3K/Akt, leading eventually to the induction of cell cycle arrest. We also demonstrated that CM-hfMSCs could enhance the therapeutic efficacy of sorafenib and sunitinib. To the best of our knowledge, this is the first report to show that CM-hfMSCs has a tumor-specific, antiproliferative effect that is not observed with normal human hepatocyte cells and patient-derived matched normal tissues. Our results thus suggest that CM-hfMSCs can provide a useful tool to design alternative/adjuvant treatment strategies for HCC, especially in related function to potentiate the effects of chemotherapeutic drugs.

HSV-1 amplicon viral vector-mediated gene transfer to human bone marrow-derived mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (BM-hMSCs) are nonhematopoietic stem cells that have the potential to differentiate into adipocytes, osteocytes and chondrocytes. Because of its propensity to migrate to the sites of injury and the ability to expand them rapidly, BM-hMSCs have been exploited as potential gene transfer vehicles to deliver therapeutic genes. Herein, we evaluated the feasibility of employing herpes simplex virus type I (HSV-1) amplicon viral vector as a gene delivery vector to BM-hMSCs. High transduction efficiencies were consistently observed in different isolates of BM-hMSCs following infection with HSV-1 amplicon viral vectors. Furthermore, we demonstrated that transduction with HSV-1 amplicon viral vector did not alter the intrinsic properties of the BM-hMSCs. The morphology and cellular proliferation of the transduced BM-hMSCs were not altered. Chromosomal stability, as confirmed by karyotyping and soft agar colony assays, of the transduced BM-hMSCs was not affected. Similarly, transduction with HSV-1 amplicon viral vectors has no effect on the pluripotent differentiation potential and the tumor tropism of BM-hMSCs. Taken together, these results demonstrated that BM-hMSCs could be transduced efficiently by HSV-1 amplicon viral vector in an ‘inert’ manner and thus enable strategies to express potential therapeutic genes in BM-hMSCs.

Matrix metalloproteinase-1-mediated mesenchymal stem cell tumor tropism is dependent on crosstalk with stromal derived growth factor 1/C-X-C chemokine receptor 4 axis

Human bone marrow‐derived mesenchymal stem cells (MSCs) have the unique ability to home toward injuries or tumor sites. We have previously shown that the tumor‐tropic property is dependent on the intrinsic expression and activity of the matrix remodeling gene, matrix metalloproteinase 1 (MMP‐1). Herein, crosstalk between MMP‐1/protease activated receptor 1 (PAR‐1) and the G‐protein coupled receptor stromal‐derived growth factor 1 (SDF‐1)/C‐X‐C chemokine receptor 4 (CXCR‐4) in facilitating cell migration was investigated. Gain‐of‐function and RNA interference (RNAi) technology were used to evaluate the interplay between the key players. The downstream effect on the tumor‐tropic migration of MSCs was investigated using modified Boyden chamber assay. Neutralizing PAR‐1 activation using monoclonal antibody and targeted knockdown of MMP‐1 using RNAi resulted in decreased expression of SDF‐1, which was not observed in control‐RNAi‐transfected cells. Over‐expression of CXCR‐4 failed to promote MSC migration; the percentage of migrated cells toward tumor cell conditioned medium was similar to the vector‐transduced and the CXCR‐4‐transduced MSCs. Furthermore, inhibition of SDF‐1/CXCR‐4 signaling using AMD3100 reduced MSC migration through the deregulation of MMP‐1 promoter activities, protein expression, and metalloproteinase activity. Collectively, our results showed that MMP‐1‐mediated MSC tumor tropism is dependent on crosstalk with the SDF‐1/CXCR‐4 axis.—Ho, I. A. W., Yulyana, Y., Sia, K. C., Newman, J. P., Guo, C. M., Hui, K. M., Lam, P. Y. P., Matrix metalloproteinase‐1‐mediated mesenchymal stem cell tumor tropism is dependent on crosstalk with stromal derived growth factor 1/C‐X‐C chemokine receptor 4 axis. FASEB J. 28, 4359–4368 (2014). www.fasebj.org