Moneeb Ehtesham

CXCR4 expression mediates glioma cell invasiveness

Glioblastoma multiforme is a highly invasive tumor bearing a dismal prognosis. Experimental strategies that focus on the specific biological cues governing the invasive capacity of these tumors may hold significant therapeutic promise. In this context, we describe the in vitro and in vivo association of the cell surface chemokine receptor, CXCR4, with the development of an invasive phenotype in malignant glioblastoma. We demonstrate that invasive populations of glioma cells overexpress CXCR4 at the message and protein levels, and that this expression ranges from 25- to 89-fold higher than that found in noninvasive tumor cells. Furthermore, neutralization of CXCR4 significantly impairs the in vitro invasive capacity of malignant glial cells. In addition, glioma cells secrete CXCL12 and demonstrate robust invasive capacity toward a CXCL12 gradient in vitro. These findings underscore the importance of CXCR4 as a potential therapeutic target for the treatment of invasive glioblastoma.

Induction of a CD4+ T Regulatory Type 1 Response by Cyclooxygenase-2-Overexpressing Glioma

PGE2, synthesized by cyclooxygenase-2 (COX-2)-overexpressing tumor, is known to contribute to cellular immune suppression in cancer patients, but the mechanism remains unclear. We report the mechanism of a CD4+ T regulatory type 1 (Tr1) induction by CD11c+ mature dendritic cells (DCs) that phagocytose allogeneic and autologous COX-2-overexpressing glioma. A human glioma cell line, U-87MG, and primary cultured glioblastoma cells (MG-377) overexpressed COX-2. We did not detect IL-10Rα expression in these gliomas, and rIL-10 did not suppress their COX-2 expression. Exposure to COX-2-overexpressing glioma induced mature DCs to overexpress IL-10 and decreased IL-12p70 production. These DCs induced a Tr1 response, which is characterized by robust secretion of IL-10 and TGF-β with negligible IL-4 secretion by CD4+ T cells, and an inhibitory effect on admixed lymphocytes. Peripheral CD4+ T cell populations isolated from an MG-377 patient also predominantly demonstrated a Tr1 response against MG-377 cells. Selective COX-2 inhibition in COX-2-overexpressing gliomas at the time of phagocytic uptake by DCs abrogated this regulatory response and instead elicited Th1 activity. COX-2 stable transfectants in LN-18 (LN-18-COX2) also induced a Tr1 response. The effect of a COX-2 inhibition in LN-18-COX2 is reversible after administration of PGE2. Taken together, robust levels of PGE2 from COX-2-overexpressing glioma, which is unresponsive to IL-10 within the local microenvironment, may cause DCs to secrete high levels of IL-10. These results indicate that COX-2-overexpressing tumors induce a Tr1 response, which is mediated by tumor-exposed, IL-10-enhanced DCs.

Generation of neural progenitor cells from whole adult bone marrow.

The efficient and large-scale generation of neural progenitor cells for neural grafting in the treatment of neurological diseases has been a challenge. Here we describe the isolation and successful propagation of neural progenitor cells from adult rat bone marrow. Unfractionated bone marrow cultured in vitro with epidermal growth factor and basic fibroblast growth factor gave rise to cellular spheres which differentiated into neurons and glia. The cellular spheres expressed nestin, a neural stem cell marker as well as CD90, a marker of hematopoietic stem cells. This methodology addresses the ethical and tissue rejection problems associated with fetal neural stem cells and would circumvent the difficulty associated with generating neural progenitors from the adult brain. We demonstrate that bone marrow may offer a renewable autologous extracranial source of neural progenitor cells.

Glioma Tropic Neural Stem Cells Consist of Astrocytic Precursors and Their Migratory Capacity Is Mediated by CXCR4

Malignant gliomas spawn disseminated microsatellites, which are largely refractory to currently employed therapies, resulting in eventual tumor recurrence and death. The use of tumor-tropic neural stem cells (NSCs) as delivery vehicles for therapeutic gene products represents an attractive strategy specifically focused at treating these residual neoplastic foci. We wished to elucidate the biological cues governing NSC tropism for glioma. In this context, we describe that tumor-tropic NSCs comprise largely of astrocytic progenitors expressing chemokine receptor 4 (CXCR4). Blocking of CXCR4 significantly inhibits NSC migration toward the tumor. These findings define specific characteristics associated with the cell populations within transplanted NSCs that demonstrate glioma-tracking behavior.

Integrating spatially resolved three-dimensional MALDI IMS with in vivo magnetic resonance imaging

We have developed a method for integrating three dimensional–volume reconstructions of spatially resolved matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) ion images of whole mouse heads with high-resolution images from other modalities in an animal-specific manner. This approach enabled us to analyze proteomic profiles from MALDI IMS data with corresponding in vivo data provided by magnetic resonance imaging.

Ligand-dependent activation of the hedgehog pathway in glioma progenitor cells.

The hedgehog (Hh) signaling pathway regulates progenitor cells during embryogenesis and tumorigenesis in multiple organ systems. We have investigated the activity of this pathway in adult gliomas, and demonstrate that the Hh pathway is operational and activated within grade II and III gliomas, but not grade IV de novo glioblastoma multiforme. Furthermore, our studies reveal that pathway activity and responsiveness is confined to progenitor cells within these tumors. Additionally, we demonstrate that Hh signaling in glioma progenitor cells is ligand-dependent and provide evidence documenting the in vivo source of Sonic hedgehog protein. These findings suggest a regulatory role for the Hh pathway in progenitor cells within grade II and III gliomas, and the potential clinical utility of monitoring and targeting this pathway in these primary brain tumors.

CXCR4 mediates the proliferation of glioblastoma progenitor cells

Increasing evidence points to a fundamental role for cancer stem cells (CSC) in the initiation and propagation of many tumors. As such, in the context of glioblastoma multiforme (GBM), the development of treatment strategies specifically targeted towards CSC-like populations may hold significant therapeutic promise. To this end, we now report that the cell surface chemokine receptor, CXCR4, a known mediator of cancer cell proliferation and invasion, is overexpressed in primary glioblastoma progenitor cells versus corresponding differentiated tumor cells. Furthermore, administration of CXCL12, the only known ligand for CXCR4, stimulates a specific and significant proliferative response in progenitors but not differentiated tumor cells. Taken together, these results implicate an important role for the CXCR4 signaling mechanism in glioma CSC biology and point to the therapeutic potential of targeting this pathway in patients with GBM.

Small interference RNA modulation of IL-10 in human monocyte-derived dendritic cells enhances the Th1 response

RNA interference technology has been used to modulate dendritic cell (DC) function by targeting the expression of genes such as IL‐12 and NF‐kB. In this paper, we demonstrate that transfectionof DC with IL‐10‐specific double strands of small interference RNA (siRNA) resulted in potent suppression of IL‐10 gene expression without inducing DC apoptosis or blocking DC maturation. Inhibition of IL‐10 by siRNA was accompanied by increased CD40 expression and IL‐12 production after maturation, which endowed DC with the ability to significantly enhance allogeneic T cell proliferation. IL‐10 siRNA transfection did not affect MHC class II, CD86, CD83, or CD54 expression in mature DC. To further test the ability of IL‐10 siRNA‐treated DC to induce a T cell response, naive CD4 T cells were stimulated by autologous DC pulsed with KLH. The results indicated that IL‐10 siRNA‐transfected DC enhanced Th1 responses by increasing IFN‐γ and decreasing IL‐4 production. These findings suggest the potential for a novel immunotherapeutic strategy of using IL‐10 siRNA‐transfected antigen‐presenting cells as vaccine delivery agents to boost the Th1 response against pathogens and tumors that are controlled by Th1 immunity.

Intratumoral dendritic cell vaccination elicits potent tumoricidal immunity against malignant glioma in rats

Dendritic cells (DC) are attractive candidates for innovative cancer immunotherapy by virtue of their ability to function as powerful antigen presenting cells and elicit potent antitumor cytotoxic immune responses. With the aim of generating antitumor immunity, the authors sought to enhance in vivo tumor antigen presentation by using an intratumoral DC vaccination strategy in the setting of partially irradiated intracranial brain tumors. Fisher rats, implanted with 9L gliomas in the right corpus striatum, were treated with freshly cultured, unpulsed syngeneic DC inoculated directly into the tumor bed. Intracranially inoculated DCs were found to drain to ipsilateral deep cervical lymph nodes. This was associated with increased local and systemic antitumor cytoxicity, as evidenced by robust infiltration of treated tumors with CD4+ and CD8+ T cells as well as by increased IFN-&ggr; protein and message levels in in vitro restimulated splenic lymphocytes. DC therapy resulted in prolonged survival and immunity to subsequent intracranial tumor re-challenge. These results demonstrate the viability of intratumoral DC vaccination as an effective therapeutic strategy for intracranial glioma.

Treatment of intracranial glioma with in situ interferon-gamma and tumor necrosis factor-alpha gene transfer

Interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα) are potent immunostimulatory cytokines with demonstrated tumoricidal effects in a variety of cancers. With the aim of investigating their ability to generate antitumor immune responses in malignant brain tumors, we describe the use of in situ adenoviral-mediated IFNγ and TNFα gene transfer in glioma-bearing rodents. Survival was prolonged in mice treated with AdmIFNγ or AdTNFα compared to AdLacZ- and saline-inoculated controls, and AdmIFNγ- or AdTNFα-treated animals revealed significantly smaller tumors. These effects were accompanied by significant up-regulation of tumor MHC-I expression in AdmIFNγ-inoculated animals, and of MHC-II in AdTNFα-treated tumors. Significantly enhanced intratumoral infiltration with CD4+ and CD8+ T cells was visible in animals treated with AdmIFNγ, AdTNFα, or a combination of AdmIFNγ and AdTNFα. In addition, AdTNFα therapy down-regulated the expression of endothelial Fas ligand, a cell membrane protein implicated as a contributor to immune privilege in cancer. These findings demonstrate the effectiveness of local IFNγ and TNFα gene transfer as a treatment strategy for glioma and illustrate possible physiological pathways responsible for the therapeutic benefit observed.