Peter Kabos

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.

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.

Live Multicellular Tumor Spheroid Models For High-Content Imagingand Screening In Cancer Drug Discovery

The multi cellular tumor spheroid (MCTS) model has been used for decades with proven superiority over monolayer cell culture models at recapitulating in vivo tumor growth. Yet its use in high-throughput drug discovery has been limited, particularly with image based screening, due to practical and technical hurdles. Here we report a significant advance in utilizing live MCTS models for high-content image based drug discovery. Using a validated GFP reporter (CK5Pro-GFP) of luminal breast cancer stem cells (CSC), we developed an algorithm to quantify changes in CK5Pro-GFP expression levels for individual Z-stack planes (local) or as maximal projections of the summed Z-stacks (global) of MCTS. From these image sets, we can quantify the cross-sectional area of GFP positive cells, the fluorescence intensity of the GFP positive cells, and the percent of spheroid cross-sectional area that expresses CK5Pro-GFP.We demonstrate that acquiring data in this manner can be done in real time and is statistically robust (Z’=0.85) for use in primary high-content screening cancer drug discovery.

Neural stem cells as delivery vehicles.

The discovery of neural stem cells (NSCs) has changed our long-held view that the adult mammalian central nervous system (CNS) is postmitotic and lacks the capability for self-repair. The role of NSCs in physiological and pathological processes in the brain is slowly emerging. We are now able to isolate, expand, genetically engineer and transplant NSCs. An important characteristic of NSCs, not fully understood so far, is their migratory ability and their tropism to brain pathology. The migratory ability of NSCs and their capacity to differentiate into all neural phenotypes gives us a potentially powerful tool for the treatment of both diffuse and localised neurologic disorders. The delivery of gene products by NSCs to specific sites in the CNS can maximise the efficiency of delivery and minimise the unwanted exposure of surrounding intact tissue. Here, the recent preclinical advances in the use of NSCs for the delivery of therapeutic products are reviewed, in particular the employment of their migratory potential and the homing ability to pathology in the nervous system.

Development of an intracranial ependymoma at the site of a pre-existing cavernous malformation.

BACKGROUND The ability of vascular anomalies to induce neoplastic transformation in normal brain parenchyma has been suggested but not demonstrated. We present a novel case in which a patient with a pre-existing cavernous malformation developed an adjacent ependymoma. CASE DESCRIPTION A 72-year-old man developed an anaplastic ependymoma at the site of a pre-existing cavernous malformation. This is the first documented instance of an ependymoma developing at the site of an existing cavernous malformation. The colocalization of both lesions and the low incidence of supratentorial ependymomas in this age group makes it unlikely that their coexistence represents a random event. Immunohistochemistry demonstrated vascular endothelial growth factor (VEGF) production by the cavernous malformation and robust VEGF receptor expression by the ependymoma. CONCLUSIONS Based on these findings, we suggest that production of VEGF by vascular malformations may play a role in the neoplastic transformation of adjacent tissue.

Use of neural stem cells as therapeutic vehicles for the treatment of malignant glioma.

The prognosis for patients with malignant glioma, the most common primary intracranial neoplasm, remains dismal despite significant progress in neuro-oncological therapies and technology. This stems from the inability of current treatment strategies to address the highly invasive nature of this disease. Malignant glial cells often disseminate throughout the brain, making it exceedingly difficult to target and treat all intracranial neoplastic foci with the result that tumor recurrence is inevitable despite aggressive surgery and adjuvant radio- and/or chemotherapy. The use of neural stem cells as delivery vehicles for tumor toxic molecules is a novel experimental strategy aimed specifically at targeting disseminated tumor pockets. It has been demonstrated that neural stem cells possess a robust tropism for infiltrating tumor cells and that they can be used to deliver therapeutic agents directly to tumor satellites with significant therapeutic benefit. The aim is to develop these findings into a clinically viable technology which would not be hindered by ethical and tissue rejection related concerns stemming from the use of fetal or embryonic tissue transplants. A novel technology whereby neural progenitors similar in morphology, phenotype and behavior to fetal neural stem cells can be isolated from adult bone marrow is also discussed. These technologies represent important progress in the development of a treatment strategy that can specifically target disseminated neoplastic pockets within the brain. However, despite encouraging results in preclinical models, there are significant impediments that must be overcome prior to clinical implementation of this strategy. The key among these is understanding the specific tropic mechanisms that govern neural stem cells migration towards tumors and refining the processes used to generate neural progenitors from adult bone marrow in a clinically implementable fashion. Despite these limitations, the use of neural stem cells for brain tumor therapy holds significant promise and may emerge as an important therapeutic modality for patients with malignant glioma.