Khalid Shah

MicroRNA-21 Knockdown Disrupts Glioma Growth In vivo and Displays Synergistic Cytotoxicity with Neural Precursor Cell–Delivered S-TRAIL in Human Gliomas

Despite the development of new glioma therapies that allow for tumor-targeted in situ delivery of cytotoxic drugs, tumor resistance to apoptosis remains a key impediment to effective treatment. Mounting evidence indicates that microRNAs (miRNA) might play a fundamental role in tumorigenesis, controlling cell proliferation and apoptosis. In gliomas, microRNA-21 (miR-21) levels have been reported to be elevated and their knockdown is associated with increased apoptotic activity. We hypothesized that suppression of miR-21 might sensitize gliomas for cytotoxic tumor therapy. With the use of locked nucleic acid (LNA)-antimiR-21 oligonucleotides, bimodal imaging vectors, and neural precursor cells (NPC) expressing a secretable variant of the cytotoxic agent tumor necrosis factor-related apoptosis inducing ligand (S-TRAIL), we show that the combined suppression of miR-21 and NPC-S-TRAIL leads to a synergistic increase in caspase activity and significantly decreased cell viability in human glioma cells in vitro. This phenomenon persists in vivo, as we observed complete eradication of LNA-antimiR-21-treated gliomas subjected to the presence of NPC-S-TRAIL in the murine brain. Our results reveal the efficacy of miR-21 antagonism in murine glioma models and implicate miR-21 as a target for therapeutic intervention. Furthermore, our findings provide the basis for developing combination therapies using miRNA modulation and cytotoxic tumor therapies.

Assessment of therapeutic efficacy and fate of engineered human mesenchymal stem cells for cancer therapy

The poor prognosis of patients with aggressive and invasive cancers combined with toxic effects and short half-life of currently available treatments necessitate development of more effective tumor selective therapies. Mesenchymal stem cells (MSCs) are emerging as novel cell-based delivery agents; however, a thorough investigation addressing their therapeutic potential and fate in different cancer models is lacking. In this study, we explored the engineering potential, fate, and therapeutic efficacy of human MSCs in a highly malignant and invasive model of glioblastoma. We show that engineered MSC retain their “stem-like” properties, survive longer in mice with gliomas than in the normal brain, and migrate extensively toward gliomas. We also show that MSCs are resistant to the cytokine tumor necrosis factor apoptosis ligand (TRAIL) and, when engineered to express secreted recombinant TRAIL, induce caspase-mediated apoptosis in established glioma cell lines as well as CD133-positive primary glioma cells in vitro. Using highly malignant and invasive human glioma models and employing real-time imaging with correlative neuropathology, we demonstrate that MSC-delivered recombinant TRAIL has profound anti-tumor effects in vivo. This study demonstrates the efficacy of diagnostic and therapeutic MSC in preclinical glioma models and forms the basis for developing stem cell-based therapies for different cancers.

Human Glioblastoma–Derived Cancer Stem Cells: Establishment of Invasive Glioma Models and Treatment with Oncolytic Herpes Simplex Virus Vectors

Glioblastoma, the most malignant type of primary brain tumor, is one of the solid cancers where cancer stem cells have been isolated, and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here, we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2, varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC), we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV, the deletion of gamma34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of alpha47. Infection with oHSV G47Delta (ICP6(-), gamma34.5(-), alpha47(-)) not only killed GBM-SCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly, despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs, intratumoral injection of G47Delta significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover, the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis.

In vivo tracking of neural progenitor cell migration to glioblastomas

The ability to noninvasively track the migration, engraftment, and proliferation of neural progenitor cells (NPCs) has significant clinical and research implications. The purpose of our study was to explore the macroscopic migratory capabilities of NPCs toward brain tumors after implantation into nude mice. We stably transfected C17.2 NPCs with the firefly luciferase gene (F-luc) and implanted cells into (1) the contralateral brain parenchyma (2 x 10(6) cells), (2) the ventricles (2 x 10(6) cells), (3) the vasculature (1 x 10(5) cells), or (4) the intraperitoneal cavity (5 x 10(6) cells) of mice bearing intracranial gliomas (Gli36). Using serial bioluminescence imaging, migration of parenchymally injected cells was observed across the corpus callosum, first detected at 1 week, with maximal density at the tumor site 2-3 weeks after implantation. Similar patterns were also observed with intraventricular injections; however, tumors were populated earlier, presumably because of the shorter distance to travel. Intravenous injections resulted in more modest tumoral NPC populations, whereas virtually no cells could be identified in tumors after intraperitoneal injection. These results confirm the migratory capability of NPCs over considerable distances and their preferential accumulation in brain tumors on CNS rather than peripheral injection.

Glioma therapy and real-time imaging of neural precursor cell migration and tumor regression.

Despite many refinements in current therapeutic strategies, the overall prognosis for a patient with glioblastoma is dismal. Neural precursor cells (NPCs) are capable of tracking glioma tumors and thus could be used to deliver therapeutic molecules. We have engineered mouse NPCs to deliver a secreted form of tumor necrosis factor–related apoptosis–inducing ligand (S‐TRAIL); S‐TRAIL is optimized to selectively kill neoplastic cells. Furthermore, we have developed means to simultaneously monitor both the migration of NSCs toward gliomas and the changes in glioma burden in real time. Using a highly malignant human glioma model expressing Renilla luciferase (Rluc), intracranially implanted NPC‐FL‐sTRAIL expressing both firefly luciferase (Fluc) and S‐TRAIL was shown to migrate into the tumors and have profound antitumor effects. These studies demonstrate the potential of NPCs as therapeutically effective delivery vehicles for the treatment of gliomas and also provide important tools to evaluate the migration of NPCs and changes in glioma burden in vivo. Ann Neurol 2005;57:34–41

Mesenchymal stem cells engineered for cancer therapy.

Recent pre-clinical and clinical studies have shown that stem cell-based therapies hold tremendous promise for the treatment of human disease. Mesenchymal stem cells (MSC) are emerging as promising anti-cancer agents which have an enormous potential to be utilized to treat a number of different cancer types. MSC have inherent tumor-trophic migratory properties, which allows them to serve as vehicles for delivering effective, targeted therapy to isolated tumors and metastatic disease. MSC have been readily engineered to express anti-proliferative, pro-apoptotic, anti-angiogenic agents that specifically target different cancer types. Many of these strategies have been validated in a wide range of studies evaluating treatment feasibility or efficacy, as well as establishing methods for real-time monitoring of stem cell migration in vivo for optimal therapy surveillance and accelerated development. This review aims to provide an in depth status of current MSC-based cancer therapies, as well as the prospects for their clinical translation.

Molecular imaging of gene therapy for cancer.

Gene therapy of cancer has been one of the most exciting and elusive areas of scientific and clinical research in the past decade. One of the most critical issues for ensuring success of this therapy is the development of technology for noninvasive monitoring of the location, magnitude and duration of vector-mediated gene expression, as well as the distribution and targeting of vector particles in vivo. In recent years many advances have been made in high-resolution, in vivo imaging methods, including: radionuclide imaging, such as positron emission tomography (PET) and single photon emission tomography (SPECT), magnetic resonance (MR) imaging and spectroscopy, bioluminescence imaging and various fluorescence imaging techniques, including fluorescence-mediated tomography (FMT) and near-infrared fluorescence (NIRF) reflectance imaging. A variety of factors determine the choice of specific imaging system, some of them are the imaging requirements (single or repeated), intended use (animal or human) and spatial requirements (organs versus cellular resolution and depth). This review provides descriptions of modalities applicable to imaging different parameters of vector-mediated gene expression in tumors and stem cell tracking in vivo.

Molecular optical imaging: Applications leading to the development of present day therapeutics

SummaryA number of advances in the molecular imaging field have led to the sensing of specific molecular targets and pathways in living animals. In the optical imaging field, these include the designing of biocompatible near-infrared fluorochromes, development of targeted and activatable “smart” imaging probes, and engineering of activatable fluorescent and bioluminescent proteins. The current advances in molecular optical imaging will help in early disease diagnoses, functioning of a number of pathways and finally help speed drug discovery. In this review, we will describe the near infrared fluorescent and bioluminescence imaging modalities and how these techniques have been employed in current research. Furthermore, we will also shed some light on the use of these imaging modalities in neurotherapeutics, for example imaging different parameters of vector-mediated gene expression in glioma tumors and stem cell trackingin vivo.

Imaging of Stem Cell Recruitment to Ischemic Infarcts in a Murine Model

Background and Purpose— Neural progenitor cells (NPC) have been reported to aid in the functional recovery from stroke and hold promise as a novel treatment for a variety of neurological diseases. There is a need for imaging tools to study the in vivo migratory behavior of these cells. Methods— C17.2 NPC, stably transfected with firefly luciferase, were serially imaged through intact skull and skin by bioluminescence imaging over 2 to 3 weeks in nu/nu mice with closed-vessel middle cerebral artery infarcts, followed by contralateral intraparenchymal or intraventricular injections of NPC. Results— NPC migrated to the site of infarct from the contraleral parenchyma, crossing the midline at 7 days. In control animals without infarcts, NPC remained at the site of administration. Intraventricular cell administration resulted in a wide distribution of cells, including the site of infarct. Within the infarct area, NPC colabeled with the neuronal marker NeuN and with astroglial marker glial fibrillary acidic protein. The time course and magnitude of NPC recruitment were longitudinally compared between the treatment groups. Conclusions— NPC recruitment to infarct can be assessed noninvasively by serial in vivo imaging. Images correlate well with histological cell distributions. NPC are recruited to infarcts with both parenchymal and cerebrospinal fluid administration, but higher initial photon counts suggest that cerebrospinal fluid administration is more efficient at early infarct seeding.

In Vivo Imaging of β-Galactosidase Activity Using Far Red Fluorescent Switch

β-Galactosidase (β-gal) has been widely used as a transgene reporter enzyme, and several substrates are available for its in vitro detection. The ability to image β-gal expression in living animals would further extend the use of this reporter. Here we show that DDAOG, a conjugate of β-galactoside and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO), is not only a chromogenic β-gal substrate but that the cleavage product has far-red fluorescence properties detectable by imaging. Importantly, the cleavage substrate shows a 50-nm red shift, enabling its specific detection in a background of intact probe, a highly desirable feature for in vivo imaging. Specifically, we show that β-gal-expressing 9L gliomas are readily detectable by red fluorescence imaging in comparison with the native 9L gliomas. We furthermore show that herpes simplex virus amplicon-mediated LacZ gene transfer into tumors can be transiently and thus serially visualized over time. The results indicated that in vivo real-time detection of β-gal activity is possible by fluorescence imaging technology.