Iron Labeling and Pre-Clinical MRI Visualization of Therapeutic Human Neural Stem Cells in a Murine Glioma Model

Treatment strategies for the highly invasive brain tumor, glioblastoma multiforme, require that cells which have invaded into the surrounding brain be specifically targeted. The inherent tumor-tropism of neural stem cells (NSCs) to primary and invasive tumor foci can be exploited to deliver therapeutics to invasive brain tumor cells in humans. Use of the strategy of converting prodrug to drug via therapeutic transgenes delivered by immortalized therapeutic NSC lines have shown efficacy in animal models. Thus therapeutic NSCs are being proposed for use in human brain tumor clinical trials. In the context of NSC-based therapies, MRI can be used both to non-invasively follow dynamic spatio-temporal patterns of the NSC tumor targeting allowing for the optimization of treatment strategies and to assess efficacy of the therapy. Iron-labeling of cells allows their presence to be visualized and tracked by MRI. Thus we aimed to iron-label therapeutic NSCs without affecting their cellular physiology using a method likely to gain United States Federal Drug Administration (FDA) approval.For human use, the characteristics of therapeutic Neural Stem Cells must be clearly defined with any pertubation to the cell including iron labeling requiring reanalysis of cellular physiology. Here, we studied the effect of iron-loading of the therapeutic NSCs, with ferumoxide-protamine sulfate complex (FE-Pro) on viability, proliferation, migratory properties and transgene expression, when compared to non-labeled cells. FE-Pro labeled NSCs were imaged by MRI at tumor sites, after intracranial administration into the hemisphere contralateral to the tumor, in an orthotopic human glioma xenograft mouse model.FE-Pro labeled NSCs retain their proliferative status, tumor tropism, and maintain stem cell character, while allowing in vivo cellular MRI tracking at 7 Tesla, to monitor their real-time migration and distribution at brain tumor sites. Of significance, this work directly supports the use of FE-Pro-labeled NSCs for real-time tracking in the clinical trial under development: "A Pilot Feasibility Study of Oral 5-Fluorocytosine and Genetically modified Neural Stem Cells Expressing Escherichia coli Cytosine Deaminase for Treatment of Recurrent High-Grade Gliomas"

Neural Stem Cells as a Novel Platform for Tumor-Specific Delivery of Therapeutic Antibodies

Recombinant monoclonal antibodies have emerged as important tools for cancer therapy. Despite the promise shown by antibody-based therapies, the large molecular size of antibodies limits their ability to efficiently penetrate solid tumors and precludes efficient crossing of the blood-brain-barrier into the central nervous system (CNS). Consequently, poorly vascularized solid tumors and CNS metastases cannot be effectively treated by intravenously-injected antibodies. The inherent tumor-tropic properties of human neural stem cells (NSCs) can potentially be harnessed to overcome these obstacles and significantly improve cancer immunotherapy. Intravenously-delivered NSCs preferentially migrate to primary and metastatic tumor sites within and outside the CNS. Therefore, we hypothesized that NSCs could serve as an ideal cellular delivery platform for targeting antibodies to malignant tumors., and can deliver antibodies to human breast cancer xenografts in mice.Taken together, these results suggest that NSCs modified to secrete HER2-targeting antibodies constitute a promising novel platform for targeted cancer immunotherapy. Specifically, this NSC-mediated antibody delivery system has the potential to significantly improve clinical outcome for patients with HER2-overexpressing breast cancer

Identification of uPAR-positive Chemoresistant Cells in Small Cell Lung Cancer

BACKGROUND: The urokinase plasminogen activator (uPA) and its receptor (uPAR/CD87) are major regulators of extracellular matrix degradation and are involved in cell migration and invasion under physiological and pathological conditions. The uPA/uPAR system has been of great interest in cancer research because it is involved in the development of most invasive cancer phenotypes and is a strong predictor of poor patient survival. However, little is known about the role of uPA/uPAR in small cell lung cancer (SCLC), the most aggressive type of lung cancer. We therefore determined whether uPA and uPAR are involved in generation of drug resistant SCLC cell phenotype. METHODS AND FINDINGS: We screened six human SCLC cell lines for surface markers for putative stem and cancer cells. We used fluorescence-activated cell sorting (FACS), fluorescence microscopy and clonogenic assays to demonstrate uPAR expression in a subpopulation of cells derived from primary and metastatic SCLC cell lines. Cytotoxic assays were used to determine the sensitivity of uPAR-positive and uPAR-negative cells to chemotherapeutic agents. The uPAR-positive cells in all SCLC lines demonstrated multi-drug resistance, high clonogenic activity and co-expression of CD44 and MDR1, putative cancer stem cell markers. CONCLUSIONS: These data suggest that uPAR-positive cells may define a functionally important population of cancer cells in SCLC, which are resistant to traditional chemotherapies, and could serve as critical targets for more effective therapeutic interventions in SCLC

Development of a Tumor-Selective Approach to Treat Metastatic Cancer

BACKGROUND: Patients diagnosed with metastatic cancer have almost uniformly poor prognoses. The treatments available for patients with disseminated disease are usually not curative and have side effects that limit the therapy that can be given. A treatment that is selectively toxic to tumors would maximize the beneficial effects of therapy and minimize side effects, potentially enabling effective treatment to be administered. METHODS AND FINDINGS: We postulated that the tumor-tropic property of stem cells or progenitor cells could be exploited to selectively deliver a therapeutic gene to metastatic solid tumors, and that expression of an appropriate transgene at tumor loci might mediate cures of metastatic disease. To test this hypothesis, we injected HB1.F3.C1 cells transduced to express an enzyme that efficiently activates the anti-cancer prodrug CPT-11 intravenously into mice bearing disseminated neuroblastoma tumors. The HB1.F3.C1 cells migrated selectively to tumor sites regardless of the size or anatomical location of the tumors. Mice were then treated systemically with CPT-11, and the efficacy of treatment was monitored. Mice treated with the combination of HB1.F3.C1 cells expressing the CPT-11-activating enzyme and this prodrug produced tumor-free survival of 100% of the mice for >6 months (P<0.001 compared to control groups). CONCLUSIONS: The novel and significant finding of this study is that it may be possible to exploit the tumor-tropic property of stem or progenitor cells to mediate effective, tumor-selective therapy for metastatic tumors, for which no tolerated curative treatments are currently available

Cellular Host Responses to Gliomas

Background: Glioblastoma multiforme (GBM) is the most aggressive type of malignant primary brain tumors in adults. Molecular and genetic analysis has advanced our understanding of glioma biology, however mapping the cellular composition of the tumor microenvironment is crucial for understanding the pathology of this dreaded brain cancer. In this study we identified major cell populations attracted by glioma using orthotopic rodent models of human glioma xenografts. Marker-specific, anatomical and morphological analyses revealed a robust influx of host cells into the main tumor bed and tumor satellites. Methodology/Principal Findings: Human glioma cell lines and glioma spheroid orthotopic implants were used in rodents. In both models, the xenografts recruited large numbers of host nestin-expressing cells, which formed a ‘network’ with glioma. The host nestin-expressing cells appeared to originate in the subventricular zone ipsilateral to the tumor, and were clearly distinguishable from pericytes that expressed smooth muscle actin. These distinct cell populations established close physical contact in a ‘pair-wise’ manner and migrated together to the deeper layers of tumor satellites and gave rise to tumor vasculature. The GBM biopsy xenografts displayed two different phenotypes: (a) low-generation tumors (first in vivo passage in rats) were highly invasive and non-angiogenic, and host nestin-positive cells that infiltrated into these tumors displayed astrocytic or elongated bipolar morphology; (b) high-generation xenografts (fifth passage) had pronounced cellularity, were angiogenic with ‘glomerulus-like’ microvascular proliferations that contained host nestin-positive cells. Stromal cell-derived factor-1 and its receptor CXCR4 were highly expressed in and around glioma xenografts, suggesting their role in glioma progression and invasion. Conclusions/Significance: Our data demonstrate a robust migration of nestin-expressing host cells to glioma, which together with pericytes give rise to tumor vasculature. Mapping the cellular composition of glioma microenvironment and deciphering the complex ‘crosstalk’ between tumor and host may ultimately aid the development of novel anti-glioma therapies

Harvey Murine Sarcoma Virus/MDR1 Retrovital Vectors: Efficient Virus Production and Foreign Gene Transduction Using MDR1 as a Selectable Marker

AbstractRetroviruses are used for a variety of applications requiring the delivery of exogenous genes to cells and animals. For many of these applications, including gene therapy, safer and more efficient retroviral vectors are needed. Vectors based on Harvey murine sarcoma virus (HaMSV) are attractive because nearly all their viral sequences outside of the LTRs are derived from rat endogenous VL30 retroviruses. These sequences are not homologous to the functional viral mRNAs in commonly used retrovirus packaging cell lines, the packaging and dimerization domains of HaMSV are small and contain no splice donor sites, and the 5′ sequences of HaMSV appear to confer efficient packaging and stability on genomic RNAs. HaMSV/MDR1 vectors use the human multidrug resistance gene as a dominant, selectable, amplifiable marker for gene delivery, but current versions of these vectors are large, with over 3300 nt of HaMSV sequences downstream of MDR1. We analyzed the requirement for these downstream sequences in HaMSV vectors and found that modified HaMSV/MDR1 vectors lacking virtually all viral sequences downstream of MDR1 support the production of high-titer retroviruses and the efficient transduction, selection, and amplification of MDR1. A reduced-size HaMSV/MDR1 vector was further modified to include a second heterologous gene under the control of an internal SV40 promoter. Using MDR 1 as a selectable marker, we obtained efficient virus production, gene transduction, and expression of MDR1 plus the heterologous gene

Bicistronic and Two-Gene Retroviral Vectors for UsingMDR1 as a Selectable Marker and a Therapeutic Gene

AbstractWe describe a series of two-gene and bicistronic retroviral vectors that use the humanMDR1 gene as a selectable marker for the overexpression of a second heterologous gene in transduced cells. The vectors use Harvey murine sarcoma virus sequences for viral expression and packaging functions and include sites for cloning foreign genes of interest under the control of either an internal promoter (two-gene vectors) or an internal ribosome entry site (bicistronic vectors). To characterize these vectors, we usedneoas a reporter gene for foreign gene expression and as an independently selectable marker for comparison withMDR1. Each of the vector constructions supported high-titer retrovirus production and transduction of mouse and human cell lines. UsingMDR1–neovirus supernatants in parallel titering assays, we found that titers based on colchicine resistance were 10- to 20-fold lower than titers based on G418 resistance, suggesting thatMDR1 is a more stringent selectable marker thanneoin NIH 3T3 and KB-3-1 cell lines. Whereasneogene expression with the two-gene vectors was subject to host-specific limitations on internal promoter activity, the bicistronic vectors were highly active in three cell lines tested. In K562 cells, using the bicistronic vector, selection with colchicine led to at least 20-fold higher expression of theMDR1 gene product than did selection with G418, suggesting that the stringentMDR1 selection system is very efficient for obtaining overexpression of foreign genes. Retroviral vectors carryingMDR1 as a selectable marker plus a second, heterologous gene of interest could have widespread utility forin vitroandin vivoapplications of gene transfer technology, including gene therapy