Margarita Gutova

Neural stem cell tropism to glioma: critical role of tumor hypoxia.

Hypoxia is a critical aspect of the microenvironment in glioma and generally signifies unfavorable clinical outcome. Effective targeting of hypoxic areas in gliomas remains a significant therapeutic challenge. New therapeutic platforms using neural stem cells (NSC) for tumor-targeted drug delivery show promise in treatment of cancers that are refractory to traditional therapies. However, the molecular mechanisms of NSC targeting to hypoxic tumor areas are not well understood. Therefore, we investigated the role of hypoxia in directed migration of NSCs to glioma and identified the specific signaling molecules involved. Our data showed that hypoxia caused increased migration of human HB1.F3 NSCs to U251 human glioma-conditioned medium in vitro. In HB1.F3 NSCs, hypoxia led to up-regulation of CXCR4, urokinase-type plasminogen activator receptor (uPAR), vascular endothelial growth factor receptor 2 (VEGFR2), and c-Met receptors. Function-inhibiting antibodies to these receptors inhibited the migration of HB1.F3 cells to glioma-conditioned medium. Small interfering RNA knockdown of hypoxia-inducible factor-1α in glioma cells blocked the hypoxia-induced migration of NSCs, which was due to decreased expression of stromal cell–derived factor-1 (SDF-1), uPA, and VEGF in glioma cells. Our in vivo data provided direct evidence that NSCs preferentially distributed to hypoxic areas inside intracranial glioma xenografts, as detected by pimonidazole hypoxia probe, as well as to the tumor edge, and that both areas displayed high SDF-1 expression. These observations indicate that hypoxia is a key factor in determining NSC tropism to glioma and that SDF-1/CXCR4, uPA/uPAR, VEGF/VEGFR2, and hepatocyte growth factor/c-Met signaling pathways mediate increased NSC-to-glioma tropism under hypoxia. These results have significant implications for development of stem cell–mediated tumor-selective gene therapies. (Mol Cancer Res 2008;6(12):1819–29)

Neural Stem Cell–Mediated Enzyme/Prodrug Therapy for Glioma: Preclinical Studies

Neural stem cells home to gliomas in mice where they convert a prodrug to 5-fluorouracil, leading to tumor regression. Cellular Assassins Derived from the supporting cells of the brain, gliomas are deadly tumors that can be only temporarily held at bay, but not cured. New ways to treat these cancers are needed. To get regulatory approval to test a new stem cell–based therapy in patients, Aboody et al. performed a series of preclinical experiments in mice with artificially implanted gliomas in their brains. By mimicking closely the treatments that they hoped to perform in humans, these authors were able to show to the satisfaction of the regulatory agency that the treatment was safe and effective enough in the mice to warrant a first-in-human trial in patients. The authors used a neural stem cell line carrying a v-myc gene and a gene for cytosine deaminase. These cells exhibit tropism to human glioma cells. When injected into mice with gliomas, they migrate to the site of the tumor, even when the mice are treated with steroids or radiation, as might be the case for human patients. The cytosine deaminase in the cells provides another anticancer weapon. This enzyme converts the prodrug 5-fluorocytosine (5-FC) to the toxic 5-flurouracil (5-FU), delivering a high concentration of the therapeutic agent directly in and around the tumor and causing it to shrink significantly. Injection of excess numbers of cells or increasing the dose of 5-FU did not result in any abnormalities in the animals; in fact, by 12 weeks after injection, no cells were to be seen in the brain or elsewhere, even when a highly sensitive polymerase chain reaction method was used to look for the v-myc DNA. This targeted cell-based approach to cancer therapy that concentrates the therapeutic agent in the vicinity of the tumor is expected to reduce toxicity to other tissues. Thus, a higher local dose is possible, potentially improving efficacy against the tumor. The phase 1 trial derived from these preclinical results is ongoing; its end will allow evaluation of how well these preclinical in vivo studies set the stage for humans. High-grade gliomas are extremely difficult to treat because they are invasive and therefore not curable by surgical resection; the toxicity of current chemo- and radiation therapies limits the doses that can be used. Neural stem cells (NSCs) have inherent tumor-tropic properties that enable their use as delivery vehicles to target enzyme/prodrug therapy selectively to tumors. We used a cytosine deaminase (CD)–expressing clonal human NSC line, HB1.F3.CD, to home to gliomas in mice and locally convert the prodrug 5-fluorocytosine to the active chemotherapeutic 5-fluorouracil. In vitro studies confirmed that the NSCs have normal karyotype, tumor tropism, and CD expression, and are genetically and functionally stable. In vivo biodistribution studies demonstrated NSC retention of tumor tropism, even in mice pretreated with radiation or dexamethasone to mimic clinically relevant adjuvant therapies. We evaluated safety and toxicity after intracerebral administration of the NSCs in non–tumor-bearing and orthotopic glioma–bearing immunocompetent and immunodeficient mice. We detected no difference in toxicity associated with conversion of 5-fluorocytosine to 5-fluorouracil, no NSCs outside the brain, and no histological evidence of pathology or tumorigenesis attributable to the NSCs. The average tumor volume in mice that received HB1.F3.CD NSCs and 5-fluorocytosine was about one-third that of the average volume in control mice. On the basis of these results, we conclude that combination therapy with HB1.F3.CD NSCs and 5-fluorocytosine is safe, nontoxic, and effective in mice. These data have led to approval of a first-in-human study of an allogeneic NSC-mediated enzyme/prodrug-targeted cancer therapy in patients with recurrent high-grade glioma.

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.

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

Background 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. Methodology 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. Conclusion 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”.

Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor Mediate Human Stem Cell Tropism to Malignant Solid Tumors

Human neural and mesenchymal stem cells have been identified for cell‐based therapies in regenerative medicine and as vehicles for delivering therapeutic agents to areas of injury and tumors. However, the signals required for homing and recruitment of stem cells to these sites are not well understood. Urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) are involved in chemotaxis and cell guidance during normal development and are upregulated in invasive tumors. Here we provided evidence that activation of uPA and uPAR in malignant solid tumors (brain, lung, prostate, and breast) augments neural and mesenchymal stem cell tropism. Expression levels of uPAR on human solid tumor cell lines correlated with levels of uPA and soluble uPAR in tumor cell‐conditioned media. Cytokine expression profiles of these tumor‐conditioned media were determined by protein arrays. Among 79 cytokines investigated, interleukin (IL)‐6, IL‐8, and monocyte chemoattractant protein‐1 were the most highly expressed cytokines in uPAR‐positive tumors. We provided evidence that human recombinant uPA induced stem cell migration, whereas depletion of uPA from PC‐3 prostate cancer cell‐conditioned medium blocked stem cell migration. Furthermore, retrovirus‐mediated overexpression of uPA and uPAR in neuroblastoma (NB1691) cells induced robust migration of stem cells toward NB1691 cell‐conditioned media, compared with media derived from wild‐type NB1691 cells. We conclude that expression of uPA and uPAR in cancer cells underlies a novel mechanism of stem cell tropism to malignant solid tumors, which may be important for development of optimal stem cell‐based therapies.

Magnetic Resonance Imaging Tracking of Ferumoxytol-Labeled Human Neural Stem Cells: Studies Leading to Clinical Use

Numerous stem cell‐based therapies are currently under clinical investigation, including the use of neural stem cells (NSCs) as delivery vehicles to target therapeutic agents to invasive brain tumors. The ability to monitor the time course, migration, and distribution of stem cells following transplantation into patients would provide critical information for optimizing treatment regimens. No effective cell‐tracking methodology has yet garnered clinical acceptance. A highly promising noninvasive method for monitoring NSCs and potentially other cell types in vivo involves preloading them with ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) to enable cell tracking using magnetic resonance imaging (MRI). We report here the preclinical studies that led to U.S. Food and Drug Administration approval for first‐in‐human investigational use of ferumoxytol to label NSCs prior to transplantation into brain tumor patients, followed by surveillance serial MRI. A combination of heparin, protamine sulfate, and ferumoxytol (HPF) was used to label the NSCs. HPF labeling did not affect cell viability, growth kinetics, or tumor tropism in vitro, and it enabled MRI visualization of NSC distribution within orthotopic glioma xenografts. MRI revealed dynamic in vivo NSC distribution at multiple time points following intracerebral or intravenous injection into glioma‐bearing mice that correlated with histological analysis. Preclinical safety/toxicity studies of intracerebrally administered HPF‐labeled NSCs in mice were also performed, and they showed no significant clinical or behavioral changes, no neuronal or systemic toxicities, and no abnormal accumulation of iron in the liver or spleen. These studies support the clinical use of ferumoxytol labeling of cells for post‐transplant MRI visualization and tracking.

Human Neural Stem Cell Tropism to Metastatic Breast Cancer

Metastasis to multiple organs is the primary cause of mortality in breast cancer patients. The poor prognosis for patients with metastatic breast cancer and toxic side effects of currently available treatments necessitate the development of effective tumor‐selective therapies. Neural stem cells (NSCs) possess inherent tumor tropic properties that enable them to overcome many obstacles of drug delivery that limit effective chemotherapy strategies for breast cancer. We report that increased NSC tropism to breast tumor cell lines is strongly correlated with the invasiveness of cancer cells. Interleukin 6 (IL‐6) was identified as a major cytokine mediating NSC tropism to invasive breast cancer cells. We show for the first time in a preclinical mouse model of metastatic human breast cancer that NSCs preferentially target tumor metastases in multiple organs, including liver, lung, lymph nodes, and femur, versus the primary intramammary fat pad tumor. For proof‐of‐concept of stem cell‐mediated breast cancer therapy, NSCs were genetically modified to secrete rabbit carboxylesterase (rCE), an enzyme that activates the CPT‐11 prodrug to SN‐38, a potent topoisomerase I inhibitor, to effect tumor‐localized chemotherapy. In vitro data demonstrate that exposure of breast cancer cells to conditioned media from rCE‐secreting NSCs (NSC.rCE) increased their sensitivity to CPT‐11 by 200‐fold. In vivo, treatment of tumor‐bearing mice with NSC.rCE cells in combination with CPT‐11 resulted in reduction of metastatic tumor burden in lung and lymph nodes. These data suggest that NSC‐mediated enzyme/prodrug therapy may be more effective and less toxic than currently available chemotherapy strategies for breast cancer metastases. STEM CELLS 2012; 30:314–325.

Intranasal Delivery of Mesenchymal Stem Cells Significantly Extends Survival of Irradiated Mice with Experimental Brain Tumors

Treatment options of glioblastoma multiforme are limited due to the blood-brain barrier (BBB). In this study, we investigated the utility of intranasal (IN) delivery as a means of transporting stem cell-based antiglioma therapeutics. We hypothesized that mesenchymal stem cells (MSCs) delivered via nasal application could impart therapeutic efficacy when expressing TNF-related apoptosis-inducing ligand (TRAIL) in a model of human glioma. ¹¹¹In-oxine, histology and magnetic resonance imaging (MRI) were utilized to track MSCs within the brain and associated tumor. We demonstrate that MSCs can penetrate the brain from nasal cavity and infiltrate intracranial glioma xenografts in a mouse model. Furthermore, irradiation of tumor-bearing mice tripled the penetration of (¹¹¹In)-oxine-labeled MSCs in the brain with a fivefold increase in cerebellum. Significant increase in CXCL12 expression was observed in irradiated xenograft tissue, implicating a CXCL12-dependent mechanism of MSCs migration towards irradiated glioma xenografts. Finally, MSCs expressing TRAIL improved the median survival of irradiated mice bearing intracranial U87 glioma xenografts in comparison with nonirradiated and irradiated control mice. Cumulatively, our data suggest that IN delivery of stem cell-based therapeutics is a feasible and highly efficacious treatment modality, allowing for repeated application of modified stem cells to target malignant glioma.

Neural Stem Cell-Mediated Delivery of Irinotecan-Activating Carboxylesterases to Glioma: Implications for Clinical Use

CPT‐11 (irinotecan) has been investigated as a treatment for malignant brain tumors. However, limitations of CPT‐11 therapy include low levels of the drug entering brain tumor sites and systemic toxicities associated with higher doses. Neural stem cells (NSCs) offer a novel way to overcome these obstacles because of their inherent tumor tropism and ability to cross the blood‐brain barrier, which enables them to selectively target brain tumor sites. Carboxylesterases (CEs) are enzymes that can convert the prodrug CPT‐11 (irinotecan) to its active metabolite SN‐38, a potent topoisomerase I inhibitor. We have adenovirally transduced an established clonal human NSC line (HB1.F3.CD) to express a rabbit carboxylesterase (rCE) or a modified human CE (hCE1m6), which are more effective at converting CPT‐11 to SN‐38 than endogenous human CE. We hypothesized that NSC‐mediated CE/CPT‐11 therapy would allow tumor‐localized production of SN‐38 and significantly increase the therapeutic efficacy of irinotecan. Here, we report that transduced NSCs transiently expressed high levels of active CE enzymes, retained their tumor‐tropic properties, and mediated an increase in the cytotoxicity of CPT‐11 toward glioma cells. CE‐expressing NSCs (NSC.CEs), whether administered intracranially or intravenously, delivered CE to orthotopic human glioma xenografts in mice. NSC‐delivered CE catalyzed conversion of CPT‐11 to SN‐38 locally at tumor sites. These studies demonstrate the feasibility of NSC‐mediated delivery of CE to glioma and lay the foundation for translational studies of this therapeutic paradigm to improve clinical outcome and quality of life in patients with malignant brain tumors.

Therapeutic targeting of melanoma cells using neural stem cells expressing carboxylesterase, a CPT-11 activating enzyme.

Neural stem cells (NSCs) have been investigated in preclinical models as delivery vehicles for therapeutic genes for treatment of tumors in the central nervous system and other organs. Melanoma at early stages is effectively treated with surgery and radiotherapy, however metastatic disease is almost universally fatal, thus novel therapeutic approaches are needed. We studied the use of HB1.F3.CD therapeutic NSCs, a well-characterized clonal cell line derived from human fetal telencephalon, for their potential of secreting prodrug-activating enzyme. HB1.F3.CD cells were transduced by adenovirus encoding rabbit carboxylesterase (rCE), which converts CPT-11 into SN-38, a potent topoisomerase 1 inhibitor. In vitro cell migration assays revealed robust migration of NSCs to conditioned media from melanoma cells. Cytokine profiles showed that IL-6, IL-8, MCP-1 and TIMP-2, known chemoattractants for stem cells, were highly expressed by melanoma cells. Exposure of melanoma cells to conditioned media from the HB1.F3.CD.rCE cells in the presence of CPT-11 increased the tumor cell-killing effect by approximately 100-fold when compared to CPT-11 alone. Our data demonstrate the rational for NSC-based enzyme/prodrug therapeutic approach to target metastatic melanoma. Future experiments will evaluate the therapeutic efficacy of NSC-mediated melanoma therapy in animal models, which will provide the basis for targeted therapy in patients with advanced melanoma.