Feasibility of intracerebrally administering multiple doses of genetically modified neural stem cells to locally produce chemotherapy in glioma patients.

Neural stem cells (NSCs) are tumor tropic and can be genetically modified to produce anti-cancer therapies locally in the brain. In a prior first-in-human study we demonstrated that a single dose of intracerebrally administered allogeneic NSCs, which were retrovirally transduced to express cytosine deaminase (CD), tracked to glioma sites and converted oral 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). The next step in the clinical development of this NSC-based anti-cancer strategy was to assess the feasibility of administering multiple intracerebral doses of CD-expressing NSCs (CD-NSCs) in patients with recurrent high-grade gliomas. CD-NSCs were given every 2 weeks using an indwelling brain catheter, followed each time by a 7-d course of oral 5-FC (and leucovorin in the final patient cohort). Fifteen evaluable patients received a median of 4 (range 2-10) intracerebral CD-NSC doses; doses were escalated from 50 × 106 to 150 × 106 CD-NSCs. Neuropharmacokinetic data confirmed that CD-NSCs continuously produced 5-FU in the brain during the course of 5-FC. There were no clinical signs of immunogenicity, and only three patients developed anti-NSC antibodies. Our results suggest intracerebral administration of serial doses of CD-NSCs is safe and feasible and identified a recommended dose for phase II testing of 150 × 106 CD-NSCs.

Quantitative Evaluation of Intraventricular Delivery of Therapeutic Neural Stem Cells to Orthotopic Glioma.

Neural stem cells (NSCs) are inherently tumor-tropic, which allows them to migrate through normal tissue and selectively localize to invasive tumor sites in the brain. We have engineered a clonal, immortalized allogeneic NSC line (HB1.F3.CD21; CD-NSCs) that maintains its stem-like properties, a normal karyotype and is HLA Class II negative. It is genetically and functionally stable over time and multiple passages, and has demonstrated safety in phase I glioma trials. These properties enable the production of an "off-the-shelf" therapy that can be readily available for patient treatment. There are multiple factors contributing to stem cell tumor-tropism, and much remains to be elucidated. The route of NSC delivery and the distribution of NSCs at tumor sites are key factors in the development of effective cell-based therapies. Stem cells can be engineered to deliver and/or produce many different therapeutic agents, including prodrug activating enzymes (which locally convert systemically administered prodrugs to active chemotherapeutic agents); oncolytic viruses; tumor-targeted antibodies; therapeutic nanoparticles; and extracellular vesicles that contain therapeutic oligonucleotides. By targeting these therapeutics selectively to tumor foci, we aim to minimize toxicity to normal tissues and maximize therapeutic benefits. In this manuscript, we demonstrate that NSCs administered via intracerebral/ventricular (IVEN) routes can migrate efficiently toward single or multiple tumor foci. IVEN delivery will enable repeat administrations for patients through an Ommaya reservoir, potentially resulting in improved therapeutic outcomes. In our preclinical studies using various glioma lines, we have quantified NSC migration and distribution in mouse brains and have found robust migration of our clinically relevant HB1.F3.CD21 NSC line toward invasive tumor foci, irrespective of their origin. These results establish proof-of-concept and demonstrate the potential of developing a multitude of therapeutic options using modified NSCs.

GMP Production and Scale-Up of Adherent Neural Stem Cells with a Quantum Cell Expansion System.

Cell-based therapies hold great promise for a myriad of clinical applications. However, as these therapies move from phase I to phase II and III trials, there is a need to improve scale-up of adherent cells for the production of larger good manufacturing practice (GMP) cell banks. As we advanced our neural stem cell (NSC)-mediated gene therapy trials for glioma to include dose escalation and multiple treatment cycles, GMP production using cell factories (CellStacks) generated insufficient neural stem cell (NSC) yields. To increase yield, we developed an expansion method using the hollow fiber quantum cell expansion (QCE) system. Seeding of 5.2 × 107 NSCs in a single unit yielded up to 3 × 109 cells within 10 days. These QCE NSCs showed genetic and functional stability equivalent to those expanded by conventional flask-based methods. We then expanded the NSCs in 7 units simultaneously to generate a pooled GMP-grade NSC clinical lot of more than 1.5 × 1010 cells in only 9 days versus 8 × 109 over 6 weeks in CellStacks. We also adenovirally transduced our NSCs within the QCE. We found the QCE system enabled rapid cell expansion and increased yield while maintaining cell properties and reducing process time, labor, and costs with improved efficiency and reproducibility.

Human Neural Stem Cell Biodistribution and Predicted Tumor Coverage by a Diffusible Therapeutic in a Mouse Glioma Model.

Engineered neural stem cells (NSCs) intrinsically migrating to brain tumors offer a promising mechanism for local therapeutic delivery. However, difficulties in quantitative assessments of NSC migration and in estimates of tumor coverage by diffusible therapeutics have impeded development and refinement of NSC-based therapies. To address this need, we developed techniques by which conventional serial-sectioned formalin-fixed paraffin-embedded (FFPE) brains can be analyzed in their entirety across multiple test animals. We considered a conventional human glioblastoma model: U251 glioma cells orthotopically engrafted in immunodeficient mice receiving intracerebral (i.c.) or intravenous (i.v.) administrations of NSCs expressing a diffusible enzyme to locally catalyze chemotherapeutic formation. NSC migration to tumor sites was dose-dependent, reaching 50%-60% of total administered NSCs for the i.c route and 1.5% for the i.v. route. Curiously, the most efficient NSC homing was seen with smaller NSC doses, implying existence of rate-limiting process active during administration and/or migration. Predicted tumor exposure to a diffusing therapeutic (assuming a 50 µm radius of action) could reach greater than 50% of the entire tumor volume for i.c. and 25% for i.v. administration. Within individual sections, coverage of tumor area could be as high as 100% for i.c. and 70% for i.v. routes. Greater estimated therapeutic coverage was observed for larger tumors and for larger tumor regions in individual sections. Overall, we have demonstrated a framework within which investigators may rationally evaluate NSC migration to, and integration into, brain tumors, and therefore enhance understanding of mechanisms that both promote and limit this therapeutic modality. Stem Cells Translational Medicine 2017;6:1522-1532.

Optimization of a Neural Stem-Cell-Mediated Carboxylesterase/Irinotecan Gene Therapy for Metastatic Neuroblastoma.

Despite improved survival for children with newly diagnosed neuroblastoma (NB), recurrent disease is a significant problem, with treatment options limited by anti-tumor efficacy, patient drug tolerance, and cumulative toxicity. We previously demonstrated that neural stem cells (NSCs) expressing a modified rabbit carboxylesterase (rCE) can distribute to metastatic NB tumor foci in multiple organs in mice and convert the prodrug irinotecan (CPT-11) to the 1,000-fold more toxic topoisomerase-1 inhibitor SN-38, resulting in significant therapeutic efficacy. We sought to extend these studies by using a clinically relevant NSC line expressing a modified human CE (hCE1m6-NSCs) to establish proof of concept and identify an intravenous dose and treatment schedule that gave maximal efficacy. Human-derived NB cell lines were significantly more sensitive to treatment with hCE1m6-NSCs and irinotecan as compared with drug alone. This was supported by pharmacokinetic studies in subcutaneous NB mouse models demonstrating tumor-specific conversion of irinotecan to SN-38. Furthermore, NB-bearing mice that received repeat treatment with intravenous hCE1m6-NSCs and irinotecan showed significantly lower tumor burden (1.4-fold, p = 0.0093) and increased long-term survival compared with mice treated with drug alone. These studies support the continued development of NSC-mediated gene therapy for improved clinical outcome in NB patients.

Neural Stem Cell-Based Anticancer Gene Therapy: A First-in-Human Study in Recurrent High-Grade Glioma Patients.

Purpose: Human neural stem cells (NSC) are inherently tumor tropic, making them attractive drug delivery vehicles. Toward this goal, we retrovirally transduced an immortalized, clonal NSC line to stably express cytosine deaminase (HB1.F3.CD.C21; CD-NSCs), which converts the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU).Experimental Design: Recurrent high-grade glioma patients underwent intracranial administration of CD-NSCs during tumor resection or biopsy. Four days later, patients began taking oral 5-FC every 6 hours for 7 days. Study treatment was given only once. A standard 3 + 3 dose escalation schema was used to increase doses of CD-NSCs from 1 × 107 to 5 × 107 and 5-FC from 75 to 150 mg/kg/day. Intracerebral microdialysis was performed to measure brain levels of 5-FC and 5-FU. Serial blood samples were obtained to assess systemic drug concentrations as well as to perform immunologic correlative studies.Results: Fifteen patients underwent study treatment. We saw no dose-limiting toxicity (DLT) due to the CD-NSCs. There was 1 DLT (grade 3 transaminitis) possibly related to 5-FC. We did not see development of anti-CD-NSC antibodies and did not detect CD-NSCs or replication-competent retrovirus in the systemic circulation. Intracerebral microdialysis revealed that CD-NSCs produced 5-FU locally in the brain in a 5-FC dose-dependent manner. Autopsy data indicate that CD-NSCs migrated to distant tumor sites and were nontumorigenic.Conclusions: Collectively, our results from this first-in-human study demonstrate initial safety and proof of concept regarding the ability of NSCs to target brain tumors and locally produce chemotherapy. Clin Cancer Res; 23(12); 2951-60. ©2016 AACR.

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.

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.

Neural stem cell-mediated enzyme/prodrug therapy for glioma: preclinical studies.

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.

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.

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.

Neural stem cells as a novel platform for tumor-specific delivery of therapeutic antibodies.

BACKGROUND: 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. METHODS AND FINDINGS: As proof-of-concept, we selected Herceptin (trastuzumab), a monoclonal antibody widely used to treat HER2-overexpressing breast cancer. HER2 overexpression in breast cancer is highly correlated with CNS metastases, which are inaccessible to trastuzumab therapy. Therefore, NSC-mediated delivery of trastuzumab may improve its therapeutic efficacy. Here we report, for the first time, that human NSCs can be genetically modified to secrete anti-HER2 immunoglobulin molecules. These NSC-secreted antibodies assemble properly, possess tumor cell-binding affinity and specificity, and can effectively inhibit the proliferation of HER2-overexpressing breast cancer cells in vitro. We also demonstrate that immunoglobulin-secreting NSCs exhibit preferential tropism to tumor cells in vivo, and can deliver antibodies to human breast cancer xenografts in mice. CONCLUSIONS: 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.

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".

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-1alpha 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.

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. Disclosure of potential conflicts of interest is found at the end of this article.

Neural stem cell targeting of glioma is dependent on phosphoinositide 3-kinase signaling.

The utility of neural stem cells (NSCs) has extended beyond regenerative medicine to targeted gene delivery, as NSCs possess an inherent tropism to solid tumors, including invasive gliomas. However, for optimal clinical implementation, an understanding of the molecular events that regulate NSC tumor tropism is needed to ensure their safety and to maximize therapeutic efficacy. We show that human NSC lines responded to multiple tumor-derived growth factors and that hepatocyte growth factor (HGF) induced the strongest chemotactic response. Gliomatropism was critically dependent on c-Met signaling, as short hairpin RNA-mediated ablation of c-Met significantly attenuated the response. Furthermore, inhibition of Ras-phosphoinositide 3-kinase (PI3K) signaling impaired the migration of human neural stem cells (hNSCs) toward HGF and other growth factors. Migration toward tumor cells is a highly regulated process, in which multiple growth factor signals converge on Ras-PI3K, causing direct modification of the cytoskeleton. The signaling pathways that regulate hNSC migration are similar to those that promote unregulated glioma invasion, suggesting shared cellular mechanisms and responses. Disclosure of potential conflicts of interest is found at the end of this article.

Novel method for visualizing and modeling the spatial distribution of neural stem cells within intracranial glioma.

Neural stem cells (NSCs) hold great promise for glioma therapy due to their inherent tumor-tropic properties, enabling them to deliver therapeutic agents directly to invasive tumor sites. In the present study, we visualized and quantitatively analyzed the spatial distribution of tumor-tropic NSCs in a mouse model of orthotopic glioma in order to predict the therapeutic efficacy of a representative NSC-based glioma therapy. U251.eGFP human glioma was established in the brain of athymic mice, followed by stereotactic injection of CM-DiI-labeled human NSCs posterior-lateral to the tumor site. Confocal microscopy, three-dimensional modeling and mathematical algorithms were used to visualize and characterize the spatial distribution of NSCs throughout the tumor. The pattern of NSC distribution showed a gradient with higher densities toward the centroid of the tumor mass. We estimate that NSC-mediated therapy would eradicate 70-90% of the primary tumor mass and the majority of invasive tumor foci. Our method may serve as a model for optimizing the efficacy of NSC-based glioma therapy.

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.

Tumor-targeted enzyme/prodrug therapy mediates long-term disease-free survival of mice bearing disseminated neuroblastoma.

Neural stem cells and progenitor cells migrate selectively to tumor loci in vivo. We exploited the tumor-tropic properties of HB1.F3.C1 cells, an immortalized cell line derived from human fetal telencephalon, to deliver the cDNA encoding a secreted form of rabbit carboxylesterase (rCE) to disseminated neuroblastoma tumors in mice. This enzyme activates the prodrug CPT-11 more efficiently than do human enzymes. Mice bearing multiple tumors were treated with rCE-expressing HB1.F3.C1 cells and schedules of administration of CPT-11 that produced levels of active drug (SN-38) tolerated by patients. Both HB1.F3.C1 cells and CPT-11 were given i.v. None of the untreated mice and 30% of mice that received only CPT-11 survived long term. In contrast, 90% of mice treated with rCE-expressing HB1.F3.C1 cells and 15 mg/kg CPT-11 survived for 1 year without detectable tumors. Plasma carboxylesterase activity and SN-38 levels in mice receiving both rCE-expressing HB1.F3.C1 cells (HB1.F3.C1/AdCMVrCE) and CPT-11 were comparable with those in mice receiving CPT-11 only. These data support the hypothesis that the antitumor effect of the described neural stem/progenitor cell-directed enzyme prodrug therapy (NDEPT) is mediated by production of high concentrations of active drug selectively at tumor sites, thereby maximizing the antitumor effect of CPT-11. NDEPT approaches merit further investigation as effective, targeted therapy for metastatic tumors. We propose that the described approach may have greatest use for eradicating minimum residual disease.

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.