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2.  Neural stem cell-mediated CE/CPT-11 enzyme/prodrug therapy in transgenic mouse model of intracerebellar medulloblastoma 
Gene therapy  2012;20(2):143-150.
Medulloblastoma is a heterogeneous diffuse neoplasm that can be highly disseminated, and is the most common malignant childhood brain tumor. Although multimodal treatments have improved survival rates for patients with medulloblastoma, these tumors are associated with high morbidity and mortality. New treatment strategies are urgently needed to improve cure rates and, importantly, to spare normal brain tissue from neurotoxicity and patients from life-long cognitive and functional deficits associated with current therapies. In numerous preclinical brain tumor models, neural stem cells (NSCs) have shown great promise as delivery vehicles for therapeutic genes. Here, we have used an established, genetically modified human NSC line (HB1.F3.CD) to deliver carboxylesterase (CE) to cerebellar tumor foci and locally activate the prodrug CPT-11 (Irinotecan) to the potent topoisomerase I inhibitor SN-38. HB1.F3.CD NSC tumor tropism, intratumoral distribution and therapeutic efficacy were investigated in clinically relevant experimental models. Magnetic resonance imaging was used for in vivo tracking of iron nanoparticle-labeled NSCs, and to assess the therapeutic efficacy of CE-expressing HB1.F3.CD cells. As compared to controls, a significant decrease in tumor growth rate was seen in mice that received both NSCs and CPT-11 as their treatment regimen. Thus, this study provides proof-of-concept for NSC-mediated CE/CPT-11 treatment of medulloblastoma, and serves as a foundation for further studies toward potential clinical application.
doi:10.1038/gt.2012.12
PMCID: PMC4149468  PMID: 22402322
carboxylesterase; CPT-11; gene therapy; irinotecan; MRI; medulloblastoma; neural stem cells; pediatric cancer; mouse model
3.  A Preclinical Evaluation of Neural Stem Cell–Based Cell Carrier for Targeted Antiglioma Oncolytic Virotherapy 
Background
Oncolytic adenoviral virotherapy (OV) is a highly promising approach for the treatment of glioblastoma multiforme (GBM). In practice, however, the approach is limited by poor viral distribution and spread throughout the tumor mass.
Methods
To enhance viral delivery, replication, and spread, we used a US Food and Drug Administration–approved neural stem cell line (NSC), HB1.F3.CD, which is currently employed in human clinical trials. HB1.F3.CD cells were loaded with an oncolytic adenovirus, CRAd-Survivin-pk7, and mice bearing various human-derived GBMs were assessed with regard to NSC migration, viral replication, and therapeutic efficacy. Survival curves were evaluated with Kaplan–Meier methods. All statistical tests were two-sided.
Results
Antiglioma activity of OV-loaded HB1.F3.CD cells was effective against clinically relevant human-derived glioma models as well as a glioma stem cell–enriched xenograft model. Median survival was prolonged by 34% to 50% compared with mice treated with OV alone (GBM43FL model median survival = 19.5 days, OV alone vs NSC + OV, hazard ratio of survival = 2.26, 95% confidence interval [CI] = 1.21 to 12.23, P = .02; GBM12 model median survival = 43.5 days, OV alone vs NSC + OV, hazard ratio of survival = 2.53, 95% CI = 1.21 to 10.38, P = .02). OV-loaded HB1.F3.CD cells were shown to effectively migrate to the contralateral hemisphere and hand off the therapeutic payload of OV to targeted glioma cells. In vivo distribution and migratory kinetics of the OV-loaded HB1.F3.CD cells were successfully monitored in real time by magnetic resonance imaging. OV-loaded NSCs retained their differentiation fate and were nontumorigenic in vivo.
Conclusions
HB1.F3.CD NSCs loaded with CRAd-Survivin-pk7 overcome major limitations of OV in vivo and warrant translation in a phase I human clinical trial for patients with GBM.
doi:10.1093/jnci/djt141
PMCID: PMC3699440  PMID: 23821758
4.  Neural Stem Cell-Mediated Delivery of Irinotecan-Activating Carboxylesterases to Glioma: Implications for Clinical Use 
Stem Cells Translational Medicine  2013;2(12):983-992.
Irinotecan (CPT-11) is a chemotherapeutic agent used for therapy of several systemic solid tumors, and has also been investigated as a treatment for malignant brain tumors; however, its efficacy has been limited. Neural stem cells (NSCs) offer a novel way to overcome these limitations. This study investigated NSC-mediated carboxylesterase (CE)/CPT-11 therapy to increase the therapeutic efficacy of irinotecan specifically at brain tumor sites. Results support the feasibility of NSC-mediated delivery of CE to glioma and lay the foundation for translational studies, potentially improving clinical outcome and quality of life in patients with malignant brain tumors.
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.
doi:10.5966/sctm.2012-0177
PMCID: PMC3841093  PMID: 24167321
Cancer therapy; Carboxylesterase; CPT-11; Glioma; Immunogenicity; Irinotecan; Microdialysis; Neural stem cells
5.  Neural Stem Cell-Mediated Enzyme-Prodrug Therapy for Glioma: Preclinical Studies 
Science translational medicine  2013;5(184):10.1126/scitranslmed.3005365.
High-grade gliomas are extremely difficult to treat because they are invasive and therefore are not curable by surgical resection; the toxicity of currently 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 that can target enzyme/prodrug therapy selectively to tumors. We have used a cytosine deaminase (CD)-expressing clonal human NSC line, HB1.F3.CD, to home to gliomas in mice and locally convert the tumor-localized prodrug 5-fluorocytosine to the active chemotherapeutic 5-fluorouracil. In vitro studies confirmed that the NSCs have normal karyotype, tumor tropism, and CD expression, indicating that these cells are genetically and functionally stable. In vivo biodistribution studies demonstrated that these NSCs retained tumor tropism, even in mice pre-treated 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 in 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 approximately 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, non-toxic and effective in mice. These data have led to approval of a first-inhuman study of an allogeneic NSC-mediated enzyme/prodrug targeted cancer therapy in patients with recurrent high-grade glioma.
doi:10.1126/scitranslmed.3005365
PMCID: PMC3864887  PMID: 23658244
6.  Magnetic Resonance Imaging Tracking of Ferumoxytol-Labeled Human Neural Stem Cells: Studies Leading to Clinical Use 
Stem Cells Translational Medicine  2013;2(10):766-775.
These preclinical studies of ferumoxytol-labeled neural stem cells (NSCs) for magnetic resonance imaging (MRI) cell tracking led to U.S. FDA approval for first-in-human use of this labeling method for NSCs transplanted into brain tumor patients. Ferumoxytol labeling of NSCs did not affect cell viability, growth kinetics, or tumor tropism, and enabled MRI visualization of NSC distribution in vivo. These studies support the clinical development of ferumoxytol labeling of cells for post-transplant MRI visualization and tracking.
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.
doi:10.5966/sctm.2013-0049
PMCID: PMC3785261  PMID: 24014682
Cell transplantation; Cellular therapy; Clinical trials; In vivo tracking; Neural stem cell; Stem cell; Stem cell transplantation
7.  The Timing of Neural Stem Cell-Based Virotherapy Is Critical for Optimal Therapeutic Efficacy When Applied With Radiation and Chemotherapy for the Treatment of Glioblastoma 
This study established a logical experimental model to recapitulate the complex clinical scenario for the treatment of glioblastoma multiforme (GBM) and tested the compatibility of neural stem cells loaded with oncolytic virus. Data from this report support the testing of CRAd-S-pk7-loaded HB1.F3-CD cells in the clinical setting and argue for a multimodality approach for the treatment of patients with GBM.
Abstract
Glioblastoma multiforme (GBM) remains fatal despite intensive surgical, radiotherapeutic, and chemotherapeutic interventions. Neural stem cells (NSCs) have been used as cellular vehicles for the transportation of oncolytic virus (OV) to therapeutically resistant and infiltrative tumor burdens throughout the brain. The HB1.F3-CD human NSC line has demonstrated efficacy as a cell carrier for the delivery of a glioma tropic OV CRAd-Survivin-pk7 (CRAd-S-pk7) in vitro and in animal models of glioma. At this juncture, no study has investigated the effectiveness of OV-loaded NSCs when applied in conjunction with the standard of care for GBM treatment, and therefore this study was designed to fill this void. Here, we show that CRAd-S-pk7-loaded HB1.F3-CD cells retain their tumor-tropic properties and capacity to function as in situ viral manufacturers in the presence of ionizing radiation (XRT) and temozolomide (TMZ). Furthermore, for the first time, we establish a logical experimental model that aims to recapitulate the complex clinical scenario for the treatment of GBM and tests the compatibility of NSCs loaded with OV. We report that applying OV-loaded NSCs together with XRT and TMZ can increase the median survival of glioma bearing mice by approximately 46%. Most importantly, the timing and order of therapeutic implementation impact therapeutic outcome. When OV-loaded NSCs are delivered prior to rather than after XRT and TMZ treatment, the median survival of mice bearing patient-derived GBM43 glioma xenografts is extended by 30%. Together, data from this report support the testing of CRAd-S-pk7-loaded HB1.F3-CD cells in the clinical setting and argue in favor of a multimodality approach for the treatment of patients with GBM.
doi:10.5966/sctm.2013-0039
PMCID: PMC3754466  PMID: 23926209
Glioma; Gene therapy; Neural stem cell; Adenovirus; Virotherapy; Radiation; Chemotherapy; Temozolomide
8.  Contact and Encirclement of Glioma Cells In Vitro Is an Intrinsic Behavior of a Clonal Human Neural Stem Cell Line 
PLoS ONE  2012;7(12):e51859.
Pathotropic neural stem and/or progenitor cells (NSCs) can potentially deliver therapeutic agents to otherwise inaccessible cancers. In glioma, NSCs are found in close contact with tumor cells, raising the possibility that specificity of NSC contact with glioma targets originates in the tumor cells themselves. Alternatively, target preferences may originate, at least in part, in the tumor microenvironment. To better understand mechanisms underlying NSC interactions with glioma cells, we examined NSC-target cell contacts in a highly simplified 3-dimensional peptide hydrogel (Puramatrix) in which cell behaviors can be studied in the relative absence of external cues. HB1.F3 is an immortalized clonal human NSC line extensively characterized in preclinical investigations. To study contact formation between HB1.F3 NSCs and glioma cells, we first examined co-cultures of eGFP-expressing HB1.F3 (HB1.F3.eGFP) NSCs and dsRed-expressing U251 glioma (U251.dsRed) cells. Using confocal microscopy, HB1.F3.eGFP cells were observed contacting or encircling U251.dsRed glioma cells, but never the reverse. Next, examining specificity of these contacts, no significant quantitative differences in either percentages of HB1.F3 NSCs contacting targets, or in the extent of target cell encirclement, were observed when HB1.F3.eGFP cells were presented with various potential target cells (human glioma and breast cancer cell lines, patient-derived brain tumor lines, non-tumor fibroblasts, primary mouse and human astroglial cells, and primary adult and newborn human dermal fibroblasts) except that interactions between HB1.F3 cells did not progress beyond establishing contacts. Finally cytoskeletal mechanisms employed by HB1.F3.eGFP cells varied with the substrate. When migrating in Puramatrix, HB1.F3 NSCs exhibited intermittent process extension followed by soma translocation, while during encirclement their movements were more amoeboid. We conclude that formation of contacts and subsequent encirclement of target cells by HB1.F3 NSCs is an intrinsic property of these NSCs, and that preferential contact formation with tumor cells in vivo must therefore be highly dependent on microenvironmental cues.
doi:10.1371/journal.pone.0051859
PMCID: PMC3519902  PMID: 23240066
9.  Pharmacokinetic study of neural stem cell-based cell carrier for oncolytic virotherapy: Targeted delivery of the therapeutic payload in an orthotopic brain tumor model 
Cancer Gene Therapy  2012;19(6):431-442.
Oncolytic virotherapy is a promising novel therapy for glioblastoma that needs to be optimized before introduced to clinic. The targeting of conditionally replicating adenoviruses (CRAds) can be improved by relying on the tumor tropic properties of neural stem cells (NSCs). Here, we report the characterization of an FDA approved NSC, HB1.F3-CD, as a cell carrier for CRAd-S-pk7, a glioma-tropic oncolytic adenovirus. We show that NSCs replicate and release infectious CRAd-S-pk7 progeny capable of lysing glioma cell lines. Moreover, ex-vivo loaded NSCs, injected intracranially in nude mice bearing human glioma xenografts (i) retained their tumor-tropism, (ii) continued to replicate CRAd-S-pk7 for more than a week after reaching the tumor site and (iii) successfully handed-off CRAd-S-pk7 to glioma cells in vivo. Delivery via carrier cells reduced non-specific adenovirus distribution in the mouse brain. Moreover, we assessed biodistribution of loaded NSCs after intracranial injection in animal models semi-permissive to adenovirus replication, the Syrian hamster and cotton rat. NSCs did not migrate to distant organs and high levels of CRAd-S-pk7 DNA were observed only in the injected hemisphere. In conclusion, this optimized carrier system, with high efficiency of adenovirus delivery and minimal systemic toxicity, poses considerable advantages for anti-glioma oncolytic virotherapy.
doi:10.1038/cgt.2012.21
PMCID: PMC3356460  PMID: 22555507
neural stem cell; carrier; oncolytic virus; adenovirus; hamster; cotton rat
10.  TWIST1 associates with NF-κB subunit RELA via carboxyl-terminal WR domain to promote cell autonomous invasion through IL8 production 
BMC Biology  2012;10:73.
Background
Metastasis is the primary cause of death for cancer patients. TWIST1, an evolutionarily conserved basic helix-loop-helix (bHLH) transcription factor, is a strong promoter of metastatic spread and its expression is elevated in many advanced human carcinomas. However, the molecular events triggered by TWIST1 to motivate dissemination of cancer cells are largely unknown.
Results
Here we show that TWIST1 induces the production of interleukin 8 (IL8), which activates matrix metalloproteinases and promotes invasion of breast epithelial and cancer cells. In this novel mechanism, TWIST1-mediated IL8 transcription is induced through the TWIST1 carboxy-terminal WR (Trp-Arg) domain instead of the classic DNA binding bHLH domain. Co-immunoprecipitation analyses revealed that the WR domain mediates the formation of a protein complex comprised of TWIST1 and the nuclear factor-kappaB (NF-κB) subunit RELA (p65/NF-κB3), which synergistically activates the transcriptional activity of NF-κB. This activation leads to increased DNA binding affinity of RELA to the IL8 promoter and thus induces the expression of the cytokine. Blockage of IL8 signaling by IL8 neutralizing antibodies or receptor inhibition reduced the invasiveness of both breast epithelial and cancer cells, indicating that TWIST1 induces autonomous cell invasion by establishing an IL8 antocrine loop.
Conclusions
Our data demonstrate that the TWIST1 WR domain plays a critical role in TWIST1-induced IL8 expression through interactions with and activation of NF-κB. The produced IL8 signals through an autocrine loop and promotes extracellular matrix degradation to enable cell invasion across the basement membrane.
doi:10.1186/1741-7007-10-73
PMCID: PMC3482588  PMID: 22891766
TWIST1; WR domain; RELA; NF-κB; IL8
11.  Cellular Host Responses to Gliomas 
PLoS ONE  2012;7(4):e35150.
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.
doi:10.1371/journal.pone.0035150
PMCID: PMC3335155  PMID: 22539956
12.  Neural Progenitor Cell-Mediated Delivery of Osteoprotegerin Limits Disease Progression in a Pre-Clinical Model of Neuroblastoma Bone Metastasis 
Journal of pediatric surgery  2009;44(1):204-211.
Purpose
Osteoprotegerin (OPG) inhibits osteoclast activation and reduces osteolysis in bone tumors. We hypothesized that tumor-tropic neural progenitor cells (NPCs) engineered to express OPG would reduce neuroblastoma disease burden in the bone.
Methods
Stable expression of green fluorescent protein (NPC-GFP) and OPG (NPC-OPG) was established in human NPCs by lentivirus-mediated transduction. Bone disease was established by intrafemoral injection of luciferase-expressing human neuroblastoma (CHLA-255) cells into 20 SCID mice. Three weeks later, mice began receiving IV injection of 2×106 NPC-OPG or NPC-GFP (control) every 10 days × 3 doses. Disease was monitored with quantitative bioluminescent imaging (BLI) and X-ray images, which were evaluated on a scale of 0 to 4. These studies were IACUC approved.
Results
OPG treatment in vitro produced no direct toxicity to tumor cells. Co-culture of tumor cells with bone marrow significantly increased activation of bone-marrow derived osteoclasts as assessed by TRAP staining (156±10.8osteoclasts/well) compared to bone marrow culture alone (91.67±4.7, p=0.005). This increase was abrogated by adding OPG-containing media (68.3±2.8, p=0.001). NPC-OPG slowed tumor progression (108-fold increase from pre-treatment) compared to mice treated with NPC-GFP (538-fold), as judged by BLI. X-rays subjectively demonstrated less bone disease in NPC-OPG-treated mice (2.27±0.25) compared to NPC-GFP-treated mice (3.25±0.22, p=0.04).
Conclusions
NPC-mediated delivery of OPG slowed disease progression in a pre-clinical model of neuroblastoma bone metastasis. The decrease in bone disease was not from direct tumor cell toxicity but likely occurred indirectly through inhibition of osteoclast-directed bone resorption. Thus, targeted delivery of OPG by NPCs may be effective in the treatment of neuroblastoma bone metastasis.
doi:10.1016/j.jpedsurg.2008.10.041
PMCID: PMC2655202  PMID: 19159744
Neuroblastoma; Osteoprotegerin; Neural progenitor cells; Osteoclast; Bone metastasis
13.  Neural Stem Cells as a Novel Platform for Tumor-Specific Delivery of Therapeutic Antibodies 
PLoS ONE  2009;4(12):e8314.
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.
doi:10.1371/journal.pone.0008314
PMCID: PMC2789379  PMID: 20016813
14.  Neural progenitor cell-mediated delivery of interferon-β improves neuroblastoma response to cyclophosphamide 
Annals of surgical oncology  2008;15(11):3259-3267.
Background
We have shown that continuous, systemic delivery of interferon-β (IFN-β) remodels dysfunctional tumor vasculature, thereby improving tumor perfusion and enhancing delivery and efficacy of chemotherapeutic drugs. We hypothesized that because of their inherent tumor-tropism, neural progenitor cells (NPCs) engineered to express IFN-β could also effect maturation of tumor vasculature without generating high systemic levels of IFN-β.
Methods
Mice with luciferase-expressing, disseminated human neuroblastoma were divided into four groups of equal tumor burden by bioluminescence imaging: (1)untreated controls (2)NPC-IFN-β only (3)cyclophosphamide only and (4)NPC-IFN-β in combination with cyclophosphamide. Two million NPC-IFN-β cells were given twice, seven days apart, starting twenty-one days after tail vein administration of tumor cells. Cyclophosphamide was given every six days for three doses. Mice were euthanized at six weeks, livers and kidneys weighed, and tissue harvested for immunohistochemistry for endothelial cells (CD34), pericytes (α-SMA), apoptosis (TUNEL), and diI-labeled NPCs.
Results
Fluorescent-labeled NPCs confirmed localization to tumors. The α-SMA/CD34 ratio, a marker for vascular maturation, significantly increased in NPC-IFN-β treated tumors compared to controls. Bioluminescent signal from luciferase-expressing tumor cells, reflecting tumor burden, was lower with combination therapy than control or either monotherapy, and combination therapy resulted in significantly less tumor burden by weight in the kidneys and liver.
Conclusions
Targeted delivery of IFN-β with NPCs produced low circulating levels of IFN-β, yet the maturing effect on the tumor vasculature and the enhanced efficacy of adjuvant therapy was maintained. Thus, combination therapy of NPC-IFN-β with cyclophosphamide warrants further investigation for the treatment of high-risk neuroblastoma patients.
doi:10.1245/s10434-008-0103-z
PMCID: PMC2587051  PMID: 18726131
Neuroblastoma; Neural progenitor cells; Interferon-beta; Cyclophosphamide; Angiogenesis
15.  Iron Labeling and Pre-Clinical MRI Visualization of Therapeutic Human Neural Stem Cells in a Murine Glioma Model 
PLoS ONE  2009;4(9):e7218.
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”.
doi:10.1371/journal.pone.0007218
PMCID: PMC2746284  PMID: 19787043
16.  Targeting of melanoma brain metastases using engineered neural stem/progenitor cells1 
Neuro-Oncology  2006;8(2):119-126.
Brain metastases are an increasingly frequent and serious clinical problem for cancer patients, especially those with advanced melanoma. Given the extensive tropism of neural stem/progenitor cells (NSPCs) for pathological areas in the central nervous system, we expanded investigations to determine whether NSPCs could also target multiple sites of brain metastases in a syngeneic experimental melanoma model. Using cytosine deaminase–expressing NSPCs (CD-NSPCs) and systemic 5-fluorocytosine (5-FC) pro-drug administration, we explored their potential as a cell-based targeted drug delivery system to disseminated brain metastases. Our results indicate a strong tropism of NSPCs for intracerebral melanoma metastases. Furthermore, in our therapeutic paradigm, animals with established melanoma brain metastasis received intracranial implantation of CD-NSPCs followed by systemic 5-FC treatment, resulting in a significant (71%) reduction in tumor burden. These data provide proof of principle for the use of NSPCs for targeted delivery of therapeutic gene products to melanoma brain metastases.
doi:10.1215/15228517-2005-012
PMCID: PMC1871940  PMID: 16524944
brain metastases; cytosine deaminase; gene therapy; melanoma; neural progenitor cells; neural stem cells; tumor targeting
17.  Identification of uPAR-positive Chemoresistant Cells in Small Cell Lung Cancer 
PLoS ONE  2007;2(2):e243.
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.
doi:10.1371/journal.pone.0000243
PMCID: PMC1800348  PMID: 17327908
18.  Development of a Tumor-Selective Approach to Treat Metastatic Cancer 
PLoS ONE  2006;1(1):e23.
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.
doi:10.1371/journal.pone.0000023
PMCID: PMC1762394  PMID: 17183650
19.  Brain Tumor Tropism of Transplanted Human Neural Stem Cells Is Induced by Vascular Endothelial Growth Factor1* 
Neoplasia (New York, N.Y.)  2005;7(6):623-629.
Abstract
The transplantation of neural stem cells (NSCs) offers a new potential therapeutic approach as a cell-based delivery system for gene therapy in brain tumors. This is based on the unique capacity of NSCs to migrate throughout the brain and to target invading tumor cells. However, the signals controlling the targeted migration of transplanted NSCs are poorly defined. We analyzed the in vitro and in vivo effects of angiogenic growth factors and protein extracts from surgical specimens of brain tumor patients on NSC migration. Here, we demonstrate that vascular endothelial growth factor (VEGF) is able to induce a long-range attraction of transplanted human NSCs from distant sites in the adult brain. Our results indicate that tumor-upregulated VEGF and angiogenic-activated microvasculature are relevant guidance signals for NSC tropism toward brain tumors.
PMCID: PMC1501284  PMID: 16036113
brain tumor; neural stem cells; migration; cell therapy; angiogenesis
20.  Concise Review: Stem Cells As an Emerging Platform for Antibody Therapy of Cancer 
Stem Cells (Dayton, Ohio)  2010;28(11):2084-2087.
Monoclonal antibodies are important tools for cancer therapy, however, three factors limit their effectiveness: toxicity, poor tumor penetration, and inability to cross the blood-brain barrier. This review discusses the emerging field of stem cell-mediated antibody delivery and how this approach may improve antibody therapy of cancer by overcoming these obstacles. STEM CELLS 2010;28:2084–2087
doi:10.1002/stem.513
PMCID: PMC3003900  PMID: 21089119
Antibody; Blood-brain barrier; Cancer therapy; α-Carcinoembryonic antigen; CD3; Diabody; EGFR; HER2; Neural stem cells; Mesenchymal stem cells; scFv

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