Intraperitoneal Administration of Neural Stem Cell-Nanoparticle Conjugates Targets Chemotherapy to Ovarian Tumors.

Ovarian cancer is particularly aggressive once it has metastasized to the abdominal cavity (stage III). Intraperitoneal (IP) as compared to intravenous (IV) administration of chemotherapy improves survival for stage III ovarian cancer, demonstrating that concentrating chemotherapy at tumor sites has therapeutic benefit; unfortunately, IP therapy also increases toxic side effects, thus preventing its completion in many patients. The ability to target chemotherapy selectively to ovarian tumors while sparing normal tissue would improve efficacy and decrease toxicities. We have previously shown that tumor-tropic neural stem cells (NSCs) dramatically improve the intratumoral distribution of nanoparticles (NPs) when given intracerebrally near an orthotopic brain tumor or into a flank xenograft tumor. Here, we show that NPs either conjugated to the surface of NSCs or loaded within the cells are selectively delivered to and distributed within ovarian tumors in the abdominal cavity following IP injection, with no evidence of localization to normal tissue. IP administration is significantly more effective than IV administration, and NPs carried by NSCs show substantially deeper penetration into tumors than free NPs. The NSCs and NPs target and localize to ovarian tumors within 1 h of administration. Pt-loaded silica NPs (SiNP[Pt]) were developed that can be transported in NSCs, and it was found that the NSC delivery of SiNP[Pt] (NSC-SiNP[Pt]) results in higher levels of Pt in tumors as compared to free drug or SiNP[Pt]. To the best of our knowledge, this work represents the first demonstration that cells given IP can target the delivery of drug-loaded NPs.

Neural stem cells improve intracranial nanoparticle retention and tumor-selective distribution.

AIM: The purpose of this work is to determine if tumor-tropic neural stem cells (NSCs) can improve the tumor-selective distribution and retention of nanoparticles (NPs) within invasive brain tumors. MATERIALS & METHODS: Streptavidin-conjugated, polystyrene NPs are surface-coupled to biotinylated human NSCs. These NPs are large (798 nm), yet when conjugated to tropic cells, they are too large to passively diffuse through brain tissue or cross the blood-tumor barrier. NP distribution and retention was quantified 4 days after injections located either adjacent to an intracerebral glioma, in the contralateral hemisphere, or intravenously. RESULTS & CONCLUSION: In all three in vivo injection paradigms, NSC-coupled NPs exhibited significantly improved tumor-selective distribution and retention over free-NP suspensions. These results provide proof-of-principle that NSCs can facilitate the tumor-selective distribution of NPs, a platform useful for improving intracranial drug delivery.

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.

Behavioral deficits and subregion-specific suppression of LTP in mice expressing a population of mutant NMDA receptors throughout the hippocampus.

The NMDA receptor (NMDAR) subunit GluN1 is an obligatory component of NMDARs without a known functional homolog and is expressed in almost every neuronal cell type. The NMDAR system is a coincidence detector with critical roles in spatial learning and synaptic plasticity. Its coincidence detection property is crucial for the induction of hippocampal long-term potentiation (LTP). We have generated a mutant mouse model expressing a hypomorph of the Grin1(N598R) allele, which leads to a minority (about 10%) of coincidence detection-impaired NMDARs. Surprisingly, these animals revealed specific functional changes in the dentate gyrus (DG) of the hippocampal formation. Early LTP was expressed normally in area CA1 in vivo, but was completely suppressed at perforant path-granule cell synapses in the DG. In addition, there was a pronounced reduction in the amplitude of the evoked population spike in the DG. These specific changes were accompanied by behavioral impairments in spatial recognition, spatial learning, reversal learning, and retention. Our data show that minor changes in GluN1-dependent NMDAR physiology can cause dramatic consequences in synaptic signaling in a subregion-specific fashion despite the nonredundant nature of the GluN1 gene and its global expression.

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.

Early Changes in Tumor Perfusion from T1-Weighted Dynamic Contrast-Enhanced MRI following Neural Stem Cell-Mediated Therapy of Recurrent High-Grade Glioma Correlate with Overall Survival.

BACKGROUND: The aim of this study was to correlate T1-weighted dynamic contrast-enhanced MRI- (DCE-MRI-) derived perfusion parameters with overall survival of recurrent high-grade glioma patients who received neural stem cell- (NSC-) mediated enzyme/prodrug gene therapy. METHODS: A total of 12 patients were included in this retrospective study. All patients were enrolled in a first-in-human study (NCT01172964) of NSC-mediated therapy for recurrent high-grade glioma. DCE-MRI data from all patients were collected and analyzed at three time points: MRI#1-day 1 postsurgery/treatment, MRI#2- day 7 ± 3 posttreatment, and MRI#3-one-month follow-up. Plasma volume (Vp), permeability (Ktr), and leakage (λtr) perfusion parameters were calculated by fitting a pharmacokinetic model to the DCE-MRI data. The contrast-enhancing (CE) volume was measured from the last dynamic phase acquired in the DCE sequence. Perfusion parameters and CE at each MRI time point were recorded along with their relative change between MRI#2 and MRI#3 (Δ32). Cox regression was used to analyze patient survival. RESULTS: At MRI#1 and at MRI#3, none of the parameters showed a significant correlation with overall survival (OS). However, at MRI#2, CE and λtr were significantly associated with OS (p < 0.05). The relative λtr and Vp from timepoint 2 to timepoint 3 (Δ32λtr and Δ32Vp) were each associated with a higher hazard ratio (p < 0.05). All parameters were highly correlated, resulting in a multivariate model for OS including only CE at MRI#2 and Δ32Vp, with an R2 of 0.89. CONCLUSION: The change in perfusion parameter values from 1 week to 1 month following NSC-mediated therapy combined with contrast-enhancing volume may be a useful biomarker to predict overall survival in patients with recurrent high-grade glioma.

Cell-mediated enzyme prodrug cancer therapies.

Cell-directed gene therapy is a promising new frontier for the field of targeted cancer therapies. Here we discuss the current pre-clinical and clinical use of cell-mediated enzyme prodrug therapy (EPT) directed against solid tumors and avenues for further development. We also discuss some of the challenges encountered upon translating these therapies to clinical trials. Upon sufficient development, cell-mediated enzyme prodrug therapy has the potential to maximize the distribution of therapeutic enzymes within the tumor environment, localizing conversion of prodrug to active drug at the tumor sites thereby decreasing off-target toxicities. New combinatorial possibilities are also promising. For example, when combined with viral gene-delivery vehicles, this may result in new hybrid vehicles that attain heretofore unmatched levels of therapeutic gene expression within the tumor.

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.

L-MYC Expression Maintains Self-Renewal and Prolongs Multipotency of Primary Human Neural Stem Cells.

Pre-clinical studies indicate that neural stem cells (NSCs) can limit or reverse CNS damage through direct cell replacement, promotion of regeneration, or delivery of therapeutic agents. Immortalized NSC lines are in growing demand due to the inherent limitations of adult patient-derived NSCs, including availability, expandability, potential for genetic modifications, and costs. Here, we describe the generation and characterization of a new human fetal NSC line, immortalized by transduction with L-MYC (LM-NSC008) that in vitro displays both self-renewal and multipotent differentiation into neurons, oligodendrocytes, and astrocytes. These LM-NSC008 cells were non-tumorigenic in vivo, and migrated to orthotopic glioma xenografts in immunodeficient mice. When administered intranasally, LM-NSC008 distributed specifically to sites of traumatic brain injury (TBI). These data support the therapeutic development of immortalized LM-NSC008 cells for allogeneic use in TBI and other CNS diseases.

Absence of Whisker-related pattern formation in mice with NMDA receptors lacking coincidence detection properties and calcium signaling.

Precise refinement of synaptic connectivity is the result of activity-dependent mechanisms in which coincidence-dependent calcium signaling by NMDA receptors (NMDARs) under control of the voltage-dependent Mg2+ block might play a special role. In the developing rodent trigeminal system, the pattern of synaptic connections between whisker-specific inputs and their target cells in the brainstem is refined to form functionally and morphologically distinct units (barrelettes). To test the role of NMDA receptor signaling in this process, we introduced the N598R mutation into the native NR1 gene. This leads to the expression of functional NMDARs that are Mg2+ insensitive and Ca2+ impermeable. Newborn mice expressing exclusively NR1 N598R-containing NMDARs do not show any whisker-related patterning in the brainstem, whereas the topographic projection of trigeminal afferents and gross brain morphology appear normal. Furthermore, the NR1 N598R mutation does not affect expression levels of NMDAR subunits and other important neurotransmitter receptors. Our results show that coincidence detection by, and/or Ca2+ permeability of, NMDARs is necessary for the development of somatotopic maps in the brainstem and suggest that highly specific signaling underlies synaptic refinement.

Noninvasive measurement of myocardial activity concentrations and perfusion defect sizes in rats with a new small-animal positron emission tomograph.

BACKGROUND: We explored the feasibility of measuring regional tracer activity concentrations and flow defects in myocardium of rats with a high spatial resolution small-animal PET system (microPET). METHODS AND RESULTS: Myocardial images were obtained after intravenous (18)F-fluorodeoxyglucose (18FDG) in 11 normal rats (group 1) and assembled into polar maps. Regional 18F activity concentrations were measured in 9 regions of interest and compared with tissue activity concentrations measured by well counting. In another 9 rats (group 2), myocardial perfusion images were acquired with 13N-ammonia at baseline and during coronary occlusion. On the polar maps recorded during coronary occlusion, the size of perfusion defects was measured as the myocardium with <50% of maximum activity and expressed as percent total myocardium and was correlated with the area at risk defined by postmortem staining. The diagnostic quality of 18FDG and 13N-ammonia microPET images was good to excellent; the images were easily assembled into polar maps. In group 1, regional (18)F concentrations by microPET and postmortem were correlated linearly (r=0.99; P<0.01 for average and r=0.97; P<0.01 for regional concentrations). In group 2, perfusion defect sizes by microPET and postmortem were correlated linearly (P<0.01; r=0.93). CONCLUSIONS: The findings indicate the feasibility of noninvasive studies of the myocardium in rats with a dedicated small-animal PET-imaging device.

Noninvasive monitoring of target gene expression by imaging reporter gene expression in living animals using improved bicistronic vectors.

UNLABELLED: Indirect, noninvasive imaging of therapeutic gene expression based on levels of reporter gene expression is a powerful tool to devise improved therapeutic strategies in cancer gene therapy. The use of bicistronic vectors carrying internal ribosome entry sites (IRESs) allows the coexpression of multiple gene products from the same promoter but leads to considerable attenuation of the downstream gene. In this study, we describe the use of 10 linked copies of the Gtx (homeodomain protein) IRES (abbreviated as SIRES) in place of the encephalomyocarditis (EMCV) IRES in mediating downstream reporter gene expression in cell culture and in vivo. METHODS: We constructed several plasmid vectors carrying different upstream and downstream reporter genes (herpes simplex virus type I thymidine kinase [tk], firefly luciferase [fl], and Renilla luciferase [rl]) placed between EMCV IRES and SIRES segments. RL, FL, and TK enzyme activities in N2a, C6, and 293 cells transiently transfected with these vectors were found to be significantly higher for the SIRES vectors than for the EMCV IRES vectors. For in vivo experiments, 4 stably transfected N2a cell lines were implanted in nude mice. The mice were imaged for rl and fl gene expression using a charged-coupled device (CCD) camera. For bioluminescence and microPET imaging of downstream gene expression of fl and tk genes, respectively, mice carrying 4 stably transfected xenografts were imaged using the CCD camera and microPET. RESULTS: In cell culture, using rl as the upstream gene, we demonstrate that the expression of the downstream tk gene is 12-fold greater using SIRES when compared with EMCV IRES. Furthermore, the expression of the 2 genes was highly correlated in N2a cells. In vivo bioluminescence imaging using 4 stably transfected N2a cell lines revealed increasing levels of rl and fl gene expression. Bioluminescence and microPET, respectively, of fl and tk reporter gene expression in nude mice bearing N2a tumor xenografts showed the gene expression mediated by SIRES to be 4- and 8-fold higher, respectively, than EMCV IRES. CONCLUSION: These findings support the use of SIRES bicistronic vectors for a better assessment of therapeutic gene expression based on reporter gene expression in living subjects.

Neural stem cell-mediated intratumoral delivery of gold nanorods improves photothermal therapy.

Plasmonic photothermal therapy utilizes biologically inert gold nanorods (AuNRs) as tumor-localized antennas that convert light into heat capable of eliminating cancerous tissue. This approach has lower morbidity than surgical resection and can potentially synergize with other treatment modalities including chemotherapy and immunotherapy. Despite these advantages, it is still challenging to obtain heating of the entire tumor mass while avoiding unnecessary collateral damage to surrounding healthy tissue. It is therefore critical to identify innovative methods to distribute an effective concentration of AuNRs throughout tumors without depositing them in surrounding healthy tissue. Here we demonstrate that AuNR-loaded, tumor-tropic neural stem cells (NSCs) can be used to improve the intratumoral distribution of AuNRs. A simple UV-vis technique for measuring AuNR loading within NSCs was established. It was then confirmed that NSC viability is unimpaired following AuNR loading and that NSCs retain AuNRs long enough to migrate throughout tumors. We then demonstrate that intratumoral injections of AuNR-loaded NSCs are more efficacious than free AuNR injections, as evidenced by reduced recurrence rates of triple-negative breast cancer (MDA-MB-231) xenografts following NIR exposure. Finally, we demonstrate that the distribution of AuNRs throughout the tumors is improved when transported by NSCs, likely resulting in the improved efficacy of AuNR-loaded NSCs as compared to free AuNRs. These findings highlight the advantage of combining cellular therapies and nanotechnology to generate more effective cancer treatments.

Conjugation of pH-responsive nanoparticles to neural stem cells improves intratumoral therapy.

Intratumoral drug delivery is an inherently appealing approach for concentrating toxic chemotherapies at the site of action. This mode of administration is currently used in a number of clinical treatments such as neoadjuvant, adjuvant, and even standalone therapies when radiation and surgery are not possible. However, even when injected locally, it is difficult to achieve efficient distribution of chemotherapeutics throughout the tumor. This is primarily attributed to the high interstitial pressure which results in gradients that drive fluid away from the tumor center. The stiff extracellular matrix also limits drug penetration throughout the tumor. We have previously shown that neural stem cells can penetrate tumor interstitium, actively migrating even to hypoxic tumor cores. When used to deliver therapeutics, these migratory neural stem cells result in dramatically enhanced tumor coverage relative to conventional delivery approaches. We recently showed that neural stem cells maintain their tumor tropic properties when surface-conjugated to nanoparticles. Here we demonstrate that this hybrid delivery system can be used to improve the efficacy of docetaxel-loaded nanoparticles when administered intratumorally. This was achieved by conjugating drug-loaded nanoparticles to the surface of neural stem cells using a bond that allows the stem cells to efficiently distribute nanoparticles throughout the tumor before releasing the drug for uptake by tumor cells. The modular nature of this system suggests that it could be used to improve the efficacy of many chemotherapy drugs after intratumoral administration.

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.