Dou Yu

Cell-Penetrating Peptide-Modified Gold Nanoparticles for the Delivery of Doxorubicin to Brain Metastatic Breast Cancer

As therapies continue to increase the lifespan of patients with breast cancer, the incidence of brain metastases has steadily increased, affecting a significant number of patients with metastatic disease. However, a major barrier toward treating these lesions is the inability of therapeutics to penetrate into the central nervous system and accumulate within intracranial tumor sites. In this study, we designed a cell-penetrating gold nanoparticle platform to increase drug delivery to brain metastatic breast cancer cells. TAT peptide-modified gold nanoparticles carrying doxorubicin led to improved cytotoxicity toward two brain metastatic breast cancer cell lines with a decrease in the IC50 of at least 80% compared to free drug. Intravenous administration of these particles led to extensive accumulation of particles throughout diffuse intracranial metastatic microsatellites with cleaved caspase-3 activity corresponding to tumor foci. Furthermore, intratumoral administration of these particles improved survival in an intracranial MDA-MB-231-Br xenograft mouse model. Our results demonstrate the promising application of gold nanoparticles for improving drug delivery in the context of brain metastatic breast cancer.

CCL2 Produced by the Glioma Microenvironment Is Essential for the Recruitment of Regulatory T Cells and Myeloid-Derived Suppressor Cells

In many aggressive cancers, such as glioblastoma multiforme, progression is enabled by local immunosuppression driven by the accumulation of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). However, the mechanistic details of how Tregs and MDSCs are recruited in various tumors are not yet well understood. Here we report that macrophages and microglia within the glioma microenvironment produce CCL2, a chemokine that is critical for recruiting both CCR4+ Treg and CCR2+Ly-6C+ monocytic MDSCs in this disease setting. In murine gliomas, we established novel roles for tumor-derived CCL20 and osteoprotegerin in inducing CCL2 production from macrophages and microglia. Tumors grown in CCL2-deficient mice failed to maximally accrue Tregs and monocytic MDSCs. In mixed-bone marrow chimera assays, we found that CCR4-deficient Treg and CCR2-deficient monocytic MDSCs were defective in glioma accumulation. Furthermore, administration of a small-molecule antagonist of CCR4 improved median survival in the model. In clinical specimens of glioblastoma multiforme, elevated levels of CCL2 expression correlated with reduced overall survival of patients. Finally, we found that CD163-positive infiltrating macrophages were a major source of CCL2 in glioblastoma multiforme patients. Collectively, our findings show how glioma cells influence the tumor microenvironment to recruit potent effectors of immunosuppression that drive progression. Cancer Res; 76(19); 5671-82. ©2016 AACR.

Multimodal Actions of Neural Stem Cells in a Mouse Model of ALS: A Meta-Analysis

A meta-analysis reports the beneficial effects of transplanting mouse or human neural stem cells into the spinal cord of the SOD1G93A mouse, a model of ALS. Stem Cells to the Rescue Amyotrophic lateral sclerosis (ALS) or Lou Gehrig’s disease is an untreatable fatal disorder characterized by rapid and unremitting degeneration of nerve cells in the spinal cord that enable movement and respiration. Multiple processes involving these neurons and other cell types have been implicated as the cause of this disease. Neural stem cells (NSCs) normally function in the nervous system to create structures during development and to restore function to damaged systems throughout life. When these cells are isolated from the nervous system, grown and expanded in a dish, and then transplanted back into a diseased or injured part of the nervous system, they are thought to be able to perform at least some of these same tasks by producing therapeutic factors, improving the milieu, rescuing dying neurons, protecting neural connections, and reducing inflammation. Transplanted NSCs might be able to ameliorate some of the pathological processes that occur in ALS. Teng et al. now test this hypothesis by performing a meta-analysis of 11 studies that have transplanted mouse or human NSCs into the spinal cord of the transgenic mutant SOD1 ALS mouse. The authors found that disease onset and progression were slowed, such that extensive, often motor symptom-reduced, survival was predictably achievable in a subset of animals. This was particularly noticeable in those mice where transplanted NSCs covered a large part of the spinal cord including regions mediating vital functions such as respiration. The benefits of transplanted NSCs seem to be derived from a number of different actions including production of trophic factors, preservation of neuromuscular function, and a reduction in astrogliosis and inflammation. Through multiple modulatory mechanisms, NSCs may have potential for treating ALS and other untreatable degenerative diseases. Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by the unremitting degeneration of motor neurons. Multiple processes involving motor neurons and other cell types have been implicated in its pathogenesis. Neural stem cells (NSCs) perform multiple actions within the nervous system to fulfill their functions of organogenesis and homeostasis. We test the hypothesis that transplanted, undifferentiated multipotent migratory NSCs may help to ameliorate an array of pathological mechanisms in the SOD1G93A transgenic mouse model of ALS. On the basis of a meta-analysis of 11 independent studies performed by a consortium of ALS investigators, we propose that transplanted NSCs (both mouse and human) can slow both the onset and the progression of clinical signs and prolong survival in ALS mice, particularly if regions sustaining vital functions such as respiration are rendered chimeric. The beneficial effects of transplanted NSCs seem to be mediated by a number of actions including their ability to produce trophic factors, preserve neuromuscular function, and reduce astrogliosis and inflammation. We conclude that the widespread, pleiotropic, modulatory actions exerted by transplanted NSCs may represent an accessible therapeutic application of stem cells for treating ALS and other untreatable degenerative diseases.

Blockade of Peroxynitrite‐Induced Neural Stem Cell Death in the Acutely Injured Spinal Cord by Drug‐Releasing Polymer

Therapeutic impact of neural stem cells (NSCs) for acute spinal cord injury (SCI) has been limited by the rapid loss of donor cells. Neuroinflammation is likely the cause. As there are close temporal‐spatial correlations between the inducible nitric oxide (NO) synthase expression and the donor NSC death after neurotrauma, we reasoned that NO‐associated radical species might be the inflammatory effectors which eliminate NSC grafts and kill host neurons. To test this hypothesis, human NSCs (hNSCs: 5 × 104 to 2 × 106 per milliliter) were treated in vitro with “plain” medium, 20 μM glutamate, or donors of NO and peroxynitrite (ONOO−; 100 and 400 μM of spermine or DETA NONOate, and SIN‐1, respectively). hNSC apoptosis primarily resulted from SIN‐1 treatment, showing ONOO−‐triggered protein nitration and the activation of p38 MAPK, cytochrome c release, and caspases. Therefore, cell death following post‐SCI (p.i.) NO surge may be mediated through conversion of NO into ONOO−. We subsequently examined such causal relationship in a rat model of dual penetrating SCI using a retrievable design of poly‐lactic‐co‐glycolic acid (PLGA) scaffold seeded with hNSCs that was shielded by drug‐releasing polymer. Besides confirming the ONOO−‐induced cell death signaling, we demonstrated that cotransplantation of PLGA film embedded with ONOO− scavenger, manganese (III) tetrakis (4‐benzoic acid) porphyrin, or uric acid (1 μmol per film), markedly protected hNSCs 24 hours p.i. (total: n = 10). Our findings may provide a bioengineering approach for investigating mechanisms underlying the host microenvironment and donor NSC interaction and help formulate strategies for enhancing graft and host cell survival after SCI. Stem Cells 2009;27:1212–1222

Nitric Oxide Stimulates γ-Aminobutyric Acid Release and Inhibits Glycine Release in Retina

Nitric oxide (NO) modulates the uptake and/or release of neurotransmitters through a variety of cellular mechanisms. However, the pharmacological and biochemical processes underlying these neurochemical effects of NO often remain unclear. In our study, we used immunocytochemical methods to study the effects of NO, cyclic guanosine monophosphate (cGMP), and peroxynitrite on the uptake and release of γ‐aminobutyric acid (GABA) and glycine in the turtle retina. In addition, we examined the involvement of glutamate receptors, calcium, and the GABA transporter in this GABA uptake and release. We also tested for interactions between the GABAergic and glycinergic systems. In general, we show that NO stimulated GABA release and inhibited glycine release. The NO‐stimulated GABA release involved calcium‐dependent or calcium‐independent synaptic release or reversal of the GABA transporter. Some effects of NO on GABA release involved glutamate, cGMP, or peroxynitrite. NO promoted glycine uptake and inhibited its release, and this inhibition of glycine release was influenced by GABAergic modulation. These findings indicate that NO modulates the levels of the inhibitory transmitters GABA and glycine through several specific biochemical mechanisms in different retinal cell types and layers. Thus it appears that some of the previously described reciprocal interactions between GABA and glycine in the retina function through specific NO signaling pathways. J. Comp. Neurol. 483:278–291, 2005.

Establishing a model spinal cord injury in the African green monkey for the preclinical evaluation of biodegradable polymer scaffolds seeded with human neural stem cells

Given the involvement of post-mitotic neurons, long axonal tracts and incompletely elucidated injury and repair pathways, spinal cord injury (SCI) presents a particular challenge for the creation of preclinical models to robustly evaluate longitudinal changes in neuromotor function in the setting in the presence and absence of intervention. While rodent models exhibit high degrees of spontaneous recovery from SCI injury, animal care concerns preclude complete cord transections in non-human primates and other larger vertebrate models. To overcome such limitations a segmental thoracic (T9-T10) spinal cord hemisection was created and characterized in the African green monkey. Physiological tolerance of the model permitted behavioral analyses for a prolonged period post-injury, extending to predefined study termination points at which histological and immunohistochemical analyses were performed. Four monkeys were evaluated (one receiving no implant at the lesion site, one receiving a poly(lactide-co-glycolide) (PLGA) scaffold, and two receiving PLGA scaffolds seeded with human neural stem cells (hNSC)). All subjects exhibited Brown-Séquard syndrome 2 days post-injury consisting of ipsilateral hindlimb paralysis and contralateral hindlimb hypesthesia with preservation of bowel and bladder function. A 20-point observational behavioral scoring system allowed quantitative characterization of the levels of functional recovery. Histological endpoints including silver degenerative staining and Iba1 immunohistochemistry, for microglial and macrophage activation, were determined to reliably define lesion extent and correlate with neurobehavioral data, and justify invasive telemetered electromyographic and kinematic studies to more definitively address efficacy and mechanism.

Alleviation of chronic pain following rat spinal cord compression injury with multimodal actions of huperzine A

Significance Neuropathic pain, one of the most debilitating sequelae of neurotrauma, is an unmet clinical need for at least 40% of patients with spinal cord injury (SCI). We demonstrate that [-]-huperzine A (HUP-A), a naturally occurring Lycopodium alkaloid isolated from the Chinese club moss, Huperzia serrata, with potent reversible inhibitory action on acetylcholinesterase and N-methyl-D-aspartate glutamate receptors, offers an exceptional prospect for multimodal treatment of SCI-induced neuropathic pain in rats. HUP-A restores homeostasis of central sensory neurocircuitry without invoking drug tolerance and dependence or respiratory suppression. We therefore conclude that multimodal actions provide a fresh translational approach to reduce chronic pain. Diverse mechanisms including activation of NMDA receptors, microglial activation, reactive astrogliosis, loss of descending inhibition, and spasticity are responsible for ∼40% of cases of intractable neuropathic pain after spinal cord injury (SCI). Because conventional treatments blocking individual mechanisms elicit only short-term effectiveness, a multimodal approach with simultaneous actions against major pain-related pathways may have value for clinical management of chronic pain. We hypothesize that [-]-huperzine A (HUP-A), an alkaloid isolated from the club moss Huperzia serrata, that is a potent reversible inhibitor of acetylcholinesterase and NMDA receptors, could mitigate pain without invoking drug tolerance or dependence by stimulating cholinergic interneurons to impede pain signaling, inhibiting inflammation via microglial cholinergic activation, and blocking NMDA-mediated central hypersensitization. We tested our hypothesis by administering HUP-A i.p. or intrathecally to female Sprague–Dawley rats (200–235 g body weight) after moderate static compression (35 g for 5 min) of T10 spinal cord. Compared with controls, HUP-A treatment demonstrates significant analgesic effects in both regimens. SCI rats manifested no drug tolerance following repeated bolus i.p. or chronic intrathecal HUP-A dosing. The pain-ameliorating effect of HUP-A is cholinergic dependent. Relative to vehicle treatment, HUP-A administration also reduced neural inflammation, retained higher numbers of calcium-impermeable GluR2-containing AMPA receptors, and prevented Homer1a up-regulation in dorsal horn sensory neurons. Therefore, HUP-A may provide safe and effective management for chronic postneurotrauma pain by reestablishing homeostasis of sensory circuits.

Functional Multipotency of Stem Cells: A Conceptual Review of Neurotrophic Factor-Based Evidence and Its Role in Translational Research

We here propose an updated concept of stem cells (SCs), with an emphasis on neural stem cells (NSCs). The conventional view, which has touched principally on the essential property of lineage multipotency (e.g., the ability of NSCs to differentiate into all neural cells), should be broadened to include the emerging recognition of biofunctional multipotency of SCs to mediate systemic homeostasis, evidenced in NSCs in particular by the secretion of neurotrophic factors. Under this new conceptual context and taking the NSC as a leading example, one may begin to appreciate and seek the “logic” behind the wide range of molecular tactics the NSC appears to serve at successive developmental stages as it integrates into and prepares, modifies, and guides the surrounding CNS micro- and macro-environment towards the formation and self-maintenance of a functioning adult nervous system. We suggest that embracing this view of the “multipotency” of the SCs is pivotal for correctly, efficiently, and optimally exploiting stem cell biology for therapeutic applications, including reconstitution of a dysfunctional CNS.

Dynamic In Vivo SPECT Imaging of Neural Stem Cells Functionalized with Radiolabeled Nanoparticles for Tracking of Glioblastoma

There is strong clinical interest in using neural stem cells (NSCs) as carriers for targeted delivery of therapeutics to glioblastoma. Multimodal dynamic in vivo imaging of NSC behaviors in the brain is necessary for developing such tailored therapies; however, such technology is lacking. Here we report a novel strategy for mesoporous silica nanoparticle (MSN)–facilitated NSC tracking in the brain via SPECT. Methods: 111In was conjugated to MSNs, taking advantage of the large surface area of their unique porous feature. A series of nanomaterial characterization assays was performed to assess the modified MSN. Loading efficiency and viability of NSCs with 111In-MSN complex were optimized. Radiolabeled NSCs were administered to glioma-bearing mice via either intracranial or systemic injection. SPECT imaging and bioluminescence imaging were performed daily up to 48 h after NSC injection. Histology and immunocytochemistry were used to confirm the findings. Results: 111In-MSN complexes show minimal toxicity to NSCs and robust in vitro and in vivo stability. Phantom studies demonstrate feasibility of this platform for NSC imaging. Of significance, we discovered that decayed 111In-MSN complexes exhibit strong fluorescent profiles in preloaded NSCs, allowing for ex vivo validation of the in vivo data. In vivo, SPECT visualizes actively migrating NSCs toward glioma xenografts in real time after both intracranial and systemic administrations. This is in agreement with bioluminescence live imaging, confocal microscopy, and histology. Conclusion: These advancements warrant further development and integration of this technology with MRI for multimodal noninvasive tracking of therapeutic NSCs toward various brain malignancies.

Defining recovery neurobiology of injured spinal cord by synthetic matrix-assisted hMSC implantation.

Significance We developed a platform technology to determine therapeutic mechanisms of human mesenchymal stromal stem cells (hMSCs) in a dorsal root ganglion coculture system and an intraspinal cord implantation model. The unique poly(lactic-co-glycolic) acid scaffolding augments hMSC stemness, engraftment, and function without neural transdifferentiation or mesenchymal lineage development, resulting in robust motosensory improvement, pain and tissue damage mitigation, and myelin preservation in adult rat spinal cord after injury. The scaffolded hMSC-derived neurotrophism, neurogenesis, angiogenesis, antiautoimmunity, and antiinflammation support the propriospinal network, neuromuscular junctions, and serotonergic reticulospinal reinnervation to activate the central pattern generator for restoring hindlimb locomotion. Our findings illuminate “recovery neurobiology”—i.e., the injured spinal cord may deploy polysynaptic neural circuits different from normal adulthood pathways for postinjury improvement. Mesenchymal stromal stem cells (MSCs) isolated from adult tissues offer tangible potential for regenerative medicine, given their feasibility for autologous transplantation. MSC research shows encouraging results in experimental stroke, amyotrophic lateral sclerosis, and neurotrauma models. However, further translational progress has been hampered by poor MSC graft survival, jeopardizing cellular and molecular bases for neural repair in vivo. We have devised an adult human bone marrow MSC (hMSC) delivery formula by investigating molecular events involving hMSCs incorporated in a uniquely designed poly(lactic-co-glycolic) acid scaffold, a clinically safe polymer, following inflammatory exposures in a dorsal root ganglion organotypic coculture system. Also, in rat T9–T10 hemisection spinal cord injury (SCI), we demonstrated that the tailored scaffolding maintained hMSC stemness, engraftment, and led to robust motosensory improvement, neuropathic pain and tissue damage mitigation, and myelin preservation. The scaffolded nontransdifferentiated hMSCs exerted multimodal effects of neurotrophism, angiogenesis, neurogenesis, antiautoimmunity, and antiinflammation. Hindlimb locomotion was restored by reestablished integrity of submidbrain circuits of serotonergic reticulospinal innervation at lumbar levels, the propriospinal projection network, neuromuscular junction, and central pattern generator, providing a platform for investigating molecular events underlying the repair impact of nondifferentiated hMSCs. Our approach enabled investigation of recovery neurobiology components for injured adult mammalian spinal cord that are different from those involved in normal neural function. The uncovered neural circuits and their molecular and cellular targets offer a biological underpinning for development of clinical rehabilitation therapies to treat disabilities and complications of SCI.