Jared Bushman

Paclitaxel in tyrosine-derived nanospheres as a potential anti-cancer agent: In vivo evaluation of toxicity and efficacy in comparison with paclitaxel in Cremophor

Paclitaxel (PTX) has gained widespread clinical use yet its administration is associated with significant toxicity. In the present study, the toxicity and anti-tumor efficacy of tyrosine-derived nanospheres (NSP) for the delivery of PTX was compared to a clinical formulation of PTX in PBS-diluted Cremophor® EL (PTX-CrEL-D). Maximum tolerated dose was determined using a concentration series of PTX in NSP and CrEL-D, with toxicity assessed by measuring changes in body weight. Healthy mice administered PTX-NSP continued to gain weight normally while treatment with PTX-CrEL-D resulted in significant weight loss that failed to recover following treatment. Even at the dose of 50mg/kg, PTX-NSP showed better tolerance than 25mg/kg of PTX-CrEL-D. Xenograft studies of breast cancer revealed that the anti-tumor efficacy of PTX-NSP was equal to that of PTX-CrEL-D in tumors originating from both MDA-MB-435 and ZR-75-1 cancer lines. Larger volume of distribution and longer half-life were measured for PTX-NSP administration compared to those reported in the literature for a CrEL formulation. This trend suggests the potential for improved therapeutic index of PTX when administered via NSP. The findings reported here confirm that the NSP formulation is an efficient method for PTX administration with significant increase in maximum tolerated dose, offering possible clinical implications in the treatment of breast tumors.

One-step synthesis of biodegradable curcumin-derived hydrogels as potential soft tissue fillers after breast cancer surgery.

A one-step synthesis of a curcumin-derived hydrogel (curcumin content of 25-75 mol %) is reported. Curcumin is incorporated into the hydrogel backbone and cross-linked through biodegradable carbonate linkages. Curcumin as a part of the polymer backbone is protected from oxidation and degradation, while hydrogel hydrolysis results in the release of active curcumin. Nontoxic poly(ethylene glycol) and desaminotyrosyl-tyrosine ethyl ester are used to tune the hydrophilic/hydrophobic hydrogel properties. In this way, hydrogels with a wide range of physical properties including water-uptake (100-550%) and compression moduli (7-100 kPa) were obtained. Curcumin release is swelling-controlled and could be extended to 80 days. In vitro, curcumin-derived hydrogels showed selective cytotoxicity against MDA-MB-231 (IC(50) 9 μM) breast cancer cells but no cytotoxicity to noncancerous quiescent human dermal fibroblasts even at high curcumin concentrations (160 μM). One possible application of these curcumin-derived hydrogels is as soft tissue filler after surgical removal of cancerous tissue.

Functionalized nanospheres for targeted delivery of paclitaxel

Targeted delivery of anti-cancer agents to cancer cells is a mature line of investigation that has yet to realize its full potential. In this study we report on the development of a delivery platform with the future goal of merging two thus far parallel methods for selective elimination of cancer cells: targeted nanospheres and pretargeted radioimmunotherapy. Several clinical trials have shown the promise of pretargeted radioimmunotherapy, which leverages the specificity of antibodies for targeted cell populations and delivers a localized dose of a biotinylated radionuclide that is most often administered following binding of a biotinylated antibody and streptavidin (StA) to the target cells. The work presented here describes the development of biotinylated nanospheres based on an ABA-type copolymer comprised of a tyrosine-derived oligomer as the B-block and poly(ethylene glycol) (PEG) A-blocks. The biotinylated nanospheres encapsulate paclitaxel (PTX) to the same extent as unbiotinylated nanospheres. Efficacy of targeting was shown on CD44 positive cells in the SUM159 breast cancer cell line by incubating the cells sequentially with a biotinylated anti-CD44 antibody, StA and the biotinylated nanospheres encapsulating PTX. Targeted nanospheres achieved the half maximal inhibitory concentration of PTX on SUM159 cells at a 5-10 fold lower concentration than that of PTX applied in either non-targeted nanospheres or free drug approaches. Moreover, targeted nanospheres selectively eliminated CD44 positive SUM159 cells compared to free PTX and untargeted nanospheres. This new generation of nano-sized carrier offers a versatile platform that can be adopted for a wide variety of drug and target specific applications and has the potential to be combined with the clinically emerging method of pretargeted radioimmunotherapy.

Poly(ethylene glycol) as a sensitive regulator of cell survival fate on polymeric biomaterials: the interplay of cell adhesion and pro-oxidant signaling mechanisms

Poly(ethylene glycol) (PEG) is one of the most widely used compounds across a variety of platforms and is increasingly found in medical applications. Polycarbonates containing varying mol% of PEG (Mw 1000) were used to probe the effects of PEG on cell adhesion, proliferation, spreading, and survival. Two contrasting PEG-mediated cell signaling elements affected these cellular behaviors: (i) integrin α5 receptor mediated cellular focal adhesions to the biomaterial surface and (ii) modulation of cellular redox and apoptosis through generation of reactive oxygen species (ROS). At lower PEG1k mol% (5% and 8%) cell attachment and spreading decreased concomitantly due to ROS, whereas at the higher PEG1k mol% studied (10% and 20%) an unusual super-adhesive behavior was observed. At higher PEG1k mol% cells exhibited greatly enhanced spreading, which was confirmed through immunolocalization of integrin α5 receptors and enhanced mRNA expression of the integrin α5 gene. These cellular responses on higher PEG1k mol% co-polymers were sufficient to overcome the ROS-driven effects on caspase activation and cell shrinkage, which dominated at lower PEG1k mol%. These studies elucidate PEG-mediated cellular signaling with the implication that the adhesion and apoptotic activity of PEG-rich materials can be sensitively controlled by anti-oxidant addition. Moreover, this study shows that biomaterials can drive the cell fate in opposing directions through concurrent property changes.

Porous and Nonporous Nerve Conduits: The Effects of a Hydrogel Luminal Filler With and Without a Neurite-Promoting Moiety.

Nerve conduits prefilled with hydrogels are frequently explored in an attempt to promote nerve regeneration. This study examines the interplay in vivo between the porosity of the conduit wall and the level of bioactivity of the hydrogel used to fill the conduit. Nerve regeneration in porous (P) or nonporous (NP) conduits that were filled with either collagen only or collagen enhanced with a covalently attached neurite-promoting peptide mimic of the glycan human natural killer cell antigen-1 (m-HNK) were compared in a 5 mm critical size defect in the mouse femoral nerve repair model. Although collagen is a cell-friendly matrix that does not differentiate between neural and nonneural cells, the m-HNK-enhanced collagen specifically promotes axon growth and appropriate motor neuron targeting. In this study, animals treated with NP conduits filled with collagen grafted with m-HNK (CollagenHNK) had the best overall functional recovery, based on a range of histomorphometric observations and parameters of functional recovery. Our data indicate that under some conditions, the use of generally cell friendly fillers such as collagen may limit nerve regeneration. This finding is significant, considering the frequent use of collagen-based hydrogels as fillers of nerve conduits.

Design of barrier coatings on kink-resistant peripheral nerve conduits:

Here, we report on the design of braided peripheral nerve conduits with barrier coatings. Braiding of extruded polymer fibers generates nerve conduits with excellent mechanical properties, high flexibility, and significant kink-resistance. However, braiding also results in variable levels of porosity in the conduit wall, which can lead to the infiltration of fibrous tissue into the interior of the conduit. This problem can be controlled by the application of secondary barrier coatings. Using a critical size defect in a rat sciatic nerve model, the importance of controlling the porosity of the nerve conduit walls was explored. Braided conduits without barrier coatings allowed cellular infiltration that limited nerve recovery. Several types of secondary barrier coatings were tested in animal studies, including (1) electrospinning a layer of polymer fibers onto the surface of the conduit and (2) coating the conduit with a cross-linked hyaluronic acid-based hydrogel. Sixteen weeks after implantation, hyaluronic acid-coated conduits had higher axonal density, displayed higher muscle weight, and better electrophysiological signal recovery than uncoated conduits or conduits having an electrospun layer of polymer fibers. This study indicates that braiding is a promising method of fabrication to improve the mechanical properties of peripheral nerve conduits and demonstrates the need to control the porosity of the conduit wall to optimize functional nerve recovery.

Oligodendrocyte/Type-2 Astrocyte Progenitor Cells and Glial-Restricted Precursor Cells Generate Different Tumor Phenotypes in Response to the Identical Oncogenes

Despite the great interest in identifying the cell-of-origin for different cancers, little knowledge exists regarding the extent to which the specific origin of a tumor contributes to its properties. To directly examine this question, we expressed identical oncogenes in two types of glial progenitor cells, glial-restricted precursor (GRP) cells and oligodendrocyte/type-2 astrocyte progenitor cells (O-2A/OPCs), and in astrocytes of the mouse CNS (either directly purified or generated from GRP cells). In vitro, expression of identical oncogenes in these cells generated populations differing in expression of antigens thought to identify tumor initiating cells, generation of 3D aggregates when grown as adherent cultures, and sensitivity to the chemotherapeutic agent BCNU. In vivo, cells differed in their ability to form tumors, in malignancy and even in the type of host-derived cells infiltrating the tumor mass. Moreover, identical genetic modification of these different cells yielded benign infiltrative astrocytomas, malignant astrocytomas, or tumors with characteristics seen in oligodendrogliomas and small-cell astrocytomas, indicating a contribution of cell-of-origin to the characteristic properties expressed by these different tumors. Our studies also revealed unexpected relationships between the cell-of-origin, differentiation, and the order of oncogene acquisition at different developmental stages in enabling neoplastic growth. These studies thus provide multiple novel demonstrations of the importance of the cell-of-origin in respect to the properties of transformed cells derived from them. In addition, the approaches used enable analysis of the role of cell-of-origin in tumor biology in ways that are not accessible by other more widely used approaches.

High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation

ABSTRACT Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high‐content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer‐mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative “imaging‐derived” parsing index, which reflects the dynamic evolution of the high‐dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions. HIGHLIGHTSHigh‐content analysis of nuclear shape and organization classify stem and progenitor cells poised for distinct lineages.Early oncogenic changes in mesenchymal stem cells (MSCs) are also detected with nuclear descriptors.A new class of cancer‐mitigating biomaterials was identified based on image informatics.Textural metrics of the nuclear structural protein NuMA are sufficient to parse emergent cell phenotypes.

Mutation of ataxia–telangiectasia mutated is associated with dysfunctional glutathione homeostasis in cerebellar astroglia

Astroglial dysfunction plays an important role in neurodegenerative diseases otherwise attributed to neuronal loss of function. Here we focus on the role of astroglia in ataxia–telangiectasia (A–T), a disease caused by mutations in the ataxia–telangiectasia mutated (ATM) gene. A hallmark of A–T pathology is progressive loss of cerebellar neurons, but the mechanisms that impact neuronal survival are unclear. We now provide a possible mechanism by which A–T astroglia affect the survival of cerebellar neurons. As astroglial functions are difficult to study in an in vivo setting, particularly in the cerebellum where these cells are intertwined with the far more numerous neurons, we conducted in vitro coculture experiments that allow for the generation and pharmacological manipulation of purified cell populations. Our analyses revealed that cerebellar astroglia isolated from Atm mutant mice show decreased expression of the cystine/glutamate exchanger subunit xCT, glutathione (GSH) reductase, and glutathione‐S‐transferase. We also found decreased levels of intercellular and secreted GSH in A–T astroglia. Metabolic labeling of l‐cystine, the major precursor for GSH, revealed that a key component of the defect in A–T astroglia is an impaired ability to import this rate‐limiting precursor for the production of GSH. This impairment resulted in suboptimal extracellular GSH supply, which in turn impaired survival of cerebellar neurons. We show that by circumventing the xCT‐dependent import of l‐cystine through addition of N‐acetyl‐l‐cysteine (NAC) as an alternative cysteine source, we were able to restore GSH levels in A–T mutant astroglia providing a possible future avenue for targeted therapeutic intervention. GLIA 2016;64:227–239

A novel mouse model for Ataxia-telangiectasia with a N-terminal mutation displays a behavioral defect and a low incidence of lymphoma but no increased oxidative burden

Ataxia-telangiectasia (A-T) is a rare multi-system disorder caused by mutations in the ATM gene. Significant heterogeneity exists in the underlying genetic mutations and clinical phenotypes. A number of mouse models have been generated that harbor mutations in the distal region of the gene, and a recent study suggests the presence of residual ATM protein in the brain of one such model. These mice recapitulate many of the characteristics of A-T seen in humans, with the notable exception of neurodegeneration. In order to study how an N-terminal mutation affects the disease phenotype, we generated an inducible Atm mutant mouse model (Atm(tm1Mmpl/tm1Mmpl), referred to as A-T [M]) predicted to express only the first 62 amino acids of Atm. Cells derived from A-T [M] mutant mice exhibited reduced cellular proliferation and an altered DNA damage response, but surprisingly, showed no evidence of an oxidative imbalance. Examination of the A-T [M] animals revealed an altered immunophenotype consistent with A-T. In contrast to mice harboring C-terminal Atm mutations that disproportionately develop thymic lymphomas, A-T [M] mice developed lymphoma at a similar rate as human A-T patients. Morphological analyses of A-T [M] cerebella revealed no substantial cellular defects, similar to other models of A-T, although mice display behavioral defects consistent with cerebellar dysfunction. Overall, these results suggest that loss of Atm is not necessarily associated with an oxidized phenotype as has been previously proposed and that loss of ATM protein is not sufficient to induce cerebellar degeneration in mice.