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Now-a-days, more concentration attention has been given for synthesizing of copper (Cu) nanoparticles due to its excellent physical and chemical properties. Different approaches are available to synthesize the Cu nanoparticles. Electrodeposition is an easier and simple approach to synthesize the Cu nanoparticles with high purity. The properties of Cu nanoparticles depend on their synthesis approach and electrolysis process parameters. In this work, response surface methodology (RSM), cuckoo search (CS) algorithm was used to optimize the electrodeposition process parameters like concentration of CuSO4 (4–6 g l−1), electrode potential difference (12–27 V) and electrode distance (3–5 cm) on mean size of Cu nanoparticles. The mean particle size of 21.55 nm was obtained with the electrodeposition parameters of (4 g l−1) CuSO4, (3 cm) electrode distance and (27 V) electrode potential difference. The particle size obtained from the experimental result was 20 nm which was very closer to the predicted result (21.5 nm). The size and morphology of Cu nanoparticle were analyzed by using scanning electron microscope and X-ray diffraction technique.

OBJECTIVEMalignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas that harbor a high potential for metastasis and have a devastating prognosis. Combination chemoradiation aids in tumor control and decreases tumor recurrence but causes deleterious side effects and does not extend long-term survival. An effective treatment with limited toxicity and enhanced efficacy is critical for patients suffering from MPNSTs.METHODSThe authors recently identified that interleukin-13 receptor alpha 2 (IL-13Rα2) is overexpressed on MPNSTs and could serve as a precision-based target for delivery of chemotherapeutic agents. In the work reported here, a recombinant fusion molecule consisting of a mutant human IL-13 targeting moiety and a point mutant variant of Pseudomonas exotoxin A (IL-13.E13 K-PE4E) was utilized to treat MPNST in vitro in cell culture and in an in vivo murine model.RESULTSIL-13.E13 K-PE4E had a potent cytotoxic effect on MPNST cells in vitro. Furthermore, intratumoral administration of IL-13.E13 K-PE4E to orthotopically implanted MPNSTs decreased tumor burden 6-fold and 11-fold in late-stage and early-stage MPNST models, respectively. IL-13.E13 K-PE4E treatment also increased survival by 23 days in the early-stage MPNST model.CONCLUSIONSThe current MPNST treatment paradigm consists of 3 prongs: surgery, chemotherapy, and radiation, none of which, either singly or in combination, are curative or extend survival to a clinically meaningful degree. The results presented here provide the possibility of intratumoral therapy with a potent and highly tumor-specific cytotoxin as a fourth treatment prong with the potential to yield improved outcomes in patients with MPNSTs.

The limited effectiveness of current therapeutics against malignant brain gliomas has led to an urgent need for development of new formulations against these tumors. Chelator Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) presents a promising candidate to defeat gliomas due to its exceptional anti-tumor activity and its unique ability to overcome multidrug resistance. The goal of this study is to develop a targeted nano-carrier for Dp44mT delivery to glioma tumors and to assess its therapeutic efficacy in vitro and in vivo. Dp44mT is loaded into poly(ethylene glycol) (PEG)ylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) decorated with glioma-targeting ligand Interlukin 13 (IL13). IL13-conjugation enhanced the NP uptake by glioma cells and also improved their transport across an in vitro blood-brain-barrier (BBB) model. This targeted formulation showed an outstanding toxicity towards glioma cell lines and patient-derived stem cells in vitro, with IC50 values less than 125 nM, and caused no significant death in healthy brain microvascular endothelial cells. In vivo, when tested on a xenograft mouse model, IL13-conjugated Dp44mT-NPs reduced the glioma tumor growth by ≈62% while their untargeted counterparts reduced the tumor growth by only ≈16%. Notably, this formulation does not cause any significant weight loss or kidney/liver toxicity in mice, demonstrating its great therapeutic potential.

Radiation is utilized in the therapy of more than 50% of cancer patients. Unfortunately, many malignancies become resistant to radiation over time. We investigated the hypothesis that one method of a cancer cell's ability to survive radiation occurs through cellular communication via exosomes. Exosomes are cell-derived vesicles containing DNA, RNA, and protein. Three properties were analyzed: 1) exosome function, 2) exosome profile and 3) exosome uptake/blockade. To analyze exosome function, we show radiation-derived exosomes increased proliferation and enabled recipient cancer cells to survive radiation in vitro. Furthermore, radiation-derived exosomes increased tumor burden and decreased survival in an in vivo model. To address the mechanism underlying the alterations by exosomes in recipient cells, we obtained a profile of radiation-derived exosomes that showed expression changes favoring a resistant/proliferative profile. Radiation-derived exosomes contain elevated oncogenic miR-889, oncogenic mRNAs, and proteins of the proteasome pathway, Notch, Jak-STAT, and cell cycle pathways. Radiation-derived exosomes contain decreased levels of tumor-suppressive miR-516, miR-365, and multiple tumor-suppressive mRNAs. Ingenuity pathway analysis revealed the most represented networks included cell cycle, growth/survival. Upregulation of DNM2 correlated with increased exosome uptake. To analyze the property of exosome blockade, heparin and simvastatin were used to inhibit uptake of exosomes in recipient cells resulting in inhibited induction of proliferation and cellular survival. Because these agents have shown some success as cancer therapies, our data suggest their mechanism of action could be limiting exosome communication between cells. The results of our study identify a novel exosome-based mechanism that may underlie a cancer cell's ability to survive radiation.

Glioblastoma (GBM) is both the most common and deadly malignant primary brain tumor with a 1-year survival of 37.2% and a 5-year survival of just 5.1%. Despite advances in imaging technology, distinguishing between pseudoprogression and pseudoresponse remains problematic and there is a dire need for additional diagnostic methods. Tumor-derived extracellular vesicles are found in a variety of biofluids and are good candidates for a liquid biopsy. A unique characteristic of GBM is the overexpression of the α2 variant of IL-13 receptor (IL13Rα2) in 78-96% of patients while virtually undetectable in normal brain tissue. We hypothesize that biofluids of patients with GBM contains sufficient IL13Rα2-positive extracellular vesicles (EV) to inform the clinical picture regarding disease status such as tumor recurrence and response to therapy. We have measured the levels of IL13Rα2 in the EV fraction of plasma from GBM patients and correlated these levels to patient survival, and patient clinical characteristics (n=35). We have demonstrated that the EV fraction of some patients with GBM is enriched for the IL13Rα2 receptor. The plasma level of IL13Rα2 could also predict the level of IL13Rα2 in the GBM tumor (n=9). Further, IL13Rα2 positive EVs from patient-derived GBM cell lines could be immunoprecipitated for further interrogation of genomic and proteomic molecular markers. These exciting findings lay the groundwork for a GBM liquid biopsy based on tumor-specific EVs in patient biofluids.

Cadmium selenide (CdSe) based quantum dots modified with polyethylene glycol and chemically linked to interleukin-13 (IL13) were prepared with the aim of identifying the high affinity receptor (IL13Rα2) which is expressed in glioma stem cells and exosomes secreted by these cancer stem cells. IL13 conjugated quantum dots (IL13QD) were thoroughly characterized for their physicochemical properties including particle size and surface morphology. Furthermore, the specific binding of the IL13QD to glioma cells and to glioma stem cells (GSC) was verified using a competitive binding study. The exosomes were isolated from the GSC conditioned medium and the expression of IL13Rα2 in the GSC and exosomes was verified. The binding property of IL13QD to the tumor associated exosomes was initially confirmed by transmission electron microscopy. The force of attraction between the quantum dots and U251 glioma cells and the exosomes was investigated by atomic force microscopy, which indicated a higher force of binding interaction between the IL13QD and IL13Rα2 expressing glioma cells and exosomes secreted by glioma stem cells. Flow cytometry of the IL13QD and exosomes from the culture media and cerebrospinal fluid (CSF) of patients with glioma tumors indicated a distinctly populated complex pattern different from that of non-targeted quantum dots and bovine serum albumin (BSA) conjugated quantum dots confirming specific binding potential of the IL13QD to the tumor associated exosomes. The results of this study demonstrate that IL13QD can serve as an ex vivo marker for glioma stem cells and exosomes that can inform diagnosis and prognosis of patients harboring malignant disease. Statement of Significance Functionalized quantum dots are flexible semiconductor nanomaterials which have an immense application in biomedical research. In particular, when they are functionalized with biomolecules like proteins or antibodies, they have the specialized ability to detect the expression of receptors and antigens in cells and tissues. In this study we designed a cytokine (interleukin-13) functionalized quantum dot to detect a cancer associated receptor expressed in cancer stem cells and the extracellular vesicles (exosomes) secreted by the cancer cells themselves. The binding pattern of these cytokine modified quantum dots to the cancer stem cells and exosomes alters the physical properties of the complex in the fixed and suspended form. This altered binding pattern can be monitored by a variety of techniques, including transmission electron microscopy, atomic force microscopy and flow cytometry, and subsequent characterization of this quantum dot binding profile provides useful data that can be utilized as a fingerprint to detect cancer disease progression. This type of functionalized quantum dot fingerprint is especially useful for invasive cancers including brain and other metastatic cancers and may allow for earlier detection of disease progression or recurrence, thus saving the lives of patients suffering from this devastating disease.

Neuroblastoma is the third most common childhood cancer, and timely diagnosis and sensitive therapeutic monitoring remain major challenges. Tumor progression and recurrence is common with little understanding of mechanisms. A major recent focus in cancer biology is the impact of exosomes on metastatic behavior and the tumor microenvironment. Exosomes have been demonstrated to contribute to the oncogenic effect on the surrounding tumor environment and also mediate resistance to therapy. The effect of genotype on exosomal phenotype has not yet been explored. We interrogated exosomes from human neuroblastoma cells that express wild-type or mutant forms of the HFE gene. HFE, one of the most common autosomal recessive polymorphisms in the Caucasian population, originally associated with hemochromatosis, has also been associated with increased tumor burden, therapeutic resistance boost, and negative impact on patient survival. Herein, we demonstrate that changes in genotype cause major differences in the molecular and functional properties of exosomes; specifically, HFE mutant derived exosomes have increased expression of proteins relating to invasion, angiogenesis, and cancer therapeutic resistance. HFE mutant derived exosomes were also shown to transfer this cargo to recipient cells and cause an increased oncogenic functionality in those recipient cells.

Glioblastoma (GBM) is both the most common and deadly malignant primary brain tumor with a 1-year survival of 37.2% and a 5-year survival of just 5.1%. The standard of care for GBM is surgical excision followed by radiation with concurrent and adjuvant temozolomide-centered chemotherapy (TMZ). Despite advances in imaging technology, early identification of treatment failure or impending resistance remains problematic. A unique characteristic of GBM is the overexpression of the α2 variant of IL-13 receptor (IL13Rα2) in 78–96% of patients while virtually undetectable in normal brain tissue. We hypothesize that the blood of patients with GBM contains sufficient IL13Rα2-positive extracellular vesicles (EV) to inform the clinical picture regarding disease status such as tumor recurrence and response to therapy. To detect IL13Rα2 as a specific molecular target for diagnostic testing, we have developed a modified version of IL-13 with increased affinity and specificity for IL13Rα2, and conjugated this ligand to fluorescent/ magnetic nanoparticles using an 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) linker. Furthermore, we demonstrated that the EV plasma fraction of patients with GBM is enriched for the IL13Rα2 receptor. We also demonstrated the ability of the ligand-bound nanoparticles to aggregate with IL13Rα2-positive cells and EVs in vitro. This ligand-based affinity purification particle has the potential to both quantify IL13Rα2 EV levels in patient blood and isolate these EVs for further interrogation of genomic and proteomic molecular markers.

Background Detection of glioma with MRI contrast agent is limited to cases in which the blood-brain barrier (BBB) is compromised as contrast agents cannot cross the BBB. Thus, an early-stage infiltrating tumor is not detectable. Interleukin-13 receptor alpha 2 (IL-13Rα2), which has been shown to be overexpressed in glioma, can be used as a target moiety. We hypothesized that liposomes conjugated with IL-13 and encapsulating MRI contrast agent are capable of passing through an intact BBB and producing MRI contrast with greater sensitivity. Methods The targeted MRI contrast agent was created by encapsulating Magnevist (Gd-DTPA) into liposomes conjugated with IL-13 and characterized by particle size distribution, cytotoxicity, and MRI relaxivity. MR image intensity was evaluated in the brain in normal mice post injection of Gd-DTPA and IL-13-liposome-Gd-DTPA one day apart. The specificity for glioma detection by IL-13-liposome-Gd-DTPA was demonstrated in an intracranial glioma mouse model and validated histologically. Results The average size of IL-13-liposome-Gd-DTPA was 137 ± 43 nm with relaxivity of 4.0 ± 0.4 L/mmole-s at 7 Tesla. No significant cytotoxicity was observed with MTS assay and serum chemistry in mice. The MRI signal intensity was enhanced up to 15% post injection of IL-13-liposome-Gd-DTPA in normal brain tissue following a similar time course as that for the pituitary gland outside of the BBB. MRI enhanced by IL-13-liposome-Gd-DTPA detected small tumor masses in addition to those seen with Magnevist-enhanced MRI. Conclusions IL-13-liposome-Gd-DTPA is able to pass through the uncompromised BBB and detect an early stage glioma that cannot be seen with conventional contrast-enhanced MRI.

OBJECTIVEMalignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas arising from peripheral nerves. MPNSTs have increased expression of the oncogene aurora kinase A, leading to enhanced cellular proliferation. This makes them extremely aggressive with high potential for metastasis and a devastating prognosis; 5-year survival estimates range from a dismal 15% to 60%. MPNSTs are currently treated with resection (sometimes requiring limb amputation) in combination with chemoradiation, both of which demonstrate limited effectiveness. The authors present the results of immunohistochemical, in vitro, and in vivo analyses of MLN8237 for the treatment of MPNSTs in an orthoxenograft murine model.METHODSImmunohistochemistry was performed on tumor sections to confirm the increased expression of aurora kinase A. Cytotoxicity analysis was then performed on an MPNST cell line (STS26T) to assess the efficacy of MLN8237 in vitro. A murine orthoxenograft MPNST model transfected to express luciferase was then developed to assess the efficacy of aurora kinase A inhibition in the treatment of MPNSTs in vivo. Mice with confirmed tumor on in vivo imaging were divided into 3 groups: 1) controls, 2) mice treated with MLN8237, and 3) mice treated with doxorubicin/ifosfamide. Treatment was carried out for 32 days, with imaging performed at weekly intervals until postinjection day 42. Average bioluminescence among groups was compared at weekly intervals using 1-way ANOVA. A survival analysis was performed using Kaplan-Meier curves.RESULTSImmunohistochemical analysis showed robust expression of aurora kinase A in tumor cells. Cytotoxicity analysis revealed STS26T susceptibility to MLN8237 in vitro. The group receiving treatment with MLN8237 showed a statistically significant difference in tumor size compared with the control group starting at postinjection day 21 and persisting until the end of the study. The MLN8237 group also showed decreased tumor size compared with the doxorubicin/ifosfamide group at the conclusion of the study (p = 0.036). Survival analysis revealed a significantly increased median survival in the MLN8237 group (83 days) compared with both the control (64 days) and doxorubicin/ifosfamide (67 days) groups. A hazard ratio comparing the 2 treatment groups showed a decreased hazard rate in the MLN8237 group compared with the doxorubicin/ifosfamide group (HR 2.945; p = 0.0134).CONCLUSIONSThe results of this study demonstrate that MLN8237 is superior to combination treatment with doxorubicin/ifosfamide in a preclinical orthoxenograft murine model. These data have major implications for the future of MPNST research by providing a robust murine model as well as providing evidence that MLN8237 may be an effective treatment for MPNSTs.

Glioblastoma (GBM) is the most lethal and incurable brain cancer. Despite maximum treatment, high recurrence rates result in median survival of less than 2 years. A subset of treatment resistant cells within GBM known as glioma initiating cells (GICs) are implicated in recurrence and exhibit ferritin overexpression. High expression of ferritin is known to be associated with poor overall survival in GBM patients. Our previous finding showed that downregulation of the ferritin heavy chain subunit (FTH1) enhanced treatment sensitivity in astrocytoma cells. Recently, functional importance of FTH1 in GICs was demonstrated by shRNA silencing leading to ablation of tumorigenic ability. We therefore posited that disrupting iron metabolism through FTH1 knockdown would radiosensitize GICs. Thus we developed multivalent cationic liposomes that enabled delivery of FTH1 siRNA to patient derived GICs isolated from pro-neural (PN) and mesenchymal (Mes) GBM. In both subtypes, FTH1 loss led to increased apoptosis and reduced cell viability. When exposed to radiation, PN GICs were unable to repair and recover from DNA damage, leading to a further decrease in cell viability and a complete loss of reproductive clonogenic potential unlike their more radioresistant Mes counterparts. Moreover, expression of stem cell marker Nestin was markedly reduced in PN but not Mes GICs. Thus, FTH1 loss is detrimental to both GIC subtypes, it seems to be critical for maintaining stemness and radiation resistance of PN GICs. These findings further support the viability of FTH1 as a therapeutic target in GBM.

Elevated expression of the iron regulatory protein, ferritin heavy chain 1 (FTH1), is increasingly being associated with high tumor grade and poor survival outcomes in glioblastoma. Glioma initiating cells (GICs), a small population of stem-like cells implicated in therapeutic resistance and glioblastoma recurrence, have recently been shown to exhibit increased FTH1 expression. We previously demonstrated that FTH1 knockdown enhanced therapeutic sensitivity in an astrocytoma cell line. Therefore, in this study we developed a liposomal formulation to enable the in vitro delivery of FTH1 siRNA in patient xenograft derived GICs from glioblastomas with pro-neural and mesenchymal transcriptional signatures to interrogate the effect of FTH1 downregulation on their radiation sensitivity. Transfection with siRNA decreased FTH1 expression significantly in both GICs. However, there were inherent differences in transfectability between pro-neural and mesenchymal tumor derived GICs, leading us to modify siRNA: liposome ratios for comparable transfection. Moreover, loss of FTH1 expression resulted in increased extracellular lactate dehydrogenase activity, executioner caspase 3/7 induction, substantial mitochondrial damage, diminished mitochondrial mass and reduced cell viability. However, only GICs from pro-neural glioblastoma showed marked increase in radiosensitivity upon FTH1 downregulation demonstrated by decreased cell viability, impaired DNA repair and reduced colony formation subsequent to radiation. In addition, the stemness marker Nestin was downregulated upon FTH1 silencing only in GICs of pro-neural but not mesenchymal origin. Using liposomes as a siRNA delivery system, we established FTH1 as a critical factor for survival in both GIC subtypes as well as a regulator of radioresistance and stemness in pro-neural tumor derived GICs. Our study provides further evidence to support the role of FTH1 as a promising target in glioblastoma.

Ginsenoside Rg3 is a natural active ingredient that is extracted from Korean red ginseng root. It elevates the therapeutic effect of radiotherapy and chemotherapy, but previous studies found that the application of Rg3 is heavily limited by its low bioavailability and poor absorption via oral administration. To overcome these problems, Rg3-loaded PEG-PLGA-NPs (Rg3-NPs) were prepared by the modified spontaneous emulsification solvent diffusion (SESD) method, and the physicochemical characteristics of Rg3-NPs were investigated. We treated primary glioblastoma with 50 mM Rg3-NPs for 48h. We then used gene expression arrays (Illumina) for genome-wide expression analysis and validated the results for genes of interest by means of real-time PCR. Functional annotations were then performed using the DAVID and KEGG online tools. The results showed that the Rg3-NPs are slick and uniform, the average diameter of the nanoparticles is 75-90 nm, and their entrapment efficiency is 89.7+1.7%. MTT showed that the growth of cells can be significantly inhibited by Rg3-NPs in a dose-dependent manner. FCM testing showed Rg3-NPs can be released from the conjugate nanoparticle and react with the genes in the cell nuclei, causing changes in the gene molecules. We also found that cancer cells treated with Rg3-NPs undergo cell-cycle arrest at different checkpoints. This arrest was associated with a decrease in the mRNA levels of core regulatory genes BUB1, CDC20, TTK, and CENPE, as determined by microarray analysis and verified by real-time PCR. Furthermore, Rg3-NPs induced the expression of the apoptotic and antimigratory protein p53 in cell lines. The results of the present study, together with the results of earlier studies, show that Rg3-NPs target genes involved in theprogressionoftheM-phaseofthecellcycle.Itisassociatedwithseveralimportant pathways, which include apoptosis (p53). Rg3-NPs may be a potent cell-cycle regulation drug targeting the M-phase in glioblastoma cell lines.

Activation of microglia is a histological feature observed in neurodegenerative diseases like ALS. The oral administration of minocycline has been demonstrated to have minimal neuroprotection ability in the animal models and is also associated with inadvertent toxicity due to non-specific oral absorption of the drug. Nonetheless, the drug itself shows promise in a number of disease models suggesting it could be effective if delivered optimally. Thus, we utilized LPS modified liposomes to target TLR4 receptor on the microglia in SOD1G93A mice and compared its efficacy with non- targeted nanoliposomes. The in vitro results indicate that targeting the TLR4 receptor on microglia significantly increases (p G93A mouse model of ALS, targeted and non-targeted minocycline treatment significantly increased (p G93A mice, the non- targeted nanovesicles significantly increased the latency to rotarod failure and both targeted and non-targeted nanovesicles significantly delayed disease endpoints.

The 16th Annual Scientific Meeting of the Society for Neuro-Oncology in Conjunction with the AANS/CNS Section on Tumors, Orange County, California, USA, 17-20 November 2011. In Neuro-Oncology, 2011, v. 13 n. Suppl. 3, p. iii111, abstract no. ET-18

Approximately half of all gliomas are resistant to chemotherapy, and new therapeutic strategies are urgently needed to treat this cancer. We hypothesized that disrupting iron homeostasis in glioma cells could block tumor growth, based on an acute requirement for high levels of iron to meet energy requirements associated with their rapid growth. Ferritin is best known as an intracellular iron storage protein, but it also localizes to tumor cell nuclei where it seems to protect DNA from oxidative damage and to promote transcription. In this study, we hypothesize that silencing the H-ferritin (heavy chain ferritin) gene could increase tumor sensitivity to chemotoxins. To test this hypothesis, H-ferritin siRNA was delivered to several human cancer cell lines by using cationic liposomes (C-liposome). H-ferritin siRNA decreased protein expression by 80% within 48 hours, and this decrease was associated with more than 50% decrease in the LD50 for DNA-alkylating agent carmustine (BCNU), which is commonly used to treat glioma in clinic. In a subcutaneous mouse model of human glioma, intratumoral injections of liposomes containing H-ferritin siRNA reduced the effective dose of BCNU needed for tumor suppression by more than 50%. A plasmid supercoil relaxation assay showed that H-ferritin specifically and directly protected DNA from BCNU treatment. H-ferritin siRNA additionally seemed to increase apoptosis in glioma cells in vitro upon H-ferritin knockdown. Overall, our results illustrate how silencing H-ferritin can effectively sensitize tumors to chemotherapy and also show the ability of C-liposomes to serve as a novel in vivo delivery tool for siRNAs. Cancer Res; 71(6); 2240–9. ©2011 AACR.

Glioblastoma (GBM) is the most common primary central nervous system (CNS) tumor in the United States and yet despite aggressive treatment, the median survival remains at 8 months. About 75% of human glioblastoma tumors selectively overexpress the α(2) subtype of interleukin 13 (IL-13Rα(2)), considered a “decoy receptor” for GBM, allowing it to evade apoptosis normally induced by IL-13 binding. Our laboratory and others have developed targeting strategies to take advantage of IL-13Rα(2) expression on GBMs. We showed that wildtype IL-13 targeted nanoliposomes delivering doxorubicin (WT-IL13-Dox), resulted in over 5-fold reduction of orthotopic tumors, with 60% of mice surviving for >200 days compared to mice treated with unconjugated liposomes. WT-IL13, however, also binds to the shared IL-13/IL-4 receptor α(1) subunit found in multiple organs, including the heart and lungs, suggesting potential cross-reactivity with surrounding tissues. To address off-target effects, a mutant version of IL-13 was developed known as Targeted Quadruple Mutant-13 (TQM), which display improved binding affinity to the IL-13Rα(2) receptor, and decreased affinity to the IL-13/IL-4 receptor α(1) subunit. Twenty mice were normalized by tumor burden into two groups, one treated with WT-IL13-Dox and the other group with TQM-13-conjugated liposomal doxorubicin (TQM-13-Lip-Dox). Each group received 5mg/kg of doxorubicin in the liposomes for 4 weeks. Mice treated with TQM-targeted liposomes had slower tumor growth, smaller tumor burden, and prolonged survival (33 vs 23 days, p=0.009) than those treated with WT-IL13 liposomes. WBC counts in WT-IL13 mice suggested they were immunocompromised in comparison to TQM mice (p=0.02), which may contribute to worse survival. These findings suggest that TQM-13 bound nanoliposomes may enable the development of targeted therapy with a decreased side effect profile for delivery of chemotherapeutic agents to GBMs.

Glioblastoma is the most common central nervous system malignancy in adults with a median survival of only 14 months. The current arsenal of treatment modalities including surgical resection and combination chemoradiation have been largely ineffective. One reason proposed for the ineffectiveness of our current therapeutic regimen is the resistance of glioma stem cells (GSCs) within the tumor and tumor borders. We investigated the hypothesis that the communication of GSCs to their microenvironment through exosomes is a key factor underlying the enhanced cellular proliferation and the development of resistance to therapeutics. Exosomes are nanometer sized vesicles released by cancer cells that contain DNA, RNA, and protein critical to the interaction of a cell with its microenvironment. Two properties of exosomes were analyzed: 1) exosome function and 2) exosome profile. Exosomes secreted by patient derived-glioma stem cells (GSC-exosomes) increased cellular proliferation, radiation resistance, temozolomide resistance, and doxorubicin resistance. We further profiled the GSC-exosomes to begin to probe the underlying mechanism of this phenomenon. Profiling showed specific changes to RNA and protein favoring therapeutic resistance and cellular proliferation. For example, GSC exosomes have increased expression of proteins involved in radiation and chemotherapeutic resistance (Ex. CDK4 and Notch), cellular proliferation (Ex. Cyclin B1 and Cyclin D2), angiogenesis (Ex. VEGF-A and EGFR), glioma cell stemness and de-differentiation (Ex. EPHA2, Cathepsin B), and cell invasion and metastasis (Ex. ITGA3, COL4A2). The results of our study suggest a novel exosome-based mechanism by which glioma stem cells alter therapeutic resistance and increase cellular proliferation. TUMOR MODELS

We documented that alpha-helices A, C, and D in human interleukin-13 (IL13) participate in interaction with its respective receptors. We hypothesized that alpha-helix D is the site II of the cytokine that binds IL13Ralpha1, a component of the normal tissue heterodimeric signaling IL13/4 receptor (IL13/4R), and that alpha-helix D independently binds a monomeric IL13Ralpha2 receptor, which is a non-signaling glioma-restricted receptor for IL13. Therefore, we alanine-scanned mutagenized helix D of IL13 to identify the residues involved in the respective receptors interaction. Recombinant muteins of IL13 were produced in Escherichia coli, and their structural integrity and identity were verified. The alanine mutants were tested in functional cellular assays, in which IL13 interaction with IL13Ralpha2 (glioma cells) or an ability to functionally stimulate IL13/4R (TF-1 cells) were examined, and also in binding assays. We found that residues 105, 106, and 109 of the d-helix of IL13 are responsible for interacting with the glioma-associated receptor. Moreover, glutamic acids at positions 92 and 110, and leucine at position 104 was found to be important for IL13/4R stimulation. Thus, alpha-helix D of IL13 is the primary site responsible for interaction with the IL13 binding proteins. We propose a model that illustrates the binding mode of IL13 with cancer-related IL13Ralpha2 and physiological IL13/4R.

Glioblastoma multiforme (GBM) is the most prevalent, lethal, malignant and refractory primary brain tumor with a very dismal survival rate. The glioma initiating cell (GIC) population within GBMs has been associated with treatment resistance and subsequently been implicated in tumor recurrence. Studies from our lab had previously shown that expression of the heavy chain subunit of the iron storage protein ferritin, H-ferritin, is essential for the survival and therapeutic resistance of non-stem glioma cells. Since H-ferritin is overexpressed in GICs, we hypothesized that downregulating its expression in these cells would lead to increased radiosensitivity. We therefore developed a novel strategy to sensitize these recalcitrant GICs to radiation therapy using a multivalent cationic liposome formulation that could efficiently transfect and deliver H-ferritin siRNA to GICs in vitro. Using patient derived T3691 CD133+ GICs we showed that downregulating H-ferritin led to a reduction in cell viability compared to controls upon being radiated at 4Gy and 8Gy dosage. Additionally these cells also showed an increased expression of the DNA damage response protein phospho-ϒH2AX upon radiation over controls. Thus, we have demonstrated that loss of H-ferritin in GICs leads to increased radiation sensitivity. Ongoing studies are focused on delineating the mechanisms through which H-ferritin loss mediates this effect in GICs.

Malignant peripheral nerve sheath tumor is a soft tissue sarcoma that poses tremendous challenges for effective therapy. Several therapeutic approaches using drugs such as doxorubicin and rapamycin have been tried in the past with minimum success. Therapeutic resistant property mediated by MDR proteins, in combination with DNA damage repair was proposed as a major cause leading to therapeutic inefficacy of chemotherapeutics in MPNST tumors. To overcome this issue, we encapsulated doxorubicin in lipid nanovesicles and targeted to IL13Rα2, a receptor that is selectively overexpressed in several malignant peripheral nerve sheath tumor cell lines. In the present investigation, a MPNST mouse model was developed using STS26T-Luc cell line stably transfected with luciferase reporter gene. These cells were implanted into the sciatic nerve that could be imaged by Intravital Imaging Spectroscopy (IVIS). After confirming the tumor formation, the mice were injected with 7 mg/kg body weight of (a) IL13 linked liposomal doxorubicin (IL3LIPDXR) (b) unconjugated liposomal doxorubicin (LIPDXR) and (c) control mice injected with equal volume of saline. The mice were treated weekly once for 7 weeks. The mice which received IL13LIPDXR and LIPDXR showed significant reduction in tumor volume compared to the control group. Among the targeted and non-targeted liposomes, IL13LIPDXR showed rapid tumor regression. However, beyond 4 weeks, the drug was ineffective in shrinking or maintaining the tumor volume. This concludes that although the targeted delivery of doxorubicin, seems to be effective initially however in the later course of treatment they are ineffective in controlling the tumor progression. Approaches such as combining doxorubicin with a small molecule Ras inhibitor or other agents in the liposome formulation which are currently underway may help to improve the therapeutic efficacy.

Glioblastoma Multiforme (GBM) is the most common CNS malignancy in adults, and even with multimodal therapies, carries a poor prognosis. These tumors are heterogeneous and modify their environment, promoting tumor growth, invasion, and therapeutic resistance. The intratumoral heterogeneity of GBM is accentuated during treatment. Even in the face of surgical resection and adjuvant chemoradiation, the unfortunate but inevitable recurrence and progression of GBM is nearly universal. This mechanism of acquired radiation resistance is not understood. We believe that acquired resistance of GBM is due to exosomes secreted from radiated GBM cells conferring resistance to the local cell population. Exosomes are nanometer cell-derived vesicles containing RNAs and proteins. Growing evidence demonstrates exosomes are instrumental to a cell’s interaction with its microenvironment, and have the ability to transfer information to recipient cells contributing to tumorigenicity. We utilized nanoparticle tracking analysis to analyze characteristics of exosome secretion after exposure to radiation. We used cell proliferation assays and cell survival assays to interrogate radiation derived exosome effects on the local cancer cell population. We assessed the potential of exosome blocking agents as a possible avenue for therapeutic manipulation to abrogate this oncogenic effect. We demonstrate that resistance and proliferative factors in GBM derived exosomes change after radiation exposure. Moreover, radiation derived exosomes have functional impact on tumor cell proliferation and radiation resistance. We also demonstrate the therapeutic potential of exosome blocking agents to inhibit acquired resistance, which develops in nearly all GBM patients. Radiation-derived exosomes cause increases in cellular proliferation and survival in the local cancer cell population and may be the hidden mediators of an ultimately radiation resistant tumor recurrence and the dire prognosis that follows. Adjuvant therapy with exosome blocking agents may be used to inhibit these oncogenic effects, leading to optimal patient outcomes.

High grade brain tumors like glioblastoma multiforme (GBM) and certain other brain metastatic cancers possess high tumor invasive and infiltrating properties making them harder to detect. Identification of cancer stem cells and exosomes secreted by them in the biofluids like cerebrospinal fluid (CSF) will be an ideal way of detecting the presence of residual cancer after therapy. In the current approach, a ligand conjugated quantum dot (QD) was utilized to specifically label the cancer stem cells and exosomes to identify the presence of IL13Rα2 a tumor invasive marker. Human glioma initiating cells were cultured in adherent form and were exposed to interleukin-13 conjugated quantum dots (IL13QD), which binds the glioma initiating cells expressing IL13Rα2 receptor. Similarly the exosomes isolated from cancer stem cell condition media and CSF was briefly incubated with IL13QD, before analyzing their complexing pattern. Exosomes that were isolated from glioma stem cells and patients CSF were demonstrated to express IL13Rα2 receptor. Fluorescent microscopy, transmission electron microscopy and flow cytometry were performed on the exosomes after complexing with IL13QD. IL13QD was demonstrated to specifically bind glioma stem cells both in the monolayer and spheroid culture. The binding affinity of the exosomes to the quantum dots were quantitatively and qualitatively confirmed by atomic force microscopy (AFM). The morphology and size of exosomes before and after complexing with exosomes were confirmed by electron microscopy and AFM. The density plot from the flow cytometry experiments with IL13QD-exosomes complex indicates a possibility to identify the tumor associated exosomes. Our experiment infers that tumor targeted quantum dots can selectively bind with glioma stem cells and extracellular vesicles (exosomes) that are secreted by cancer cells. This binding profile could be utilized to identify the expression of certain tumor promoting receptors and antigens on the surface of exosomes present in the CSF and cancer stem cells from the glioma patients. Experiments are in progress to identify the tumor initiating cells in an orthotopic mouse glioma tumor model after intravenous administration of wild type IL13 and certain high affinity mutated IL13 conjugated quantum dots, by non-invasive way. Citation Format: Achuthamangalam B. Madhankumar, Oliver Mrowczynski, Brad Zacharia, Michael Glantz, Lichong Xu, Becky Webb, Christopher Siedlecki, Akiva Mintz, James R. Connor. Interleukin-13 conjugated quantum dots to identify glioma initiating cells and exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 745. doi:10.1158/1538-7445.AM2017-745

Human glioblastoma tumors selectively express receptors for interleukin 13 (IL-13). In a previous study, we showed that liposomes, when conjugated with IL-13, will deliver chemotherapeutics to a subcutaneous glioma tumor model in mice much more effectively than conventional unconjugated liposomes. Based on this observation, we developed an intracranial brain tumor model in nude mice using human U87 glioma cells. Mice receiving weekly i.p. injections of 15 mg/kg of doxorubicin encapsulated in IL-13–conjugated liposomes had a 5-fold reduction in the intracranial tumor volume over 6 weeks and four of seven animals survived >200 days after tumor implantation. In contrast, the animals receiving unconjugated liposomes with the same doxorubicin concentration did not survive beyond 35 days and there was no evidence of tumor size reduction. The presence of liposomes with doxorubicin in the tumor was shown by taking advantage of the selective expression of IL-13 receptors on the tumor cells and the endogenous fluorescence of doxorubicin. There was no increase in the indices of toxicity in animals receiving the doxorubicin-containing liposomes. Finally, a model of the blood-brain barrier was used to show that the nanovesicles do not harm the endothelial cells yet maintain their toxicity to astrocytoma cells. This approach is necessary to show the efficacy of this targeting platform for tumors in which the blood-brain barrier is not compromised and as a potential use of the nanovesicle system as a surveillance mechanism to prevent recurrence. These data show that IL-13 targeted nanovesicles are a viable option for the treatment of brain tumors. [Mol Cancer Ther 2009;8(3):648–54]

High-grade astrocytoma (HGA) is an invariably fatal malignancy with a mean survival of 14 months despite surgery, radiation, and chemotherapy. We have found that a restricted receptor for interleukin-13 (IL-13), IL-13 receptor alpha 2 (IL13Ralpha2), is abundantly overexpressed in the vast majority of HGAs but is not appreciably expressed in normal tissue, with the exception of the testes. Therefore, IL-13Ralpha2 is a very attractive target for anti-HGA immunotherapy. In order to test protein and genetic vaccines that target IL13Ralpha2, we developed a G26-IL13Ralpha2-expressing syngeneic immunocompetent murine glioma model. Using this glioma model, mice were immunized with recombinant extracellular IL13Ralpha2 protein (IL13Ralpha2ex) or a DNA expression vector containing the gene for IL13Ralpha2 and were subsequently challenged with IL13Ralpha2( + ) G26 tumors. Mice immunized with either recombinant or genetic IL13Ralpha2, but not mock-immunized controls, demonstrated complete protection against IL13Ralpha2( + ) glioma growth and mortality. Of interest, only the recombinant-protein-based vaccines generated detectable anti-IL13Ralpha2 antibodies. These studies demonstrate the in vivo efficiency of protein- and DNA-based immunotherapy strategies that target IL13Ralpha2 that may play a clinical role to eradicate the residual microscopic HGA cells that inevitably cause disease recurrence and mortality.

Traditionally, transferrin has been considered the primary mechanism for cellular iron delivery, despite suggestive evidence for additional iron delivery mechanisms. In this study we examined ferritin, considered an iron storage protein, as a possible delivery protein. Ferritin consists of H- and L-subunits, and we demonstrated iron uptake by ferritin into multiple organs and that the uptake of iron is greater when the iron is delivered via H-ferritin compared with L-ferritin. The delivery of iron via H-ferritin but not L-ferritin was significantly decreased in mice with compromised iron storage compared with control, indicating that a feedback mechanism exists for H-ferritin iron delivery. To further evaluate the mechanism of ferritin iron delivery into the brain, we used a cell culture model of the blood-brain barrier to demonstrate that ferritin is transported across endothelial cells. There are receptors that prefer H-ferritin on the endothelial cells in culture and on rat brain microvasculature. These studies identify H-ferritin as an iron transport protein and suggest the presence of an H-ferritin receptor for mediating iron delivery. The relative amount of iron that could be delivered via H-ferritin could make this protein a predominant player in cellular iron delivery.

The difficulties associated with treatment of malignant brain tumors are well documented. For example, local infiltration of high-grade astrocytomas prevents the complete resection of all malignant cells. It is, therefore, critical to develop delivery systems for chemotherapeutic agents that ablate individual cancer cells without causing diffuse damage to surrounding brain tissue. Here, we describe sterically stable human interleukin-13 (IL-13)–conjugated liposomes, which efficiently bind to the brain cancer cells that overexpress the IL-13 receptor α2 protein. The conjugated liposomes bind to glioblastoma multiforme tissue specimens but not to normal cortex. Conjugating the liposomes with human IL-13 allows for specific binding to glioma cells and uptake of the liposomes via endocytosis. Delivering doxorubicin to glioma cells by IL-13–conjugated liposomes results in enhanced cytotoxicity and increased accumulation and retention of drug in the glioma cells compared with delivery of free drug. The therapeutic potential and targeting efficacy of the IL-13–conjugated liposomes carrying doxorubicin was tested in vivo using a s.c. glioma tumor mouse model. Animals receiving i.p. injections of IL-13–conjugated liposomes carrying doxorubicin for 7 weeks had a mean tumor volume of 37 mm3 compared with a mean volume of 192 mm3 in animals injected with nontargeted liposomes. These results strongly suggest that IL-13–conjugated liposomes carrying cytotoxic agents are a feasible approach for creating a nanovesicle drug delivery system for brain tumor therapy. [Mol Cancer Ther 2006;5(12):3162–9]

Gliomas, especially glioblastoma multiforme are resistant to radiation and chemotherapy. Therefore, efforts to develop new course of therapy to enhance efficacy of treatment strategies are obviously critical. We hypothesized that disrupting iron homeostasis in malignant tumor cells could prove effective at negatively affecting tumor growth because cancer cells have a robust iron requirement consistent with their rapid growth and high metabolic rates. The high levels of iron uptake require a mechanism to limit iron induced oxidative damage. This function is the responsibility of ferritin; an iron storage protein with 24 subunits of two types of chains light and heave chains. Heavy chain ferritin (H-ferritin) has been found in the nucleus of tumor cells where it appears to protect DNA from oxidative damage and promote transcription. We hypothesized that silencing the H-ferritin gene could increase the sensitivity of tumors cells to radiation. On the other hand, our previous data already demonstrated that silencing the H-ferritin gene increased chemotherapeutic efficacy for treating gliomas. To test our present hypothesis, the H-ferritin gene will be silenced via a cationic liposome carrying siRNA. We have chosen glioma cell lines (U251, U87 and MNSF) as our in vitro model. Subcutaneous glioma tumor in mouse model (athymic nude mice) was chosen as our in vivo model. Our preliminary in vitro data demonstrated silencing the H-ferritin gene in U251 cells leads to a 50% increase in cell death at 20 Gy for 24, 48 and 72 hours post radiation. Intratumoral injections of cationic liposomes containing H-ferritin siRNA reduced the effective in vivo dose of radiation for tumor suppression by more than 90% at 7 weeks. A supercoil relaxation assay demonstrated that H-ferritin, but not L-ferritin directly protects DNA from radiation by maintaining DNA in a relaxed from. We will further test our hypothesis in glioma-U87, neurofibroma-sNF96.2 and breast cancer-MCF-7 cells. As of now our data shows that silencing the H-ferritin gene appears to be an effective way to increase the sensitivity of the glioma tumors to radiation, which sheds light on increasing the radiation therapeutic efficacy for treatment of malignant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5410. doi:10.1158/1538-7445.AM2011-5410

We have previously demonstrated the therapeutic effect of interleukin-13 (IL13) linked liposomal doxorubicin (IL13LDXR) on glioma tumors. These tumors selectively overexpress IL13 receptor α2, a receptor for IL13. In our subsequent study, we found that malignant peripheral nerve sheath tumors (MPNST) also express the receptor for IL13. This observation prompted us to investigate the therapeutic value of IL13LDXR in a MPNST model. IL13LDXR was formulated and characterized in our laboratory using our previously established method. In vitro binding and internalization studies and subsequent fluorescence microscopy demonstrated the effective nuclear delivery of the doxorubicin in the MPNST cells expressing IL13Rα2 receptor. The cytotoxic potential of IL13LDXR was demonstrated in MPNST cell lines sNF96.2, ST88-14 and #215. Immunohistochemistry was performed on malignant and benign peripheral nerve sheath tumor (PNST) sections ex vivo to determine the expression of IL13Rα2 receptor. In addition we also demonstrated the expression of P-glycoprotein, a multidrug resistant protein in MPNST cell lines by immunocytochemistry. We also studied the effect of IL-13 protein in altering the expression of Pgp expression in established MPNST cell lines. We confirmed by an immunoblot that IL13 protein at 5µM concentration was able to decrease the Pgp expression in ST88-14 and sNF96.2 cells. This may explain the mechanism by which the IL-13 conjugated liposomes can deliver the encapsulated doxorubicin in a much more effective manner to drug resistant MPNST cell lines. We investigated the correlation of IL13Rα2 receptor expression with P-glycoprotein a drug resistance associated protein by immunocytochemistry. We confirmed the expression of IL13Rα2 in PNST tumors and established MPNST cell lines. We attempted to develop a MPNST tumor model in athymic nude mice by matrigel implantation method. We administered a single injection of 15 mg/kg of IL13 linked liposomal doxorubicin in a mouse tumor model by intraperitoneal route and observed the selective accumulation of doxorubicin in the tumor by endogenous fluorescence of doxorubicin, whereas no fluorescence was observed in the normal skin region as observed under fluorescence microscope. In conclusion consistent expression of IL13Ra2 receptors in PNST and MPNST cells and tissues indicate a viable approach to use this receptor as a target for MPNST therapy. The doxorubicin accumulation property and its cytotoxic potential on MPNST cells when delivered though the targeted liposomes envisage IL13LDXR as a potential drug for MPNST. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5521.