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Background & AimsAnti-tumour necrosis factor-alpha (anti-TNFα) agents have been used for inflammatory bowel disease (IBD), however, it has up to 30% non-response rate. Identifying molecular pathways and finding reliable diagnostic biomarkers for patient response to anti-TNFα treatment are clearly needed.MethodsPublicly available transcriptomic data from IBD patients receiving anti-TNFα therapy was systemically collected and integrated. In silico flow cytometry approaches and MetaScape were applied to evaluate immune cell populations and to perform gene enrichment analysis, respectively. Genes identified within enrichment pathways validated in neutrophils were tracked in an anti-TNFα-treated animal model (with lipopolysaccharide (LPS)-induced inflammation). The receiver operating characteristic (ROC) curve was applied to all genes to identify the best prediction biomarkers.ResultsA total of 449 samples were retrieved from control, baseline and after primary anti-TNFα therapy or placebo. No statistically significant differences were observed between anti-TNFα treatment responders and non-responders at baseline in immune microenvironment scores. Neutrophils, endothelial and B cell populations were higher in baseline non-responders and chemotaxis pathways may contribute to the treatment resistance. Genes related to chemotaxis pathways were significantly up-regulated in LPS-induced neutrophils but no statistically significant changes were observed in neutrophils treated with anti-TNFα. Interleukin 13 receptor subunit alpha 2 (IL13RA2) is the best predictor (ROC: 80.7%, 95% CI: 73.8% - 87.5%) with a sensitivity of 68.13% and specificity of 84.93%, and significantly higher in non-responders compared to responders (p < 0.0001).ConclusionsHyperactive chemotaxis influences responses to anti-TNFα treatment and IL13RA2 is a potential biomarker to predict anti-TNFα treatment response.

Aim/Objective: Ulcerative colitis (UC) is a chronic inflammatory bowel disease. In UC, a wide range of criteria are used for disease remission, with few studies investigating the differences between disease remission and normal control groups. This paper compares known inflammatory and healing mediators in the mucosa of UC in clinical remission and normal controls, in order to better describe the remission state. Method: Mucosal biopsies from 72 study participants (48 UC and 24 normal controls) were included from the Advanced Study of Inflammatory Bowel Disease (ASIB Study), Arctic University of Norway, Norway. Clinical remission was defined as Mayo clinical score ≤ 2, with endoscopic subscores of ≤ 1. Targeted gene transcription analyses were performed using hydrolysis probes and SYBR-green. Results: Among the mucosal transcripts examined, 10 genes were regulated in remission versus normal controls, 8 upregulated pro-inflammatory transcripts (IL1B, IL33, TNF, TRAF1, CLDN2, STAT1, STAT3 and IL13Ra2) and 2 downregulated (pro-inflammatory TBX21 and anti-inflammatory TGFB1). In total, 14 transcripts were regulated between the investigated groups. Several master transcription factors for T-cell development were upregulated in patients with Mayo endoscopic score of 1 in comparison to 0. Conclusions: The mucosa of UC in clinical and endoscopic remission differs from normal mucosa, suggesting a remaining dysregulation of inflammatory and wound healing mechanisms.

Background: In this study, we aimed to detect the changes in the level of interleukin (IL)-4 and IL-13 cytokines and their downstream genes including interleukin-13 receptor subunit alpha-2 (IL13Ra2), interleukin-4 receptor subunit alpha-1 (IL4Ra1), dual oxidase 1 (DUOX1) and dual oxidase 2 (DUOX2). The protective effects of Selenium-L-methionine on radiation-induced histopathological damages and changes in the level of these cytokines and genes were detected. Methods: Four groups of 20 rats (5 rats in each) namely, control; Selenium-L-methionine, radiation and radiation plus Selenium-L-methionine were used in this study. 4 mg/kg of Selenium-Lmethionine was administered 1 day before irradiation and five consecutive days after irradiation. Irradiation was done using a dose of 15 Gy 60Co gamma rays at 109 cGy/min. All rats were sacrificed 10 weeks after irradiation for detecting changes in IL-4 and IL-13 cytokines, the expressions of IL13Ra2, IL4Ra1, Duox1 and Duox2 and histopathological changes. Results: The level of IL-4 but not IL-13 increased after irradiation. This was associated with increased expression of IL4Ra1, Duox1 and Duox2, in addition to changes in morphological properties. Selenium-L-methionine could attenuate all injury markers following lung irradiation. Conclusion: Selenium-L-methionine can protect lung tissues against toxic effects of ionizing radiation. It is possible that the modulation of immune responses and redox interactions are involved in the radioprotective effect of this agent.

Background Non-functional pituitary adenoma (NFPA) is highly heterogeneous with different hormone expression subtypes. Of them, follicle-stimulating hormone (FSH)-positive expression is an important subtype of NFPAs. It is well-known that FSH exerted its functions through binding its receptor. However, the expression rate of FSH receptor was significantly higher in aggressive pituitary adenomas. This study aimed to investigate the molecular characteristics of FSH-positive NFPAs for effective stratification of patient, target treatment, prognostic assessment, and personalized treatment of FSH-positive NFPAs. Methods Tandem mass tag (TMT)-based quantitative proteomics was used to investigate differentially expressed proteins (DEPs) between FSH-positive and negative NFPAs. Gene ontology and KEGG pathway enrichment analyses were used to analyze the DEPs. Differentially expressed genes (DEGs) between invasive and non-invasive NFPAs from GEO database were analyzed with pathway enrichment analysis. Protein-protein interaction (PPI) networks were constructed based on DEPs in excetral cellular matrix (ECM)-receptor interaction, focal adhesion, and PI3K-Akt pathways. Cytoscape was used to obtain most significant modules. Western blot was used to validate the expressions of upregulated proteins (ITGA1, ITGA6, and ITGB4), the expression and phosphorylated status of Akt in PI3K-Akt pathway, and the expression of FSH receptors in FSH-positive relative to negative NFPAs. Results A total of 594 DEPs (374 upregulated and 220 downregulated) were identified between FSH-positive and negative NFPAs. Nineteen KEGG pathway networks were identified to involve DEPs, and reveal molecular differences between FSH-positive and negative NFPAs, including three important pathways that were significantly associated with tumor invasiveness and aggressiveness: ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathways. Further, focal adhesion pathway was also confirmed with invasiveness-related NFPA DEG data that were derived from GEO database. Moreover, the significantly upregulated DEPs (ITGA1, ITGA6, and ITGB4) that were associated with tumor invasiveness and aggressiveness were confirmed by immunoaffinity analysis in FSH-positive vs. negative NFPAs. Also, the phosphorylation level but not its expression level of AKT in PI3K-AKT signaling was significantly increased, and the expression level of FSH receptor was significantly increased in FSH-positive relative to negative NFPAs. Also, overlapping analysis of 594 DEPs and 898 DEGs revealed 45 invasiveness-related DEPs, including 11 upregulated DEPs (ITGA6, FARP1, PALLD, PPBP, LIMA1, SCD, UACA, BAG3, CLU, PLEC, and GATM) that were also upregulated genes in invasive NFPAs, and 8 downregulated DEPs (ALCAM, HP, FSTL4, IL13RA2, NPTX2, DPP6, CRABP2, and SLC27A2) that were also downregulated genes in invasive NFPAs. Conclusions FSH-positive expression was an important NFPA subtype. It was the first time for this study to reveal FSH-related proteomic variations and the corresponding molecular network alterations in FSH-positive relative to negative NFPAs. Also, three signaling pathways (ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathways) and involved upregulated proteins (ITGA1, ITGA6, ITGB4, pAKT, and FSHR) were significantly associated with tumor invasiveness and aggressiveness, and a set of invasiveness-related DEPs were identified with overlapping analysis of 594 DEPs in FSH-positive vs. negative NFPAs and 898 DEGs in invasive vs. non-invasive NFPAs. These findings offered the scientific evidence to in-depth understand molecular characteristics of FSH-positive NFPAs, and effectively stratify the post-surgery patients for personalized prognostic assessment and targeted treatment of FSH-positive NFPAs. Electronic supplementary material The online version of this article (10.1007/s13167-019-00187-w) contains supplementary material, which is available to authorized users.

Introduction: Redox interactions play a key role in radiation injury including heart diseases. In present study, we aimed to detect the possible protective role of selenium-L-methionine on infiltration of immune cells and Duox1&2 upregulation in rat’s heart tissues. Methods: In this study, 20 rats were divided into 4 groups (5 rats in each) namely: irradiation; irradiation plus Selenium-L-methionine; control; and Selenium-L-methionine treatment. Irradiation (15 Gy to chest) was performed using a cobalt-60 gamma ray source while 4 mg/kg of selenium-L-methionine was administered intraperitoneally. Ten weeks after irradiation, rats were sacrificed for detection of IL-4 and IL-13 cytokines, infiltration of macrophages and lymphocytes as well as the expressions of IL4Ra1, Duox1, IL13Ra2 and Duox2. Results: Results showed an increase in the level of IL-4 as well as the expressions of IL4Ra1, Duox1 and Duox2. Similarly, there was an increase in the infiltration of lymphocytes and macrophages. There was significant attenuation of all these changes following treatment with selenium-L-methionine. Conclusion: Selenium-L-methionine has the potential to protect heart tissues against radiation injury. Downregulation of pro-oxidant genes and modulation of some cytokines such as IL-4 are involved in the radioprotective effect of selenium-L-methionine on heart tissues.

Background: Heart injury is one of the most important concerns after exposure to a high dose of radiation in chest cancer radiotherapy or whole body exposure to a radiation disaster. Studies have proposed that increased level of inflammatory and pro-fibrotic cytokines following radiotherapy or radiation events play a key role in the development of several side effects such as cardiovascular disorders. In the current study, we aimed to evaluate the expression of IL-4 and IL-13 cytokines as well as signaling pathways such as IL4Ra1, IL13Ra2, Duox1 and Duox2. In addition, we detected the possible protective effect of curcumin on the expression of these factors and infiltration of inflammatory cells. Materials and Methods: Twenty rats were divided into 4 groups including control; curcumin treated; radiation; and radiation plus curcumin. After 10 weeks, rats were sacrificed for evaluation of mentioned parameters. Results: Results showed an increase in the level of IL-4 and all evaluated genes, as well as increased infiltration of lymphocytes and macrophages. Treatment with curcumin could attenuate these changes. Conclusion: Curcumin could reduce radiation-induced heart injury markers in rats.

Background. The interleukin-13 receptor alpha 2 (IL13RA2) and ephrin type A receptor 2 (EPHA2) are attractive therapeutic targets, being expressed in similar to 90% of canine and human gliomas, and absent in normal brain. Clinical trials using an earlier generation IL-13 based cytotoxin showed encouraging clinical effects in human glioma, but met with technical barriers associated with the convection-enhanced delivery (CED) method. In this study, IL-13 mutant and ephrin A1 (EFNA1)-based bacterial cytotoxins targeted to IL13RA2 and EPHA2 receptors, respectively, were administered locoregionally by CED to dogs with intracranial gliomas to evaluate their safety and preliminary efficacy. Methods. In this phase I, 3 + 3 dose escalation trial, cytotoxins were infused by CED in 17 dogs with gliomas expressing IL13RA2 or EPHA2 receptors. CED was performed using a shape-fitting therapeutic planning algorithm, reflux-preventing catheters, and real-time intraoperative MRI monitoring. The primary endpoint was to determine the maximum tolerated dose of the cytotoxic cocktail in dogs with gliomas. Results. Consistent intratumoral delivery of the cytotoxic cocktail was achieved, with a median target coverage of 70% (range, 40-94%). Cytotoxins were well tolerated over a dose range of 0.012-1.278 mu g/mL delivered to the target volume (median, 0.099 mu g/mL), with no dose limiting toxicities observed. Objective tumor responses, up to 94% tumor volume reduction, were observed in 50% (8/16) of dogs, including at least one dog in each dosing cohort >0.05 mu g/mL. Conclusions. This study provides preclinical data fundamental to the translation of this multireceptor targeted therapeutic approach to the human clinic. Wake Forest University Translational Sciences Institute, National Cancer Institute [R01CA139099, R01CA74145, P01CA207206]; Comprehensive Cancer Center of Wake Forest University Brain Tumor Center of Excellence; Hearn Fund for Brain Tumor Research; Pratto Fund for Brain Tumor Research; Dallas Ray Swing Brain Tumor Fund Published version This work was supported by grants from the Wake Forest University Translational Sciences Institute, National Cancer Institute (R01CA139099, R01CA74145, P01CA207206), the Comprehensive Cancer Center of Wake Forest University Brain Tumor Center of Excellence, Hearn Fund for Brain Tumor Research, Pratto Fund for Brain Tumor Research, and Dallas Ray Swing Brain Tumor Fund.

Background: It has been proposed that increased levels of pro-inflammatory and pro-fibrotic cytokines play a key role in radiation-induced lung injury. Interleukin-4 (IL-4) and IL-13 are two pro-fibrotic cytokines that promote the production of free radicals through stimulation of Duox1 and Duox2. In this experimental study, we aimed to evaluate the expression of IL4Ra1, Duox1, IL13Ra2, and Duox2 genes following rat’s lung irradiation. Objectives: Also, we detected the modulatory effect of a combination of curcumin and L-selenomethionine on the expression of these genes. Methods: Twenty male rats were divided into four groups as G1: control (no treatment or radiation); G2: treatment with a combination of curcumin and L-selenomethionine; G3: radiation; G4: radiation plus a combination of curcumin and L-selenomethionine. sixty-seven days after irradiation, rats were killed for detecting the expression of IL4Ra1, IL13Ra2, Duox1, and Duox2. Results: The results showed no detectable expression for IL13Ra2, while the expression of IL4Ra1, Duox1, and Duox2 was increased. Treatment with a combination of curcumin and L-selenomethionine could attenuate the expression of these genes. Conclusions: This study proposes that upregulation of Duox1 and Duox2 may be involved in radiation-induced lung injury. Treatment with a combination of curcumin and L-selenomethionine may be useful for the mitigation of lung injury through modulation of these genes.

Papillary renal cell carcinoma (PRCC) is the second most common type of renal carcinoma following clear cell renal cell carcinoma, and the role of immune-related genes (IRGs) in tumorigenesis and metastasis is evident; its prognostic value in PRCC remains unclear. In this study, we downloaded the gene expression profiles and clinical data of patients with PRCC from The Cancer Genome Atlas (TCGA) database and obtained IRGs from the ImmPort database. A total of 371 differentially expressed IRGs (DEIRGs) were discovered between PRCC and normal kidney tissues. Prognostic DEIRGs (PDEIRGs) were identified by univariate Cox regression analysis. Then, we screened the four most representative PDEIRGs (IL13RA2, CCL19, BIRC5, and INHBE) and used them to construct a risk model to predict the prognosis of patients with PRCC. This model precisely stratified survival outcome and accurately identified mutation burden in PRCC. Thus, our results suggest that these four PDEIRGs are available prognostic predictors for PRCC. They could be used to assess the prognosis and to guide individualized treatments for patients with PRCC.

// Anirudh Sattiraju 1, 2, * , Kiran Kumar Solingapuram Sai 1, 2, * , Ang Xuan 4, * , Darpan N. Pandya 2 , Frankis G. Almaguel 1 , Thaddeus J. Wadas 1, 3 , Denise M. Herpai 2, 3 , Waldemar Debinski 2, 3 and Akiva Mintz 1, 2 1 Department of Radiology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 2 Brain Tumor Center of Excellence, Wake Forest University Comprehensive Cancer Center, Winston-Salem, NC 27157, USA 3 Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 4 Department of Nuclear Medicine and Radiology, The People’s Hospital of Zhengzhou University, Zhengzhou, Henan 450003, China * These authors contributed equally to this work Correspondence to: Akiva Mintz, email: amintz@wakehealth.edu Keywords: glioblastoma, IL13RA2, alpha particle therapy, Actinium-225, Copper-64 Received: January 10, 2017 Accepted: April 15, 2017 Published: May 11, 2017 ABSTRACT Glioblastoma (GBM) is the most aggressive primary malignant brain cancer that invariably results in a dismal prognosis. Chemotherapy and radiotherapy have not been completely effective as standard treatment options for patients due to recurrent disease. We and others have therefore developed molecular strategies to specifically target interleukin 13 receptor alpha 2 (IL13RA2), a GBM restricted receptor expressed abundantly on over 75% of GBM patients. In this work, we evaluated the potential of Pep-1L, a novel IL13RA2 targeted peptide, as a platform to deliver targeted lethal therapies to GBM. To demonstrate GBM-specificity, we radiolabeled Pep-1L with Copper-64 and performed in vitro cell binding studies, which demonstrated specific binding that was blocked by unlabeled Pep-1L. Furthermore, we demonstrated real-time GBM localization of [ 64 Cu]Pep-1L to orthotopic GBMs using small animal PET imaging. Based on these targeting data, we performed an initial in vivo safety and therapeutic study using Pep-1L conjugated to Actinium-225, an alpha particle emitter that has been shown to potently and irreversibly kill targeted cells. We infused [ 225 Ac]Pep-1L into orthotopic GBMs using convection-enhanced delivery and found no significant adverse events at injected doses. Furthermore, our initial data also demonstrated significantly greater overall, median and mean survival in treated mice when compared to those in control groups ( p < 0.05). GBM tissue extracted from mice treated with [ 225 Ac]Pep-1L showed double stranded DNA breaks, lower Ki67 expression and greater propidium iodide internalization, indicating anti-GBM therapeutic effects of [ 225 Ac]Pep-1L. Based on our results, Pep-1L warrants further investigation as a potential targeted platform to deliver anti-cancer agents.

Peptides that target cancer cell surface receptors are promising platforms to deliver diagnostic and therapeutic payloads specifically to cancer but not normal tissue. IL13RA2 is a tumor-restricted receptor found to be present in several aggressive malignancies, including in the vast majority of high-grade gliomas and malignant melanoma. This receptor has been successfully targeted for diagnostic and therapeutic purposes using modified IL-13 ligand and more recently using a specific peptide, Pep-1L. In the current work, we establish the in vitro and in vivo tumor binding properties of radiolabeled Pep-1L, designed for tumor imaging. We radiolabeled Pep-1L with Copper-64 and demonstrated specific cell uptake in the IL13RA2-over expressing G48 glioblastoma cell line having abundant IL13RA2 expression. [64Cu]Pep-1L binding was blocked by unlabeled ligand, demonstrating specificity. To demonstrate in vivo tumor uptake, we intravenously injected into tumor-bearing mice and demonstrated that [64Cu]Pep-1L specifically bound tumors at 24 hours, which was significantly blocked (3-fold) by pre-injecting unlabeled peptide. To further demonstrate specificity of Pep-1L towards IL13RA2 in vivo, we exploited an IL13RA2-inducible melanoma tumor model that does not express receptor at baseline but expresses abundant receptor after treatment with doxycycline. We injected [64Cu]Pep-1L into mice bearing IL13RA2-inducible melanoma tumors and performed in vivo PET/CT and post-necropsy biodistribution studies and found that tumors that were induced to express IL13RA2 receptor by doxycycline pretreatment bound radiolabeled Pep-1L 3-4 fold greater than uninduced tumors, demonstrating receptor specificity. This work demonstrates that [64Cu]Pep-1L selectively binds hIL13RA2-expressing tumors and validates Pep-1L as an effective platform to deliver diagnostics and therapeutics to IL13RA2-expressing cancers.

INTRODUCTION AND OBJECTIVE:There are some treatment strategies using up-front chemotherapy or androgen receptor axis targeting therapies for metastatic hormone-naive prostate cancer (mHNPC). Howeve...

Background: Mucosal IL-13 Receptor alpha 2 (IL13RA2) mRNA expression is one of the best predictive markers for primary non-responsiveness to infliximab therapy in patients with inflammatory bowel disease (IBD). The objective of this study was to understand how IL-13Rα2, a negative regulator of IL-13 signaling, can contribute to IBD pathology. Methods: IL13RA2 knockout (KO) and wild type (WT) mice were exposed to dextran sodium sulfate (DSS) in drinking water to induce colitis. Furthermore, mucosal biopsies and resection specimen of healthy individuals and IBD patients before the start of anti-tumor necrosis factor (anti-TNF) therapy were obtained for immunohistochemistry and gene expression analysis. Results: After induction of DSS colitis, IL13RA2 KO mice had similar disease severity, but recovered more rapidly than WT animals. Goblet cell numbers and mucosal architecture were also more rapidly restored in IL13RA2 KO mice. In mucosal biopsies of active IBD patients, immunohistochemistry revealed that IL-13Rα2 protein was highly expressed in epithelial cells, while expression was restricted to goblet cells in healthy controls. Mucosal IL13RA2 mRNA negatively correlated with mRNA of several goblet cell-specific and barrier genes, and with goblet cell numbers. Conclusions: The data suggest that IL-13Rα2 on epithelial cells contributes to IBD pathology by negatively influencing goblet cell recovery, goblet cell function and epithelial restoration after injury. Therefore, blocking IL-13Rα2 could be a promising target for restoration of the epithelial barrier in IBD. ispartof: Frontiers in Immunology vol:9 pages:1-9 ispartof: location:Switzerland status: published

BACKGROUND The outstanding efficacy of bi-specific T-cell engagers (BiTE against hematological malignancies offers hope that they can similarly target solid tumors like GBM. In this study, we have designed a BiTE protein with specificity to the tumor-associated antigen, IL13Rα2, and investigated how BiTE protein engages a host’s T cell immune response to promote anti-glioma activity in pre-clinical models of GBM. MATERIAL AND METHODS BiTE molecule consisting of two single chain variable regions (scFv) of antibodies against either murine or human CD3ε and scFv47 against human IL13Rα2 connected through a flexible linker (BiTEIL13Rα2) were sub-cloned in a lentiviral expression cassette. The BiTE molecule (BiTEIL13Rα2off) modified to abrogate the interaction of scFv47 with IL13Rα2 served as a negative control. The BiTE proteins were isolated from the supernatants of HEK293T cells using His-Tag affinity chromatography and validated in SDS-PAGE, Western Blot, and ELISA assays. The BiTEIL13Rα2-induced T cells activation was measured in (i) cytotoxicity assay against IL13Rα2+ glioma cells, (ii) flow cytometry measuring for CD69 and CD25 T cells’ activation markers, and (iii) the production of cytokines, IFNγ and TNFα. For in vivo analysis, VmDk and C57Bl/6 mice bearing established intracranial glioma were treated systemically with BiTE proteins. The survival of the mice was recorded and analyzed using the log-rank test. RESULTS Here we show that BiTEIL13Rα2 specifically binds to IL13Rα2 but not to IL13Rα1, whereas BiTEIL13Rα2off has no binding activity to both IL13 receptors. The co-culture of naïve murine or donor’s human CD3+ T cells with IL13Rα2+ glioma cells in the presence of BiTEIL13Rα2 but not with BiTEIL13Rα2off (i) activates CD3+CD8+ T cells as judged by upregulation of CD69, CD25, and production of IFNγ and TNFα and (ii) results in concentration- and antigen-dependent cytotoxicity in glioma cells. Furthermore, a direct comparison of CD3+ T cells obtained from the peripheral blood and tumor tissue of GBM patients revealed that BiTEIL13Rα2 induces a potent cytotoxic activity of CD3+ T cells against IL13Rα2+ glioma cells. Finally, treatment of immunocompetent mice bearing IL13Rα2+ murine glioma with BiTEIL13Rα2 resulted in a higher frequency of intratumoral CD8+ T cells, and significant (p CONCLUSION Our data demonstrate that BiTEIL13Rα2 protein activates CD3+ T cells in an antigen-specific fashion. Furthermore, systemic treatment with BiTEIL13Rα2 protein confers a significant survival benefit in pre-clinical syngeneic glioma models, warranting investigations in other IL13Rα2-expressing cancers and translation to clinical settings.

Asthma is a chronic (long-term) lung disease that inflames and narrows the airways. Asthma causes recurring periods of wheezing (a whistling sound when you breathe), chest tightness, shortness of breath, and coughing. Asthma affects people of all ages, but it most often starts during childhood. IL-13 receptor (IL-13R) is a heterodimer composed of the IL-13Rα1 chain (IL-13Rα1) and the IL-4Rα chain (IL-4Rα), both IL-13 and IL-4 activate STAT6 and STAT3. IL-13Rα2 chain (IL-13Rα2) binds to IL-13 with high affinity and it does not contain the Box1 motif which is an important domain for intracellular signaling. IL13Rα2 was the target protein because it does not as- sociate with signaling molecules and instead acts as a "decoy" receptor. The studies were performed on IL13Rα2 to inhibit the protein that expressed during the causes for asthma disease. The potential drug with IUPAC name "5-(2-(tert-butylamino)-1-hydroxyethyl)-2-(hydroxymethyl)" was designed successfully in inhibit the actions of IL13Rα2 protein.

TUMOR MICROENVIRONMENT TMIC-01. TUMOR TARGETED QUANTUM DOTS TO IDENTIFY GLIOMA INITIATING CELLS AND EXOSOMES Achutha Madhankumar, Oliver Mrowczynski, Suhag Patel, Elias Rizk, Cody Weston, Michael Glantz, Lichong Xu, Christopher Siedlecki, and James Connor; Pennsylvania State University College of Medicine, Hershey, PA, USA GBM and certain brain metastatic cancers possess invasiveness and high infiltrating potential making them harder to detect at the earlier stage of the disease progression. Identification of cancer stem cells and exosomes secreted by them in the biofluids like CSF will be an ideal way to determine the presence of residual cancer after therapy. We utilize ligand conjugated quantum dots (QD) to specifically label the cancer stem cells and exosomes to identify the presence of certain invasive markers. 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 IL13Ra2 receptor and also the exosomes secreted by the glioma stem cells. Exosomes that were isolated from glioma stem cells and patients CSF were demonstrated to express IL13Ra2 receptor. Fluorescent microscopy, electron microscopy and flow cytometry were performed on the exosomes after complexing with nano quantum dots (QD). 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 also confirmed by atomic force microscopy. The morphology and size of exosomes were confirmed by electron microscopy and dynamic light scattering method. The density plot from the flow cytometer experiments with IL13QD-exosomes complex indicates a possibility to identify the tumor associated exosomes. Our experiment infers that in ex vivo, tumor targeted quantum dots can bind with tumor initiating glioma stem cells and extracellular vesicles (exosomes) that are secreted by cancer cells. There is a future possibility to differentiate the metastatic tumor associated exosomes from the non-metastatic exosomes based on their QD binding profile Neuro-Oncology 17:v221–v225, 2015. doi:10.1093/neuonc/nov236.1 Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2015.

INTRODUCTION: Astrocytomas are common brain tumors. Increased expression levels of Interleukin-13 Receptor α2 (IL-13RA2) have been reported in astrocytomas. The Interleukin-13 signaling pathway may be associated with cell migration when binding to Interleukin-13 Receptor α1. OBJECTIVE: To investigate Interleukin-13 Receptor α1 (IL-13RA1) and IL13RA2 expression levels in human diffusely infiltrative astrocytomas and test the involvement of Interleukin-13 levels in cell migration in two glioblastoma cell lines. METHODS: IL13RA expression levels were accessed by quantitative real time PCR in 128 samples of astrocytomas and 18 samples of non-neoplastic brain tissues from temporal lobe epilepsy surgery. The impact of IL-13 levels (10 and 20 ng/mL) on cell migration was analyzed by the wound assay in U87MG and A172 cells. RESULTS: Glioblastoma presented higher IL13RA1 and IL13RA2 expression levels compared to lower grades astrocytomas and to non-neoplastic cases. U87MG and A172 cells presented higher expression levels of IL-13RA1 vs. IL-13RA2. A significant difference in migration rate was observed in A172 cells treated with 10 ng/mL of IL-13 vs. control: treated cells presented slower migration than non-treated cells. U87MG cells treated with IL-13 20ng/mL presented slower migration than non-treated cells. This indicates that the IL13Rα1 signaling pathway was not activated, indeed inhibited by the decoy IL-13Rα2, slowing cell migration. This impact occurred with a lesser concentration of IL-13 on the A172 than on the U87MG cell line, because A172 cells have a higher IL-13RA2/A1 ratio. CONCLUSION: The present results suggest IL-13 receptors as possible targets to decrease tumor cell migration.

The success of chimeric antigen receptor (CAR)expressing T cells as therapy for refractory hematologic malignancies (1) has spurred ongoing efforts to extend their application toward other difficult-totreat tumors. High-grade gliomas, for example, are prime candidates. These tumors—often difficult to reach by means of surgery and systemic chemotherapy—can greatly benefit from therapies that can actively home to the site of disease. Several surface proteins overexpressed by high-grade gliomas have been identified, all of which, in theory, can be targeted by CAR T cells. Interleukin (IL)13Ra2, for example, has been demonstrated to be overexpressed in high-grade gliomas (2), with very little to no expression in healthy cells. However, as the study by Krebs et al. (3) reminds us, specificity cannot be taken for granted. In the August issue of Cytotherapy, the group from Baylor College of Medicine show how CAR T cells expressing IL13 muteins (that should theoretically bind preferentially to IL13Ra2) are also capable of recognizing and responding to the closely related IL13Ra1. IL13-mutein CAR T cells produced significant levels of interferon (IFN)-g in response to both IL13Ra1 and IL13Ra2 proteins (and not to IL4R proteins). Higher levels of IFN-g (P < 0.001) were seen secreted by IL13-mutein CAR T cells after culture in plates that were pre-coated with IL13Ra1 or IL13Ra2. These IL13-mutein CART cells are also able to recognize and kill cells that express either IL13Ra1 or IL13Ra2 on their surface. Tumor cells genetically modified to express only IL13Ra1 or IL13Ra2 were able to stimulate IFN-g secretion from IL13-mutein CAR T cells, but unmodified cells or green fluorescent proteinemodified cells were not able to do so (P < 0.001). More importantly

Item does not contain fulltext OBJECTIVE: Pheochromocytomas and paragangliomas (PPGL) are relatively rare and mostly benign tumours. Approximately 10% of PPGL are malignant, as defined by the presence of metastases, i.e chromaffin tissue at a location that usually does not contain chromaffin cells. However, up to 35% of tumours in patients carrying an SDHB mutation appears to be malignant. Nowadays, no reliable marker allows to predict whether a PPGL is, or will become malignant. In addition, there are no curative treatments if metastases occur. The aim of the present study was to dentify genetic markers allowing to distinguish benign from malignant tumours. DESIGN AND METHOD: An mRNA expression array was performed on benign and malignant PPGL. The genes showing a different expression between the benign and malignant tumours were selected to be confirmed and validated by qRT-PCR. Finally, the remaining genes were stained by immunohistochemistry on Tissue MicroArray including a large series of PPGL. RESULTS: Forty benign and 12 malignant PPGL were investigated for differences in mRNA expression with Affymetrix arrays. Expression data were normalized according to Affymetrix recommendations. Then, using Pomelo II (http://pomelo2.bioinfo.cnio.es/), a Limma t-test was performed, to assess which genes were differentially expressed between benign and malignant PPGL. First, a non-clustered analysis was performed and 10 genes with a False Discovery Rate (FDR) below 0.05 and a relative overexpression ratio of at least 4 were found, including Interleukin 13 Receptor alpha 2 (IL13RA2) and Monooxygenase DBH-like 1 (MOXD1). Secondly, a supervised cluster analysis was performed (based on HIF target genes), resulting in 2 groups, which were both investigated for differences in mRNA expression between benign and malignant tumours. Five genes showed an FDR below 0.01 and were overexpressed in malignant tumours with a ratio higher than 4, including Contactin 4 (CNTN4), Iroquois Homeobox 3 (IRX3), and Sulfatase 2 (SULF2). These genes were further investigated using qRT-PCR, and immunohistochemistry on Tissue Micro Array including 91 benign and 12 malignant PPGL. CONCLUSIONS: Significant overexpression of Contactin 4 was shown in malignant compared to benign tumours, and may therefore contribute to distinguish malignant from benign PPGL.

e15151 Background: We examined whether 133 germline polymorphisms (SNPs) in 15 candidate genes (CSF1R, IL8RA, TLR4, IL10, IL10RA, CTLA4, IL2, IL2RA, TGF b1, ICOS, IL13, IL13RA2, IFNgR, IL15 and IL15RA) would predict clinical outcome in the ACCORD-12 phase III trial which randomly compared neoadjuvant radiotherapy (RT) plus capecitabine (CAP45) with dose-intensified RT plus capecitabine and oxaliplatin (CAPOX50) in T3-4 Nx M0 resectable rectal cancer. Methods: A candidate-gene association study was conducted in 316 patients (n = 161 in the CAPOX50 and n = 155 in the CAP45 arm). The primary end-point was tumor response according to the Dworak score in each arm. Logistic regressions were used to assess univariate/multivariate associations. The Storey and Tibshirani method based on the control of false discovery rate was used ( q-value < 0.10 (adjusted p-value) considered as true discovery). Multivariate models adjusted on treatment arm and T stage were performed to determine prognostic and predictive values of SNPs for tumor response. Results: In univariate analysis, two SNPs in IL2RA (rs11256456: OR = 5.1 [2.38; 11] and rs706781: OR = 4.2 [1.98 ; 8.74]) were significantly associated with the Dworak score in the CAP45 arm, and one in IL2RA the CAPOX50 arm (rs2104286: OR = 0.11 [0.01 ; 0.90]. All were confirmed in the multivariate analysis. A significant haplotypic effect was observed in the CAP-45 arm (p = 0.0001). Interaction was significant for IL2RA rs11256456 ( p= 0.03) and rs706781 ( p= 0.002) and no significant for IL2RA rs2104286 ( p= 0.722), suggesting a predictive deleterious effect the first two ones for response to oxaliplatin-based chemoRT, and a prognostic effect of the third one for response to chemoRT (+/- oxaliplatin). None of the three IL2RA SNPs were correlated with survival in the multivariate analysis. Conclusions: This pharmacogenetic analysis shows that SNPs in IL2RA have a significant association with response to chemoRT with capecitabine in patients with locally advanced rectal cancer. Their predictive effect may identify patients in whom oxaliplatin addition to chemoRT is deleterious.

BACKGROUND: IL13Rα2 is a promising target for novel therapies in glioblastoma (GBM) due to its overexpression in tumor cells as well as glioma-initiating cells, which are responsible for the growth and recurrence of GBM. The role of IL13Ra2, found heterogeneously expressed in GBMs, is not fully understood and better targeting strategies are necessary for its clinical application. This study investigated the effectiveness of different patterns of IL13Rα2 expression on the survival of syngeneic rodent glioma models. This study also aims to act as a blueprint for further tests of the level of immune response to the target immunotherapy in immunocompetent mice bearing GBMs with different levels of IL13Rα2 expression. METHODS: G26 murine glioma cell line, with transfected to express hIL13Rα2 (G26-H2) and vector control (G26-V2) were intracranially injected in two groups of C57Bl/6 mice respectively. The metric analyzed for outcome was survival. Histological analysis were used to evaluate the level of IL13Ra2 expression in primary tissue obtained from patients with high gliomas. RESULTS: Two populations of G26 glioma cell lines were established per hIL13Rα2 expression: the hIL13Rα2+ and the hIL13Rα2−. In vivo, the cell lines showed a significant difference in survival: the group that received intracranial injection of G26-H2 cells (100% positive for hIL13Rα2) had a median survival of 29.5 days compared to 49 days for group receiving G26-V2 cells (100% negative for h IL13Rα2), p

METHODS: We tested efficacy of the MET TKI crizotinib, the HSP90 inhibitor ganetespib and their combination in MKN45, wild-type MET-overexpressed cell line and MET-Y1248H, TKI-resistant mutant MET-overexpressed cell line in vitro. The antitumor efficacy of HSP90 inhibition in vivo was verified in xenograft models. RESULTS: MET is an HSP90-dependent kinase, and we show that HSP90 preferentially interacts with and stabilizes activated MET, whether the activation is ligand-dependent or is a consequence of kinase domain mutation. In contrast, many MET TKI show a preference for the inactive form of the kinase and activating mutations in MET can confer resistance. Combining the HSP90 inhibitor ganetespib with the MET TKI crizotinib achieves synergistic inhibition of MET, its downstream signaling pathways, and tumor growth in TKI-sensitive METdriven tumor models. Unexpectedly, low-dose ganetespib also partially restores crizotinib sensitivity, in vitro and in vivo, to cells and tumors expressing TKI-resistant MET mutants. CONCLUSIONS: Our findings support the use of HSP90 inhibitors to overcome or delay the initiation of resistance to MET TKI, and they provide the basis for clinical evaluation of this combination in patients with MET-driven papillary renal cancer.

The gene expression profiles of benign and malignant adrenocortical tumors are different.Genomewide gene expression profiling and validation.Tertiary medical center.Eighty-five patients with benign adrenocortical tumors (n = 74) and adrenocortical carcinoma (n = 11).Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) in 89 adrenocortical tissue samples (11 malignant and 78 benign). The criteria for differentially expressed genes between benign and malignant adrenocortical tumors were a false discovery rate of less than 5% and an adjusted P.01. Genes differentially expressed by 8-fold higher or lower were validated by RT-PCR.The diagnostic accuracy of differentially expressed genes as determined by the area under the receiver operating characteristic curve (AUC).We found 37 genes differentially expressed by 8-fold higher or lower. Fifteen genes were downregulated and 22 were upregulated in adrenocortical carcinoma. Of the 37 genes, 29 differentially expressed by microarray correlated with the gene expression levels by quantitative RT-PCR (Por = .01). Of the 37 genes validated by RT-PCR, 22 were significantly differentially expressed between benign and malignant adrenocortical tumors (P.05). Five of these 22 genes had an AUC of 0.80 or greater (the AUC for IL13RA2 was 0.90; HTR2B, 0.87; CCNB2, 0.86; RARRES2, 0.86; and SLC16A9, 0.80), indicating high diagnostic accuracy for distinguishing benign from malignant adrenocortical tumors.We identified 37 genes that are dysregulated in adrenocortical carcinoma, and several of the differentially expressed genes have excellent diagnostic accuracy for distinguishing benign from malignant adrenocortical tumors.

Objective/purpose The airway epithelium serves as a physical defense barrier to the external environment for the underlying tissue and suffers frequent injury. The response to injury is inflammation followed by debris clearance and repair. Although IL-13 is known to be a key cytokine in mediating inflammatory and remodeling responses via signal transducer and activator of transcription 6 (STAT6) and early growth response protein 1 (Egr1), our laboratory has demonstrated that IL-13 is critical to airway epithelial repair via the release of heparin-binding epidermal growth factor (HB-EGF) and activation of epidermal growth factor receptor (EGFR). IL-13 signals through two receptors, IL-13Ra1/IL-4R and IL-13Ra2. IL-13Ra2 has previously been thought to act exclusively as a decoy receptor, however our findings show that IL13Ra2 can act as a signaling receptor and is involved in mediating airway epithelial repair. Differential signaling via IL-13Ra1 or IL-13Ra2 may determine a remodeling versus repair response to injury in airway epithelium.

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.

Melanoma is one of the most prevalent cancers. While early detection and surgical excision can cure melanoma, patients with advanced disseminated disease have a very poor prognosis due to its extremely aggressive growth characteristics and high resistance to chemotherapy and radiation. Improvement of our understanding of the mechanisms that lead to progression of metastatic melanoma, as well as development of novel therapeutic strategies are urgently required for patients. In our study, we applied PCR arrays and bioinformatics analysis to evaluate gene expression of metastatic melanoma cells and normal skin cells. We used the Human Inflammatory Cytokine and Receptors RT2 Profiler™ PCR Array to analyze the differential expression of genes in metastatic melanoma cell lines (A375) and primary normal epidermal melanocytes (NHEM), which are the precursor cell of melanoma. We found the 43 genes were highly expressed in A375 which were upregulated by more than 20 fold in comparison to NHEM. We next selected the top-ten differentially expressed genes (CD70, IL-1β, MGLL, SERPINA3, MDK, IL13RA2, CD74, VEGFA, CXCL1, and IL8), whose expression levels in A375 cells were > 250-fold higher than in the primary normal NHEM, for further validation. The results from our RT-PCR, immunoblot, immunocytochemistry (ICC) and tissue microarray (TMA) analysis showed that CD70, SERPINA3, IL13RA2 and CD74 genes were highly expressed at mRNA and protein levels in various melanoma cell lines and metastatic melanoma tumor tissues. A much lower level of expression of each gene was detected in normal human skin cell lines and normal skin tissues. These results thus demonstrated that the four genes identified by our PCR array are likely of molecular markers and should be pursued as therapeutic targets for melanoma treatment. To further verify their potentials as potential melanoma therapeutic targets, we also evaluated the effects of siRNAs targeting CD70 and CD74 genes and the monoclonal antibodies against CD70 and CD74 proteins for regulation of melanoma growth. Treatment with CD70 or CD74-specific siRNAs and also monoclonal antibodies dramatically inhibited cell growth in various melanoma cell lines, indicating that CD70 and CD74 are also attractive molecular targets in the treatment of human metastatic melanoma. Our findings have important implications in understanding the metastatic process of melanoma and discovering novel therapeutic strategies for melanoma treatment. The molecular markers and therapeutic targets developed in this study will have a direct and immediate translational application. If successful, our study will represent a significant advance in the diagnosis and treatment of melanoma. (This study was partially supported by the grant from the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and the Melanoma SPORE grant from the NCI, P50, CA093459). 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 1175.