Therapy In Mouse Model Research Articles

ET-65 * THE KETOGENIC DIET REDUCES CARBONIC ANHYDRASE 9 AND ALTERS THE EXPRESSION OF PROTEINS INVOLVED WITH GLIOMA GROWTH AND PROGRESSION IN HYPOXIC CONDITIONS

Patients with malignant gliomas have a median survival of approximately 15 months following diagnosis, regardless of currently available treatments which include surgery, radiation and chemotherapy. Improvement in the survival of these patients requires the design of new therapeutic modalities that take advantage of common phenotypes. One such phenotype is metabolic dysregulation - a hallmark of cancer cells. It has been postulated that one approach to treating gliomas may be by metabolic alteration such as that which occurs through the use of the ketogenic diet (KD). The KD is high-fat, low-carbohydrate diet that induces ketosis and has been utilized for the non-pharmacologic treatment of refractory epilepsy. We and others have shown that this diet enhances survival and potentiates standard therapy in mouse models of glioma, yet the anti-tumor mechanisms are not fully understood. We have previously shown that the KD reduces peritumoral edema and tumor vasculature in a mouse model of glioma. We now show that tumors from animals fed the KD show reduced expression of the hypoxia marker carbonic anhydrase 9 (CA9) when compared to animals fed a standard rodent diet (SD). Protein analyses showed that the KD alters the expression of the tight junction protein zona occludens 1 (ZO-1), and the membrane water channel aquaporin-4 (AQPN4), which are regulated by tumor hypoxia and have been implicated in peritumoral edema. We also see a reduction in phosphorylated NF-κB which may result in reduced transcription of hypoxia-related genes. Further analysis showed that the KD reduced expression of vascular endothelial growth factor receptor 2 (VEGFR2), which plays a key role in hypoxia-induced angiogenesis. Taken together our data offers insight into the anti-edema and anti-angiogenic mechanisms underlying the KD. A greater understanding of the effects of the KD as an adjuvant therapy will allow for a more rational approach to its clinical use.

Abstract 1440: Metabolic therapy reduces expression of PECAM-1/CD31 and decreases peritumoral edema in a mouse model of malignant glioma

Abstract Patients with malignant brain tumors have a median survival of approximately one year following diagnosis, regardless of currently available treatments which include surgery followed by radiation and chemotherapy. Improvement in the survival of brain cancer patients requires the design of new therapeutic modalities that take advantage of common phenotypes. One such phenotype is the metabolic dysregulation that is a hallmark of cancer cells. It has therefore been postulated that one approach to treating brain tumors may be by metabolic alteration such as that which occurs through the use of the ketogenic diet (KD). The KD is high-fat, low-carbohydrate diet that induces ketosis and has been utilized for the non-pharmacologic treatment of refractory epilepsy. We and others have shown that this diet enhances survival and potentiates standard therapy in mouse models of malignant gliomas, yet the anti-tumor mechanisms are not fully understood. It has been previously shown that caloric restriction, which induces ketosis, reduces microvessel density in mouse and human brain tumor models, suggesting an anti-angiogenic effect. We now report that in animals fed KetoCal® (KC) (4:1 fat:protein/carbohydrates) ad libitum, peritumoral edema is significantly reduced early in tumor progression when compared to those fed a standard rodent diet (SD). Western blot analysis showed a reduction of platelet endothelial cell adhesion molecule 1 (PECAM1/CD31) in tumors from animals maintained on KC. These results were supported by immunohistochemical staining for CD31 which also revealed abnormal vessel structure in the tumors from animals fed SD but not in those fed KC, suggesting a normalizing effect by the KC. Furthermore gene expression profiling demonstrated that KC decreases expression of a group of genes involved in angiogenesis and vessel structuring including the genes encoding vascular endothelial growth factor B (VEGFB) and angiopoetin 1 receptor (TEK), integrin beta 1 (ITGB1), urokinase-type plasminogen activator (PLAU) and tissue inhibitor of metalloproteases 1 (TIMP1). Taken together our data suggests that KC alters the angiogenic processes involved in malignant progression of gliomas. A greater understanding of the effects of the ketogenic diet as an adjuvant therapy will allow for a more rational approach to its clinical use. Citation Format: Eric C. Woolf, Julie A. Charlton, Qingwei Liu, Gregory Turner, Mark C. Preul, Adrienne C. Scheck. Metabolic therapy reduces expression of PECAM-1/CD31 and decreases peritumoral edema in a mouse model of malignant glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1440. doi:10.1158/1538-7445.AM2014-1440

Induced pluripotent stem cell-derived neural stem cells transduced with baculovirus encoding CD40 ligand for immunogene therapy in mouse models of breast cancer.

The interaction between CD40 ligand (CD40L) and CD40 can directly inhibit growth of CD40-positive carcinoma cells and may indirectly inhibit tumor growth through coordination of immune responses. Many efforts in CD40L cancer gene therapy have been focused on direct CD40L gene transfer into malignant target cells. This in vivo gene therapy approach relies on high-efficiency gene transfer and could be technically challenging for the treatment of certain cancers, especially multisite metastases. We report herein an alternative means of using the tumor-homing property of neural stem cells (NSCs) to deliver CD40L molecules into tumor tissues. NSCs were derived from human induced pluripotent stem cells, transduced in vitro with a baculoviral vector encoding CD40L, and intravenously injected into immunocompetent mice with orthotopic and metastatic breast cancers. Through a bystander mechanism of intercellular transfer of CD40L from the donor NSCs to tumor target cells, the treatment impeded tumor growth, leading to prolonged survival of the tumor-bearing mice. We further showed that compared with the stem cell-based gene therapy that employed a suicide gene, the CD40L immunogene therapy did not cause liver and kidney injury in the treated mice. This new approach may be particularly valuable for metastatic cancer treatments after systemic stem cell administration.

Indomethacin Prevents the Progression of Thoracic Aortic Aneurysm in Marfan Syndrome Mice.

Marfan syndrome (MFS), an inherited disorder of connective tissue characterized by abnormalities in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The high mortality in untreated patients is primarily due to aneurysm and dissection of the ascending aorta. The complex pathogenesis of MFS involves changes in transforming growth factor β (TGF-β) signaling, increased matrix metalloproteinase (MMP) expression, and fragmentation of the extracellular matrix. A number of studies have demonstrated increased counts of macrophages and T cells in the ascending aorta of persons or mouse models of MFS, but the efficacy of anti-inflammatory therapy in mouse models of MFS has not yet been assessed. FBN1 underexpressing mgR/mgR Marfan mice were treated with oral indomethacin. Treatment was begun at the age of three weeks and continued for 8 weeks, following which the aorta of wild type as well as treated and untreated mgR/mgR mice was compared. Indomethacin treatment led to a statistically significant reduction of aortic elastin degeneration and macrophage infiltration, as well as a lessening of MMP-2, MMP-9, and MMP-12 upregulation. Additionally, indomethacin decreased both cyclooxygenases 2 (COX-2) expression and activity in the aorta of mgR/mgR mice. COX-2-mediated inflammatory infiltrate contributes to the progression of aortic aneurysm in mgR/mgR mice, providing evidence that COX-2 is a relevant therapeutic target in MFS-associated aortic aneurysmal disease. COX-2 mediated inflammatory infiltration plays an important role in the pathogenesis of aortic aneurysm disease in MFS.

Abstract 5661: Nanoparticle based combinatorial siRNA therapy against human hepatocellular carcinoma (HCC) .

Abstract Background: We have previously demonstrated the therapeutic effect of lipid nanoparticles (LNP) loaded with single siRNA targeting CSN5 and WEE1 against human HCC in mouse models. Aim: To test the benefit of a combinatorial versus single siRNA therapy in mouse models of human HCC and to identify molecular mechanism(s) involved in therapeutic response by extensive microarray analyses. Materials and Methods: LNP formulations of chemically modified siRNAs targeting CSN5 and WEE1 were produced by Tekmira® Pharmaceuticals. SCID-beige mice were used for subcutaneous (Hep3B) and intra-hepatic (Huh7-luciferase) tumor transplantation. Mice with established tumors were treated intravenously with 4 mg/kg single agent siRNA or 2 mg/kg + 2 mg/kg siCSN5:siWEE1 siRNA co-encapsulated in the same LNP. Tumors were assayed following 1 to 9 injected doses. Tumor progression in the Huh7 orthotopic model was monitored by bioluminescence imaging and metastases were evaluated at endpoint. Results: Significant target silencing was observed in tumors after single or repeat administration with no antagonism between siRNAs occurring in the CSN5:WEE1 combination. We observed significant inhibition of tumor growth and metastases in mice treated with active siRNAs compared to LNP containing a non-targeting control sequence. Potency was not lost with siCSN5:siWEE1 LNP, where the concentration of each siRNA is halved in combination, relative to the most efficacious single agent. Data from preliminary microarray analyses demonstrate a strong transcriptome difference between each treatment group. Conclusion: We demonstrate a clear efficacy of a LNP based combinatorial siRNA therapy in human mouse models of HCC. Overall this therapy led to a significant decrease of tumor size induced by mRNA inhibition Citation Format: Thomas Decaens, Valentina M. Factor, Iva Kulic, Jesper B. Andersen, Daekwan Seo, Yun-Han Lee, Adam D. Judge, Elizabeth A. Conner, Ian MacLachlan, Snorri S. Thorgeirsson. Nanoparticle based combinatorial siRNA therapy against human hepatocellular carcinoma (HCC) . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5661. doi:10.1158/1538-7445.AM2013-5661

Abstract 3926: Molecular imaging of VEGFR expression modulation by antiangiogenic therapy in mouse model of human breast cancer.

Abstract Antiangiogenic therapy targeting VEGF/VEGFR signaling is an important strategy for cancer treatment, which is currently used in mono-therapy or combination therapy settings. However, the emerging resistance to antiangiogenic therapy as well as varied clinical response calls for the development of early, preferably noninvasive, markers of treatment response. In this study we used a recently developed molecular imaging tracer, site-specifically 99mTc-labeled engineered single-chain VEGF derivative, to monitor the VEGF receptor expression during the course of sunitinib antiangiogenic therapy in orthotopic human breast cancer model MDA-MB-231. The treatment protocol included two cycles of therapy delivered daily for 5 days (80mg/kg, orally) with a 5-day vacation between the cycles. Micro SPECT/CT imaging was performed longitudinally up to day 16. Animals in the control group received vehicle solution only. Tumors were excised and stained for the expression of CD31 endothelial cell marker. Experimental results suggest a highly heterogeneous vascular regression response to the first round of treatment, with significantly decreased tracer uptake and CD31 prevalence in some but not all tumors. As judged by the increase in tracer uptake and CD31 prevalence, a significant vascular rebound took place in all tumors after the vacation period. Judging by the same criteria, the second round of treatment was significantly less effective than the first round. Taken together, these results suggests that noninvasive molecular imaging of VEGF receptors reflects vascular regression and rebound in individual tumors and can provide clinically relevant information for monitoring and scheduling antiangiogenic therapy. Citation Format: Wenlian Zhu, Yoshinori Kato, Ronnie C. Mease, Susanta Sarkar, Marina V. Backer, Joseph M. Backer, Dmitri Artemov. Molecular imaging of VEGFR expression modulation by antiangiogenic therapy in mouse model of human breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3926. doi:10.1158/1538-7445.AM2013-3926

Abstract 933: Modeling acquired resistance to EGFR-directed therapies in mouse models of lung cancer.

Abstract Seventy percent of patients with Epidermal Growth Factor Receptor (EGFR) mutant lung cancer respond to treatment with the tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib. Despite this high response rate, patients almost inevitably develop resistance to these drugs on average within a year of starting drug treatment. Acquired resistance to EGFR TKIs is most commonly due to the emergence of a secondary mutation (T790M) in EGFR (in 50% of cases), amplification of the genes encoding the ERBB2 and MET receptor tyrosine kinases in 12 and 5% of cases, respectively, and phenotypic transformation of the adenocarcinomas to small cell lung cancer (5% of cases). Previously, in an effort to develop strategies to overcome T790M-mediated resistance, we generated tetracycline-inducible transgenic mice that express the EGFRL858R+T790M in the lung epithelium. Upon administration of doxycycline these mice develop lung adenocarcinomas that are resistant to TKIs. However, the combination of the irreversible TKI afatinib and the EGFR antibody cetuximab showed dramatic responses in these transgenic mice. These preclinical studies led to a clinical trial of these agents, which has showing a promising 30% response rate in patients with EGFR mutant tumors resistant to TKIs. However, tumors also acquire resistance to this drug combination and the mechanisms of resistance to afatinib+cetuximab are currently unknown. We set out to identify these mechanisms using xenograft and transgenic mouse models of EGFR mutant lung cancer. Transgenic mice with EGFRL858R+T790M-induced tumors were treated with afatinib+cetuximab using an intermittent dosing strategy that we had previously used to generate erlotinib-resistant tumors in mice with EGFRL858R and EGFRDEL-induced tumors. 75% of mice develop afatinib+cetuximab resistant tumors after three month-long rounds of treatment. The same treatment strategy applied to xenografts harboring subcutaneous tumors induced by EGFRDEL+T790M gave rise to resistant tumors in 20% of cases. Analysis of the afatinib+cetuximab resistant tumors performed to date has not revealed additional mutations in the EGFR transgene, the ERBB2 kinase domain or KRAS. Additional sequencing studies and examination of signaling pathway alterations in the resistant tumors are ongoing. Uncovering mechanisms of resistance to this drug combination will allow the development of strategies to treat tumors that acquire resistance to EGFR-directed therapies. Citation Format: Valentina Pirazzoli, Elisa de Stanchina, Jungfeng Xia, Zhongming Zhao, William Pao, Katerina A. Politi. Modeling acquired resistance to EGFR-directed therapies in mouse models of lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 933. doi:10.1158/1538-7445.AM2013-933

Abstract 5462: MEDI3185, a potent anti-CXCR4 antibody, inhibits tumor cell migration, signaling and tumor growth in preclinical models.

Abstract The chemokine receptor CXCR4 is a seven-transmembrane G-protein coupled receptor that mediates chemotaxis and cell migration upon stimulation via its ligand, stromal-derived factor 1 (SDF-1), also called CXCL12. CXCR4 is normally expressed on bone marrow stem and progenitor cells, various circulating lymphocytes, endothelial precursor cells, tissue macrophages and fibroblasts but the aberrant overexpression of CXCR4 is linked to various hematological malignancies, solid tumors and metastatic neoplasms. Moreover, CXCR4 overexpression is correlated with poor prognosis in many types of cancer, including breast, ovarian, colon, pancreatic, AML and glioblastomas. CXCR4 inhibition using siRNA, small-molecule and peptide inhibitors has demonstrated that it can inhibit tumor growth by blocking tumor cell survival/proliferation, metastasis, angiogenesis and tumor immune infiltrates. Here we describe a novel, fully human, antagonistic antibody to CXCR4, MEDI3185, which blocks SDF-1 binding to CXCR4. MEDI3185 has picomolar binding affinity to human CXCR4 and exhibits no significant binding to other chemokine receptors such as CCR4 or CXCR3. In vitro studies demonstrated that MEDI3185 inhibited tumor cell migration, blocked SDF-1 induced tumor cell signaling and induced apoptosis of tumor cells. In preclinical human tumor xenograft models in mouse, MEDI3185 showed single-agent tumor growth inhibition in multiple myeloma and B-cell Burkitt's lymphoma models and had combination activity in ovarian models. In addition, MEDI3185 extended survival as combination therapy in mouse models of CLL and also blocked lung tumor burden in a disseminated ovarian model. Combined, these data suggest that MEDI3185 is a potent CXCR4 antibody for the treatment of both hematological and solid tumors because it has pleiotropic effects on tumor biology that may enhance the efficacy of the current standard of care. Citation Format: Adeela Kamal, Youzhen Wang, Philipp Steiner, Anne-Marie Mazzola, Leslie Wetzel, Melissa Passino, Brenda McDermott, Keven Huang, Vahe Bedian, Norman Greenberg. MEDI3185, a potent anti-CXCR4 antibody, inhibits tumor cell migration, signaling and tumor growth in preclinical models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5462. doi:10.1158/1538-7445.AM2013-5462

Oncogenic RAS pathway activation promotes resistance to anti-VEGF therapy through G-CSF–induced neutrophil recruitment

Granulocyte-colony stimulating factor (G-CSF) promotes mobilization of CD11b(+)Gr1(+) myeloid cells and has been implicated in resistance to anti-VEGF therapy in mouse models. High G-CSF production has been associated with a poor prognosis in cancer patients. Here we show that activation of the RAS/MEK/ERK pathway regulates G-CSF expression through the Ets transcription factor. Several growth factors induced G-CSF expression by a MEK-dependent mechanism. Inhibition of G-CSF release with a MEK inhibitor markedly reduced G-CSF production in vitro and synergized with anti-VEGF antibodies to reduce CD11b(+)Ly6G(+) neutrophil mobilization and tumor growth and led to increased survival in animal models of cancer, including a genetically engineered mouse model of pancreatic adenocarcinoma. Analysis of biopsies from pancreatic cancer patients revealed increased phospho-MEK, G-CSF, and Ets expression and enhanced neutrophil recruitment compared with normal pancreata. These results provide insights into G-CSF regulation and on the mechanism of action of MEK inhibitors and point to unique anticancer strategies.

Evaluation of TorsinA as a target for Parkinson disease therapy in mouse models.

Parkinson disease (PD) is a common and disabling disorder. No current therapy can slow or reverse disease progression. An important aspect of research in this field is target validation, a systematic approach to evaluating the likelihood that modification of a certain molecule, mechanism or biological pathway may be useful for the development of pharmacological or molecular treatments for the disease. TorsinA, a member of the AAA+ family of chaperone proteins, has been proposed as a potential target of neuroprotective therapy. TorsinA is found in Lewy bodies in human PD, and can suppress toxicity in cellular and invertebrate models of PD. Here, we evaluated the neuroprotective properties of torsinA in mouse models of PD based on intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as well as recombinant adeno associated virus (rAAV) induced overexpression of alpha-synuclein (α-syn). Using either transgenic mice with overexpression of human torsinA (hWT mice) or mice in which torsinA expression was induced using an rAAV vector, we found no evidence for protection against acute MPTP intoxication. Similarly, genetic deletion of the endogenous mouse gene for torsinA (Dyt1) using an rAAV delivered Cre recombinase did not enhance the vulnerability of dopaminergic neurons to MPTP. Overexpression of α-syn using rAAV in the mouse substantia nigra lead to a loss of TH positive neurons six months after administration, and no difference in the degree of loss was observed between transgenic animals expressing forms of torsinA and wild type controls. Collectively, we did not observe evidence for a protective effect of torsinA in the mouse models we examined. Each of these models has limitations, and there is no single model with established predictive value with respect to the human disease. Nevertheless, these data do seem to support the view that torsinA is unlikely to be successfully translated as a target of therapy for human PD.

JMJD2A Promotes Cellular Transformation by Blocking Cellular Senescence through Transcriptional Repression of the Tumor Suppressor CHD5

Senescence is a cellular response preventing tumorigenesis. The Ras oncogene is frequently activated or mutated in human cancers, but Ras activation is insufficient to transform primary cells. In a search for cooperating oncogenes, we identify the lysine demethylase JMJD2A/KDM4A. We show that JMJD2A functions as a negative regulator of Ras-induced senescence and collaborates with oncogenic Ras to promote cellular transformation by negatively regulating the p53 pathway. We find CHD5, a known tumor suppressor regulating p53 activity, as a target of JMJD2A. The expression of JMJD2A inhibits Ras-mediated CHD5 induction leading to a reduced activity of the p53 pathway. In addition, we show that JMJD2A is overexpressed in mouse and human lung cancers. Depletion of JMJD2A in the human lung cancer cell line A549 bearing an activated K-Ras allele triggers senescence. We propose that JMJD2A is an oncogene that represents a target for Ras-expressing tumors.

VraT/YvqF Is Required for Methicillin Resistance and Activation of the VraSR Regulon in Staphylococcus aureus

Staphylococcus aureus infections caused by strains that are resistant to all forms of penicillin, so-called methicillin-resistant S. aureus (MRSA) strains, have become common. One strategy to counter MRSA infections is to use compounds that resensitize MRSA to methicillin. S. aureus responds to diverse classes of cell wall-inhibitory antibiotics, like methicillin, using the two-component regulatory system VraSR (vra) to up- or downregulate a set of genes (the cell wall stimulon) that presumably facilitates resistance to these antibiotics. Accordingly, VraS and VraR mutations decrease resistance to methicillin, vancomycin, and daptomycin cell wall antimicrobials. vraS and vraR are encoded together on a transcript downstream of two other genes, which we call vraU and vraT (previously called yvqF). By producing nonpolar deletions in vraU and vraT in a USA300 MRSA clinical isolate, we demonstrate that vraT is essential for optimal expression of methicillin resistance in vitro, whereas vraU is not required for this phenotype. The deletion of vraT also improved the outcomes of oxacillin therapy in mouse models of lung and skin infection. Since vraT expressed in trans did not complement a vra operon deletion, we conclude that VraT does not inactivate the antimicrobial. Genome-wide transcriptional microarray experiments reveal that VraT facilitates resistance by playing a necessary regulatory role in the VraSR-mediated cell wall stimulon. Our data prove that VraTSR comprise a novel three-component regulatory system required to facilitate resistance to cell wall agents in S. aureus. We also provide the first in vivo proof of principle for using VraT as a sole target to resensitize MRSA to β-lactams.

Convection-Enhanced Delivery of Neprilysin: A Novel Amyloid-β-Degrading Therapeutic Strategy

Enzymatic degradation contributes to the control of intracerebral amyloid-β (Aβ) peptide levels. Previous studies have demonstrated the therapeutic potential of viral vector-mediated neprilysin (NEP) gene therapy in mouse models of Alzheimer's disease (AD). However, clinical translation of NEP gene therapy is limited by ethical and practical considerations. In this study we have assessed the potential of convection-enhanced delivery (CED) as a means of elevating intracerebral NEP level and activity and degrading endogenous Aβ. We analyzed the interstitial and perivascular distribution of NEP following CED into rat striatum. We measured NEP protein level, clearance, activity, and toxicity by ELISA for NEP and synaptophysin, NEP-specific activity assay, and immunohistochemistry for NEP, NeuN, glial fibrillary acidic protein and Iba1. We subsequently performed CED of NEP in normal aged rats and measured endogenous Aβ by ELISA. CED resulted in widespread distribution of NEP, and a 20-fold elevation of NEP protein level with preservation of enzyme activity and without evidence of toxicity. CED in normal, aged rats resulted in a significant reduction in endogenous Aβ(40) (p = 0.04), despite rapid NEP clearance from the brain (half-life ~3 h). CED of NEP has therapeutic potential as a dynamically controllable Aβ(40)-degrading therapeutic strategy for AD. Further studies are required to determine the longer term effects on Aβ (including Aβ(42)) and on cognitive function.

Combined In Vivo Molecular and Anatomic Imaging for Detection of Ovarian Carcinoma-Associated Protease Activity and Integrin Expression in Mice

Most patients with epithelial ovarian cancer (EOC) experience drug-resistant disease recurrence. Identification of new treatments is a high priority, and preclinical studies in mouse models of EOC may expedite this goal. We previously developed methods for magnetic resonance imaging (MRI) for tumor detection and quantification in a transgenic mouse model of EOC. The goal of this study was to determine whether three-dimensional (3D) fluorescence molecular tomography (FMT) and fluorescent molecular imaging probes could be effectively used for in vivo detection of ovarian tumors and response to therapy. Ovarian tumor-bearing TgMISIIR-TAg mice injected with fluorescent probes were subjected to MRI and FMT. Tumor-specific probe retention was identified in vivo by alignment of the 3D data sets, confirmed by ex vivo fluorescent imaging and correlated with histopathologic findings. Mice were treated with standard chemotherapy, and changes in fluorescent probe binding were detected by MRI and FMT. Ovarian tumors were detected using probes specific for cathepsin proteases, matrix metalloproteinases (MMPs), and integrin αvβ3. Cathepsin and integrin αvβ3 probe activation and retention correlated strongly with tumor volume. MMP probe activation was readily detected in tumors but correlated less strongly with tumor volume. Tumor regression associated with response to therapy was detected and quantified by serial MRI and FMT. These results demonstrate the feasibility and sensitivity of FMT for detection and quantification of tumor-associated biologic targets in ovarian tumors and support the translational utility of molecular imaging to assess functional response to therapy in mouse models of EOC.

AAV-Mediated Gene Therapy in Mouse Models of Recessive Retinal Degeneration

In recent years, more and more mutant genes that cause retinal diseases have been detected. At the same time, many naturally occurring mouse models of retinal degeneration have also been found, which show similar changes to human retinal diseases. These, together with improved viral vector quality allow more and more traditionally incurable inherited retinal disorders to become potential candidates for gene therapy. Currently, the most common vehicle to deliver the therapeutic gene into target retinal cells is the adenoassociated viral vector (AAV). Following delivery to the immuno-privileged subretinal space, AAV-vectors can efficiently target both retinal pigment epithelium and photoreceptor cells, the origin of most retinal degenerations. This review focuses on the AAV-based gene therapy in mouse models of recessive retinal degenerations, especially those in which delivery of the correct copy of the wild-type gene has led to significant beneficial effects on visual function, as determined by morphological, biochemical, electroretinographic and behavioral analysis. The past studies in animal models and ongoing successful LCA2 clinical trials, predict a bright future for AAV gene replacement treatment for inherited recessive retinal diseases.

Inhibitory Monoclonal Antibodies against Mouse Proteases Raised in Gene-Deficient Mice Block Proteolytic Functions in vivo

Identification of targets for cancer therapy requires the understanding of the in vivo roles of proteins, which can be derived from studies using gene-targeted mice. An alternative strategy is the administration of inhibitory monoclonal antibodies (mAbs), causing acute disruption of the target protein function(s). This approach has the advantage of being a model for therapeutic targeting. mAbs for use in mouse models can be obtained through immunization of gene-deficient mice with the autologous protein. Such mAbs react with both species-specific epitopes and epitopes conserved between species. mAbs against proteins involved in extracellular proteolysis, including plasminogen activators urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA), their inhibitor PAI-1, the uPA receptor (uPAR), two matrix metalloproteinases (MMP9 and MMP14), as well as the collagen internalization receptor uPARAP, have been developed. The inhibitory mAbs against uPA and uPAR block plasminogen activation and thereby hepatic fibrinolysis in vivo. Wound healing, another plasmin-dependent process, is delayed by an inhibitory mAb against uPA in the adult mouse. Thromboembolism can be inhibited by anti-PAI-1 mAbs in vivo. In conclusion, function-blocking mAbs are well-suited for targeted therapy in mouse models of different diseases, including cancer.

Hyperactive behaviour in the mouse model of mucopolysaccharidosis IIIB in the open field and home cage environments

Mucopolysaccharidosis IIIB (MPS IIIB) is a lysosomal storage disorder characterized by severe behavioural disturbances and progressive loss of cognitive and motor function. There is no effective treatment, but behavioural testing is a valuable tool to assess neurodegeneration and the effect of novel therapies in mouse models of disease. Several groups have evaluated behaviour in this model, but the data are inconsistent, often conflicting with patient natural history. We hypothesize that this discrepancy could be due to differences in open field habituation and home cage behaviour. Eight-month-old wild-type and MPS IIIB mice were tested in a 1-h open field test, performed 1.5 h after lights on, and a 24-h home cage behaviour test performed after 24 h of acclimatization. In the 1-h test, MPS IIIB mice were hyperactive, with increased rapid exploratory behaviour and reduced immobility time. No differences in anxiety were seen. Over the course of the test, differences became more pronounced with maximal effects at 1 h. The 24-hour home cage test was less reliable. There was evidence of increased hyperactivity in MPS IIIB mice, however, immobility was also increased, suggesting a level of inconsistency in this test. Performance of open field analysis within 1-2 h after lights on is probably critical to achieving maximal success as MPS IIIB mice have a peak in activity around this time. The open field test effectively identifies hyperactive behaviour in MPS IIIB mice and is a significant tool for evaluating effects of therapy on neurodegeneration.

Immunotherapeutic role of Ag85B as an adjunct to antituberculous chemotherapy

BackgroundImmunotherapy to enhance the efficiency of the immune response in tuberculosis patients and to eliminate the persisters could be an additional valuable strategy to complement anti-mycobacterial chemotherapy. This study was designed to assess the immunotherapeutic potential of Ag85B as an adjunct to chemotherapy and its effect against active and persister bacteria left after therapy in mouse model of tuberculosis.Methods6-8 week old female Balb/c mice were infected with Mycobacterium tuberculosis and treated with chemotherapy or immunotherapy. Protective efficacy was measured in terms of bacterial counts in lungs and spleen. Immune correlates of protection in terms of Th1 and Th2 cytokines were measured by ELISA.ResultsTherapeutic effect of Ag85B was found to be comparable to that of short term dosage of antituberculous drugs (ATDs). The therapeutic effect of ATDs was augmented by the simultaneous treatment with rAg85B and moreover therapy with this protein allowed us to reduce ATD dosage. This therapy was found to be effective even in case of drug persisters. The levels of antigen specific IFNγ and IL-12 were significantly increased after immunotherapy as compared to the basal levels; moreover antigen specific IL-4 levels were depressed on immunotherapy with Ag85B.ConclusionWe demonstrated in this study that the new combination approach using immunotherapy and concurrent chemotherapy should offer several improvements over the existing regimens to treat tuberculosis. The therapeutic effect is associated not only with initiating a Th1 response but also with switching the insufficient Th2 immune status to the more protective Th1 response.

Regulation of the ubiquitin proteasome and calpain systems in a dog model of duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an incurable and lethal genetic disorder characterized by progressive muscle wasting caused by the loss of functional dystrophin. Activation of both calpain and the proteasome degradation systems has been reported in the pathogenesis of DMD in skeletal muscle. In fact, calpain and proteasome inhibitors have been used as therapies in mouse models. Here we investigated specific calpain and proteasome activities of 5 skeletal muscles and the left ventricle (LV) in a more severe canine model of DMD (golden retriever muscular dystrophy [GRMD]). The expression of ubiquitin proteasome system components and calpain 1 and 2 were increased in GRMD skeletal muscle generally, but decreased in the LV. We identified significant increases in all three proteasome activities (trypsin‐, chymotrypsin‐, and caspase‐like) in only 1 GRMD muscle and no activation of any proteasome activities in the LV. Calpain 1&2 activities were increased in 2 GRMD skeletal muscles and the LV compared to controls. Furthermore, protease activity of most GRMD skeletal muscles studied correlate with the degree of atrophy and hypertrophy found in the individual muscles. As proteasome and calpain inhibitors continue to be considered as potential therapies for DMD, this work provides evidence for the variable activation of these systems in different skeletal muscles and the heart. Supported by NIH R01HL104129.

G-CSF-initiated myeloid cell mobilization and angiogenesis mediate tumor refractoriness to anti-VEGF therapy in mouse models

Recent studies suggest that tumor-associated CD11b(+)Gr1(+) myeloid cells contribute to refractoriness to antiangiogenic therapy with an anti-VEGF-A antibody. However, the mechanisms of peripheral mobilization and tumor-homing of CD11b(+)Gr1(+) cells are unclear. Here, we show that, compared with other cytokines [granulocyte-macrophage colony stimulating factor (GM-CSF), stromal derived factor 1alpha, and placenta growth factor], G-CSF and the G-CSF-induced Bv8 protein have preferential expression in refractory tumors. Treatment of refractory tumors with the combination of anti-VEGF and anti-G-CSF (or anti-Bv8) reduced tumor growth compared with anti-VEGF-A monotherapy. Anti-G-CSF treatment dramatically suppressed circulating or tumor-associated CD11b(+)Gr1(+) cells, reduced Bv8 levels, and affected the tumor vasculature. Conversely, G-CSF delivery to animals bearing anti-VEGF sensitive tumors resulted in reduced responsiveness to anti-VEGF-A treatment through induction of Bv8-dependent angiogenesis. We conclude that, at least in the models examined, G-CSF expression by tumor or stromal cells is a determinant of refractoriness to anti-VEGF-A treatment.