Browsing by Subject "in vivo study"

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    Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment
    (American Association for Cancer Research Inc., 2014) Cekic, C.; Linden J.
    Adenosine A2A receptor (A2AR) blockade enhances innate and adaptive immune responses. However, mouse genetic studies have shown that A2AR deletion does not inhibit the growth of all tumor types. In the current study, we showed that growth rates for ectopic melanoma and bladder tumors are increased in Adora2a-/- mice within 2 weeks of tumor inoculation. A2AR deletion in the host reduced numbers of CD8+ T cells and effector-memory differentiation of all T cells. To examine intrinsic functions in T cells, we generated mice harboring a T-cell-specific deletion of A2AR. In this host strain, tumor-bearing mice displayed increased growth of ectopic melanomas, decreased numbers of tumor-associated T cells, reduced effector-memory differentiation, and reduced antiapoptotic IL7Rα (CD127) expression on antigen-experienced cells. Intratumoral pharmacologic blockade similarly reduced CD8+ T-cell density within tumors in wild-type hosts. We found that A2AR-proficient CD8+ T cells specific for melanoma cells displayed a relative survival advantage in tumors. Thus, abrogating A2AR signaling appeared to reduce IL7R expression, survival, and differentiation of T cells in the tumor microenvironment. One implication of these results is that the antitumor effects of A2AR blockade that can be mediated by activation of cytotoxic T cells may be overcome in some tumor microenvironments as a result of impaired T-cell maintenance and effector-memory differentiation. Thus, our findings imply that the efficacious application of A2AR inhibitors for cancer immunotherapy may require careful dose optimization to prevent activation-induced T-cell death in tumors. ©2014 AACR.
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    Herpes simplex virus 1 amplicon vector-mediated siRNA targeting epidermal growth factor receptor inhibits growth of human glioma cells in vivo
    (2005) Saydam O.; Glauser, D.L.; Heid I.; Turkeri G.; Hilbe, M.; Jacobs, A.H.; Ackermann, M.; Fraefel, C.
    In primary glioblastomas and other tumor types, the epidermal growth factor receptor (EGFR) is frequently observed with alterations, such as amplification, structural rearrangements, or overexpression of the gene, suggesting an important role in glial tumorigenesis and progression. In this study, we investigated whether posttranscriptional gene silencing by vector-mediated RNAi to inhibit EGFR expression can reduce the growth of cultured human gli36 glioma cells. To "knock down" EGFR expression, we have created HSV-1-based amplicons that contain the RNA polymerase III-dependent H1 promoter to express double-stranded hairpin RNA directed against EGFR at two different locations (pHSVsiEGFR I and pHSVsiEGFR II). We demonstrate that both pHSVsiEGFR I and pHSVsiEGFR II mediated knock-down of transiently transfected full-length EGFR or endogenous EGFR in a dose-dependent manner. The knock-down of EGFR resulted in the growth inhibition of human glioblastoma (gli36-luc) cells both in culture and in athymic mice in vivo. Cell cycle analysis and annexin V staining revealed that siRNA-mediated suppression of EGFR induced apoptosis. Overall HSV-1 amplicons can mediate efficient and specific posttranscriptional gene silencing. Copyright © The American Society of Gene Therapy.
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    Identification of novel neutralizing single-chain antibodies against vascular endothelial growth factor receptor 2
    (2011) Erdag, B.; Koray Balcioglu, B.; Ozdemir Bahadir, A.; Serhatli, M.; Kacar O.; Bahar, A.; Seker, U.O.S.; Akgun, E.; Ozkan, A.; Kilic, T.; Tamerler, C.; Baysal, K.
    Human vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2/kinase domain receptor [KDR]) play a crucial role in angiogenesis, which makes the VEGFR-2 signaling pathway a major target for therapeutic applications. In this study, a single-chain antibody phage display library was constructed from spleen cells of mice immunized with recombinant human soluble extracellular VEGFR-2/KDR consisting of all seven extracellular domains (sKDR D1-7) to obtain antibodies that block VEGF binding to VEGFR-2. Two specific single-chain antibodies (KDR1.3 and KDR2.6) that recognized human VEGFR-2 were selected; diversity analysis of the clones was performed by BstNI fingerprinting and nucleotide sequencing. The single-chain variable fragments (scFvs) were expressed in soluble form and specificity of interactions between affinity purified scFvs and VEGFR-2 was confirmed by ELISA. Binding of the recombinant antibodies for VEGFR-2 receptors was investigated by surface plasmon resonance spectroscopy. In vitro cell culture assays showed that KDR1.3 and KDR2.6 scFvs significantly suppressed the mitogenic response of human umbilical vein endothelial cells to recombinant human VEGF 165 in a dose-dependent manner, and reduced VEGF-dependent cell proliferation by 60% and 40%, respectively. In vivo analysis of these recombinant antibodies in a rat cornea angiogenesis model revealed that both antibodies suppressed the development of new corneal vessels (p < 0.05). Overall, in vitro and in vivo results disclose strong interactions of KDR1.3 and KDR2.6 scFvs with VEGFR-2. These findings indicate that KDR1.3 and KDR2.6 scFvs are promising antiangiogenic therapeutic agents. © 2011 International Union of Biochemistry and Molecular Biology, Inc.
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    MST1 is a multifunctional caspase-independent inhibitor of androgenic signaling
    (2011) Cinar, B.; Collak F.K.; Lopez, D.; Akgul, S.; Mukhopadhyay, N.K.; Kilicarslan, M.; Gioeli, D.G.; Freeman, M.R.
    The MST1 serine - threonine kinase, a component of the RASSF1-LATS tumor suppressor network, is involved in cell proliferation and apoptosis and has been implicated in cancer. However, the physiologic role of MST1 in prostate cancer (PCa) is not well understood. Here, we investigated the possibility of a biochemical and functional link between androgen receptor (AR) and MST1 signaling. We showed that MST1 forms a protein complex with AR and antagonizes AR transcriptional activity as shown by coimmunoprecipitation (co-IP), promoter reporter analysis, and molecular genetic methods. In vitro kinase and site-specific mutagenesis approaches indicate that MST1 is a potent AR kinase; however, the kinase activity of MST1 and its proapoptotic functions were shown not to be involved in inhibition of AR. MST1 was also found in AR - chromatin complexes, and enforced expression of MST1 reduced the binding of AR to a well-characterized, androgen-responsive region within the prostate-specific antigen promoter. MST1 suppressed PCa cell growth in vitro and tumor growth in mice. Because MST1 is also involved in regulating the AKT1 pathway, this kinase may be an important new link between androgenic and growth factor signaling and a novel therapeutic target in PCa. ©2011 AACR.
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    Myeloid expression of adenosine a2A receptor suppresses T and NK cell responses in the solid tumor microenvironment
    (American Association for Cancer Research Inc., 2014) Cekic, C.; Day, Y.-J.; Sag, D.; Linden J.
    High concentrations of adenosine in tumor microenvironments inhibit antitumor cytotoxic lymphocyte responses. Although T cells express inhibitory adenosine A2A receptors (A2AR) that suppress their activation and inhibit immune killing of tumors, a role for myeloid cell A2ARs in suppressing the immune response to tumors has yet to be investigated. In this study, we show that the growth of transplanted syngeneic B16F10 melanoma or Lewis lung carcinoma cells is slowed in Adora2af/f-LysMCre+/- mice, which selectively lack myeloid A2ARs. Reduced melanoma growth is associated with significant increases in MHCII and IL12 expression in tumor-associated macrophages and with >90% reductions in IL10 expression in tumor-associated macrophages, dendritic cells (DC), and Ly6C+ or Ly6G+ myeloid-derived suppressor cells (MDSC). Myeloid deletion of A2ARs significantly increases CD44 expression on tumor-associated T cells and natural killer (NK) cells. Depletion of CD8+ T cells or NK cells in tumor-bearing mice indicates that both cell types initially contribute to slowing melanoma growth in mice lacking myeloid A2A receptors, but tumor suppression mediated by CD8+ T cells is more persistent. Myeloid-selective A2AR deletion significantly reduces lung metastasis of melanomas that express luciferase (for in vivo tracking) and ovalbumin (as a model antigen). Reduced metastasis is associated with increased numbers and activation of NK cells and antigen-specific CD8+ T cells in lung in filtrates. Overall, the findings indicate that myeloid cell A2ARs have direct myelosuppressive effects that indirectly contribute to the suppression of T cells and NK cells in primary and metastatic tumor microenvironments. The results indicate that tumor-associated myeloid cells, including macrophages, DCs, and MDSCs all express immunosuppressive A2ARs that are potential targets of adenosine receptor blockers to enhance immune killing of tumors. ©2014 AACR.
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    Reduced field-of-view DWI with robust fat suppression and unrestricted slice coverage using tilted 2D RF excitation
    (John Wiley and Sons Inc., 2016) Banerjee, S.; Nishimura, D. G.; Shankaranarayanan, A.; Saritas, E. U.
    Purpose: Reduced field-of-view (rFOV) diffusion-weighted imaging (DWI) using 2D echo-planar radiofrequency (2DRF) excitation has been widely and successfully applied in clinical settings. The purpose of this work is to further improve its clinical utility by overcoming slice coverage limitations without any scan time penalty while providing robust fat suppression. Theory and Methods: During multislice imaging with 2DRF pulses, periodic sidelobes in the slice direction cause partial saturation, limiting the slice coverage. In this work, a tilting of the excitation plane is proposed to push the sidelobes out of the imaging section while preserving robust fat suppression. The 2DRF pulse is designed using Shinnar-Le Roux algorithm on a rotated excitation k-space. The performance of the method is validated via simulations, phantom experiments, and high in-plane resolution in vivo DWI of the spinal cord. Results: Results show that rFOV DWI using the tilted 2DRF pulse provides increased signal-to-noise ratio, extended coverage, and robust fat suppression, without any scan time penalty. Conclusion: Using a tilted 2DRF excitation, a high-resolution rFOV DWI method with robust fat suppression and unrestricted slice coverage is presented. This method will be beneficial in clinical applications needing large slice coverage, for example, axial imaging of the spine, prostate, or breast. Magn Reson Med 76:1668–1676, 2016. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine