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Immunological Characterization of Glioblastoma Cells for Immunotherapy

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Immunological Characterization of Glioblastoma Cells for Immunotherapy ANTICANCER RESEARCH 33: 2525-2534 (2013) Immunological Characterization of Glioblastoma Cells for Immunotherapy TAE-YOUNG JUNG1,5,6, YOO-DUK CHOI2,6, YOUNG-HEE KIM5, JE-JUNG LEE3,6, HYUNG-SEOK KIM2,6, JU-SUN KIM7, SANG-KI KIM7, SHIN JUNG1,5,6 and DUCK CHO4,6 Departments of 1Neurosurgery, 2Pathology, 3Hematology, 4Laboratory Medicine, and 5Brain Tumor Research Laboratory, Chonnam National University Hwasun Hospital, Jeollanam-do, Republic of Korea; 6Research Institute of Medical Sciences, Chonnam National University, Gwangju, Republic of Korea; 7Department of Companion & Laboratory Animal Science, Kongju National University, Yesan, Republic of Korea Abstract. The aim of this study was the immunological CD99 and ERBB2 in U251, U87 and U343 cell lines and characterization of glioblastoma cells. Glioblastoma cell lines tissues. These highly-expressed TAAs such as BIRC5, CD99 were cultured in serum and serum-free neurobasal (NBE) and ERBB2 in glioblastoma tissue could be the targets for medium conditions. These cell lines were characterized by immunotherapy. U87 and U343 cell lines could be useful for flow cytometry, reverse transcription-polymerase chain studying the efficacy of immunotherapy related to various reaction (RT-PCR), western blot and natural killer (NK) cell- TAAs and NK cell immunotherapy. cytotoxicity assays. A previously described NK cell expansion method that uses K562 cells expressing interleukin (IL)-15 Glioblastoma is a lethal malignant brain tumor and the and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) was used. RT- current standard treatment of glioblastoma consists of surgical PCR and western blots for the expression of tumor-associated resection followed by concomitant chemoradiation therapy antigens (TAAs), were carried out in 32 glioblastoma and with temozolomide (1). Even with aggressive treatment, the seven normal brain tissues. U87 and U343 tumor cell lines median survival of patients is 14.6 months. Such failure leads showed increased expression for major histocompatibility many to believe that an aggressive combination of standard complex (MHC)-I and -II molecules. No significant therapies, along with other biologically-based therapies, could differences in the levels of CD133, MHC class I/II, MHC be necessary to improve the survival of patients with class I-related chain A (MICA), MICB, UL16 binding protein glioblastoma. Intra-tumoural immune response occurred 1-3 (ULBP 1-3) expression in these cell lines and in NK cell frequently in glioblastoma and there was a consistent cytotoxicity were observed between serum and NBE response, even after conventional treatment (2). conditions. Regardless of culture conditions, U87 and U343 One of the most promising strategies of immunotherapy cell lines were sensitive to expanded NK cells, with median was dendritic cells (DCs) pulsed with tumor-derived whole- cytotoxicities at 4:1 effector/target ratio of 43.2% and 46.5%, lysate/peptides to initiate T cell-mediated antitumor immunity respectively. In RT-PCR, U343 and U87 showed the (3-5). One study discussed the safety and immunogenicity of expression of most TAAs at a high ratio compared with U251. a novel vaccination with α-type 1 polarized DCs loaded with Western blots demonstrated positive expression for BIRC5, synthetic peptides for glioma-associated antigen epitopes with EPH receptor-A2 (EPHA2), interleukin-13 receptor α2 (IL13RA2), Chitinase 3-like 1 (CHI3L1, also known as human cartilage-glycoprotein-39 or YKL40) and premelanosome Correspondence to: Duck Cho, MD, Ph.D., Department of Laboratory Medicine, Chonnam National University Hwasun protein (PMEL, also known as glycoprotein 100) for recurrent Hospital, 160, Ilsim-ri, Hwasun-eup, Hwasun-gun, Jeollanam-do, malignant glioma, which achieved progression-free status 519-809, Republic of Korea. Tel: +82 613797951, Fax: +82 lasting at least 12 months (3). Human glial tumors express a 613797984, e-mail: [email protected] and Yoo-Duk Choi, MD, variety of tumor-associated antigens (TAAs), which can be the Ph.D., Department of Pathology, Chonnam National University targets for immunotherapy (6-8). Hwasun Hospital, 160, Ilsim-ri, Hwasun-eup, Hwasun-gun, In addition to DC-based immunotherapy, another one of Jeollanam-do, 519-809, Republic of Korea. Tel: +82 622205688, strategies was the use of activated NK cells for Fax: +82 613797099, e-mail: [email protected] immunotherapy of patients with glioblastoma. It was Key Words: Characterization, glioblastoma, immunotherapy, tumor- reported that NK cells recognized and killed human associated antigens, U251, U87, U343 cells. glioblastoma cells with stem cell-like properties and these 0250-7005/2013 $2.00+.40 2525 ANTICANCER RESEARCH 33: 2525-2534 (2013) cells, although resistant to freshly-isolated NK cells, were antibodies, trastuzumab, or fluorescence-tagged isotype control highly susceptible to lysis mediated by both allogeneic and antibody for 15 min on ice. Trastuzumab-stained cells were washed autologous IL-2 (or IL-15)-activated NK cells (9). In recent and incubated with 1×PBS (1% BSA) containing fluorescein isothiocyanate (FITC) -conjugated or phycoerythrin (PE)-conjugated years there has been a remarkable improvement in a method antibody to human IgG1 for 15 min on ice. After washing all samples for highly efficient ex vivo NK cell expansion and activation. with 1xPBS (1% BSA), stained cells were then fixed in 2% Remarkable rates of highly cytotoxic NK cell expansion paraformaldehyde and analyzed by a FACSAria (BD Biosciences, San were reported using the protocol that employs irradiated, Jose, CA,USA). The experiment was repeated twice. Values are genetically engineered K562 cell line modified to express indicated as mean relative fluorescence intensity (MRFI), that is the membrane-bound IL-21 (mbIL21) or membrane bound IL- ratio of the mean of fluorescence intensity of cells stained with the 15 (mbIL15) and 4-1BB ligand (K562-mb15-41BBL) (10- selected mAb and that of the negative control; values of MRFI ≥2 were considered significant. 12). However, little has been reported on the immunological characterization of glioblastoma cell lines for the possible Flow cytometry-based NK cytotoxicity assay. Flow cytometric usefulness of expanded NK cells-based immunotherapy for cytotoxicity assay using Calcein AM-stained expanded NK cells glioblastoma. were performed as previously described (14). Target (T) cells Here, we investigated the expressions of TAAs in (U251, U343, U87) were cultured in serum and serum-free glioblastoma cell lines and human glioblastoma tissues to find neurobasal (NBE) medium conditions. To expand NK cells [effector appropriate targets for immunotherapy and the immunological (E) cells], peripheral blood mononuclear cells were isolated and co- characterization of glioblastoma cell lines for assessing the cultured with 100 Gy-irradiated K562 leukemia cells that had been modified to express 4-1BB ligand and membrane-bound interleukin efficacy of immunotherapy related with various TAAs and (IL)-15 (K562-mb15-41BBL cells), kindly provided by Dr. NK cell immunotherapy. Campana (National University of Singapore, Singapore), for 3 weeks in the presence of IL-2 and IL-15. The target cells and Materials and Methods effector cells were co-cultured at effector-to-target (E:T) ratios ranging from 4:1 to 1:1 for 4 h at 37˚C in a humidified incubator Cell lines and cell culture. Human glioma cell lines U87, U343 and containing 5% CO2. Cells were acquired and the date were analyzed U251 were obtained from the Korean Cell Line Bank, Seoul, Korea, by BD CellQuest™ Pro Software. and from the Brain Tumor Research Center, University of California, San Francisco, CA, USA, respectively. All cell lines Reverse transcription-polymerase chain reaction (RT-PCR) for were routinely grown in Dulbecco’s modified Eagle’s medium TAAs. Total RNA was isolated from U251, U87 and U343 cell lines (DMEM; GibcoBRL, Gaithersburg, MD., USA) supplemented with cultured under serum and NBE conditions, glioblastoma tissues 10% fetal bovine serum (FBS; GibcoBRL) at 37˚C in a humidified (n=32) and seven samples of brain from trauma patients using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). 95% air and 5% CO2 atmosphere, termed here ‘serum’ conditions. All cell lines were also cultured with serum-free neurobasal medium A total of 1μg of RNA was reverse transcribed to synthesize the (Invitrogen, Carlsbad, CA, USA) containing 50 ng/ml of both basic cDNA for RT-PCR. For the first-strand synthesis, 1μg of the purified fibroblast growth factor (FGF; Peprotech, Rocky Hill, NJ, USA) and total RNA was incubated with oligo (dT) (0.5 μg/μl; Promega, epidermal growth factor (EGF; Peprotech) supplemented with N2 Madison, WI, USA) for 5 min at 70˚C, followed by the addition of and B27 supplement without vitamin A and L-glutamine, termed buffer containing 4 μl of 5× Reaction Buffer (Promega), 1μl of dNTP ‘NBE’ conditions. (10 mM each), 3.5 μl of 25 mM MgCl2, 2 μl of RNase Inhibitor (40 Brain tissue specimens were obtained from patients who U/μl; Promega), and 1 μl of reverse transcriptase (200 U/μl; Promega). underwent surgery in the Department of Neurosurgery at Chonnam Reactions with differing numbers of PCR cycles were run for each National University Hwasun Hospital. Tissues were fixed in transcript and β-actin (ACTB) was used as an internal control. formalin and embedded in paraffin or immediately frozen in liquid RT-PCR was carried out for the expression of TAAs (EPHA2, nitrogen. Sections from all samples were evaluated histologically CHI3L1, AIM2, PMEL, MAGEA1, EGFR, ERBB2, TRNAP2, and graded according to the World Health Organization grading ABCC3, SOX2, CD99 type 1, CD99 type 2, IL13RA2 and BIRC5). system by two board-certified neuropathologists (13). The Primers and PCR conditions used for RT-PCR are summarized in Institutional Review Board of Chonnam National University Table I. Hwasun Hospital approved this study. Western blotting. Surgically-resected tumor samples were Flow cytometric analysis for the expression of CD133, Major homogenized with lysis buffer [50 mM Tris (pH 8.0), 5 mM EDTA, histocompatibility complex (MHC) class I and II, and NKG2D ligands 150 mM sodium chloride, 0.5% deoxycholic acid, 0.1% sodium (NKG2DLs).
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