ANTICANCER RESEARCH 33: 347-354 (2013) Characterization of Structure and Direct Antigen Presentation by Dendritic/Tumor-fused Cells as Cancer Vaccines SHIGEO KOIDO2,3 and JIANLIN GONG1,3 1Department of Medicine, Boston University School of Medicine, Boston, MA, U.S.A.; 2Division of Gastroenterology and Hepatology, Department of Internal Medicine, The Jikei University School of Medicine, Kashiwa, Chiba, Japan; 3Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, U.S.A. Abstract. Background: Previous work has shown that fusion immune responses (2-5). However, a major drawback of these of dendritic cells (DCs) and tumor cells induces potent strategies comes from the limited number of known tumor antitumor immune responses. However, little is known on peptides available in many human leukocyte antigen (HLA) whether fused cells directly present tumor-associated contexts. An alternative strategy for inducing antitumor antigens (TAAs) through major histocompatibility complex immunity is the use of cells derived from fusion of DCs and (MHC) class I and II pathways in the context of co- tumor cells (6). The fusion of DCs and tumor cells through stimulatory molecules. Materials and Methods: Fusion cells chemical (7-10), electrical (11-13), physical (14-16), or viral were generated between DCs and MC38 carcinoma cells (17-19) means creates heterokaryons, combining the stably expressing mucin-1 (MUC1) by polyethylene glycol. machinery needed for immune stimulation with presentation The characterization of structure and antigen presentation of a large repertoire of TAAs. The fusion approach offers by fused cells was examined by immunoelectron microscopic several advantages for tumor-peptide presentation and and flow cytometric analyses. Results: The cytoplasm from subsequent induction of antitumor immunity including (20- both cellular entities was integrated, while their nuclei were 22): (i) processing and presentation of unidentified TAAs, thus independently preserved. Short-term culture gave fused cells circumventing the daunting task of identifying individual sufficient time to integrate and directly display MUC1 TAAs; (ii) presentation of multiple TAAs, thus increasing the through MHC class I and II pathways in the context of co- frequency of responding T-cells and maximizing antitumor stimulatory molecules. Conclusion: DC-derived molecules immunity; (iii) presentation of TAAs in the context of and TAAs are presumably synthesized at separate sites of abundant co-stimulatory molecules, thus avoiding the potential fused cells, to converge and complex with each other. induction of tolerance; and (iv) activation of polyclonal CD4+ and CD8+ T-cells, thus providing T-cell help for the induction Dendritic cells (DCs) are most potent antigen-presenting cells of CTL responses. In DC/tumor-fused cells, efficient CTL (APCs) that have been used in cancer vaccines because of induction is closely correlated with the level of the fusion their ability to initiate Cluster of Differentiation (CD)8+ efficiency (23). Therefore, we attempted to apply simple cytotoxic T-lymphocyte (CTL)-mediated immune responses techniques to enrich DC and tumor cell fusion and examined (1). Thus, DC-based cancer vaccines are considered to be a the events surrounding the direct presentation of TAAs by promising approach for boosting antitumor responses (1). immunoelectron microscopic analysis. Various strategies have been developed to deliver tumor- associated antigens (TAAs) into DCs with tumor RNA, tumor Materials and Methods lysates, or apoptotic tumor cells to elicit and boost antitumor Cell culture. Murine MC38 colon adenocarcinoma (C57BL/6) cell line stably transfected with a MUC1 cDNA (MC38/MUC1) (6, 24) was maintained in Dulbecco's modified Eagle’s minimal essential Correspondence to: Shigeo Koido, Department of Gastroenterology medium (DMEM), supplemented with 10% heat-inactivated (Fetal and Hepatology, The Jikei University School of Medicine, 163-1 Calf Serum) FCS, 2 mM L-glutamine, 100 U/ml penicillin, Kashiwa-shita, Kashiwa, Chiba 277-8564, Japan. Tel: +81 100 μg/ml streptomycin, and 400 μg/ml geneticin (G418; Life 471641111, Fax: +81 471633488, e-mail: [email protected] Technologies, Tokyo, Japan). DCs were generated from the bone marrow of wild-type C57BL/6 mice by culture in 20 ng/ml Key Words: Dendritic cell, cancer vaccine, antigen presentation, granulocyte macrophage colony-stimulating factor (GM-CSF) (Sigma, fusion. St. Louis, MO, USA) for five days, as described elsewhere (25). 0250-7005/2013 $2.00+.40 347 ANTICANCER RESEARCH 33: 347-354 (2013) Fusion of DCs and tumor cells. Fused cells were generated with BD Pharmingen), or CD86 (GL1; BD Pharmingen) and then with purified DCs and MC38/MUC1 in the presence of polyethylene streptavidin-gold-anti-rat IgG (AuroProbe EM-G10, 1:10 dilution; glycol (PEG) (molecular weight=1,450) in dimethyl sulfoxide Amersham Life Sciences). The specimens were processed, sectioned, (DMSO) solution (Sigma-Aldrich, St. Louis, MO, USA), as and examined with a JEOL100CX TEM to determine gold particle- described elsewhere (6). Briefly, DCs and MC38/MUC1 cells were labeled molecules on the cell surface. For subcellular localization of mixed at a 10:1 ratio in serum-free pre-warmed RPMI-1640. After MHC class II and MUC1 antigenic peptides, the sorted DC/MUC1 centrifugation, the mixed cell pellets were gently resuspended in cells were prepared for ultrathin cryosectioning and immunogold pre-warmed 50% PEG solution (1 ml per 5×107 cells) for 5 min at labeling (30). Briefly, cells were fixed in 2% paraformaldehyde and room temperature. Subsequently, the PEG solution was diluted by 1% acrolein for 3-4 days, washed twice in PBS with 0.15 M glycine, slow addition and mixing with 1, 2, 4, 8, and 16 ml of serum-free and finally embedded in 10% gelatin, which was solidified on ice. pre-warmed medium to a volume of 50 ml. Cell pellets obtained Small gelatin blocks were infiltrated with 2.3 M sucrose for 3 h at after centrifuging at 170 ×g (1,000 rpm) were resuspended in RPMI- 4˚C and then frozen in liquid nitrogen. Ultrathin cryosections were 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM cut and picked-up on carbon-coated gold grids. Immunogold labeling glutamine, 10 mM nonessential amino acids, 1 mM sodium was then performed as described elswhere (18). Briefly, the ultrathin pyruvate, 10% NCTC-109 medium, 10 U/ml penicillin, 100 μg/ml cryosections were carefully washed with PBS containing 0.5% bovine streptomycin, and 10 ng/ml recombinant murine GM-CSF, and serum albumin (BSA) and 0.15% glycine, pH 7.4, blocked with 1% cultured for eight days. Unfused tumor cells grow firmly attached egg albumin in PBS, incubated with a 1:100 dilution of anti-MUC1 to the plates, whereas fused DC/tumor cells (DC/MUC1) grow mAb for 30 min, and then washed and incubated with a 1:10 dilution loosely in the wells and are suspended in the medium. Fused cells of gold-conjugated anti-mouse IgG (5-nm particles). The sections were selected and purified by gentle pipetting, and firmly attached were washed six times with PBS and stained with a 1:100 dilution of tumor cells were discarded. biotinylated-mAb against MHC class II mAb for 30 min, and incubated with a 1:10 dilution of gold-conjugated-streptavidin mAb Flow cytometry. DCs, MC38/MUC1, and DC/MUC1 were incubated against rat IgG (10-nm particles). The cryosections were washed and with fluorescein isothiocyanate (FITC)-conjugated monoclonal mounted on a thin film of 1.25% methylcellulose, and examined with antibodies (mAb) against MUC1 (HMPV; BD Pharmingen, San a JEOL 100 CX TEM. Diego, CA, USA) (26-28) and phycoerythrin (PE)-conjugated mAb against MHC class II (M5/114; BD Pharmingen) for 45 min on ice. Results Cells were washed, fixed, and analyzed by flow cytometry (FACScan; BD Immunocytometry System, NJ, USA) with the Characterization of DC/MUC1 cell preparations. Bone CellQuest software (BD Biosciences, NJ, USA). The cell aggregates were eliminated by gating-out before flow cytometric analysis. The marrow-derived murine DCs were generated in the presence fusion efficiency was determined by dual expression of tumor of GM-CSF for five days. DCs displayed a characteristic marker, MUC1, and DC marker, MHC class II molecules. phenotype with expression of MHC class II molecules (Figure 1A). Moreover, MC38/MUC1 cells expressed high Transmission electron microscopy (TEM). For observation of cell levels of MUC1 antigen (Figure 1B). In this study, fusions morphology and intracellular structure, DC/MUC1 cells were stained of DCs and MC38/MUC1 cells were generated through PEG with FITC-conjugated mAb against MUC1 (HMPV; BD Pharmingen) treatment, which is a straightforward procedure (31). and PE-conjugated mAb against MHC class II (M5/114; BD Generation of DCs with GM-CSF for more than seven days’ Pharmingen), then sorted by MoFlo (Cytomation, Fort Collins, CO, USA) with Summit v3.0 analysis software. DCs, MC38/MUC1, and culture resulted in cell death and consequently reduced sorted DC/MUC1 fused cells were fixed with 1.5% glutaraldehyde in fusion efficiency through PEG treatment (data not shown). 0.1 M cacodylate buffer, pH 7.4, for 1 h at 48˚C. The specimens were Therefore, DCs were generated in five days’ culture and washed, treated with 1% osmium tetroxide in 0.1 M cacodylate buffer, immediately used for fusion in this study. After initiation of and passed through an alcohol gradient. They were further treated the fusion process, some DCs were fused to MC38/MUC1 with propylene oxide and then embedded in plastic resin. Ultrathin cells, while most of the PEG-treated cells remained as cell sections were cut with an MT2 Sorvall ultra microtome (Thermo aggregates (Figure 1C). When the cell aggregates were gated Fisher Scientific, MA, USA) and examined with a JEOL 100 CX TEM (JEOL USA, Inc., MA, USA) (29). out, a small number of cells were double-positive for MUC1 and MHC class II.