ANTICANCER RESEARCH 33: 347-354 (2013)

Characterization of Structure and Direct Presentation by Dendritic/Tumor-fused Cells as 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 (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- 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, , antigen presentation, granulocyte macrophage colony-stimulating factor (GM-CSF) (Sigma, fusion. St. Louis, MO, USA) for five days, as described elsewhere (25).

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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. However, after eight days’ culture, the Immunoelectron microscopic analysis. Pre-embedded immunogold proportion of cell aggregates had decreased, whereas the labeling techniques were used to determine the antigen-presentation double-positive populations were drastically increased on the sorted DC/MUC1 cell surface. Cells were pelleted in (Figure 1D), suggesting that most of the cell aggregates are phosphate-buffered saline (PBS) containing 0.5 ml 1% egg albumin precursors of fused cells. After eight days’ culture, for 30 min and then incubated with a 1:100 dilution of mAb against DC/MUC1 cells were integrated to a single entity and were MUC1 (HMPV; BD Pharmingen) for 45 min at 4˚C. Gold-conjugated loosely adherent to the culture dish, whereas tumor/tumor mAb against mouse IgG (AutoProbe EM-G5, 1:10 dilution; Amersham Life Sciences, Arlington Heights, IL, USA) was added for fusions and unfused tumor cells were attached firmly to the 30 min at 4˚C. After washing twice with PBS, the cells were dish. Thus, it was possible to select the DC/MUC1 cells by incubated with a 1:100 dilution of biotinylated mAb against MHC gentle pipetting and then collecting the loosely adherent class II mAb (M5/114; BD Pharmingen), MHC class I (M1/42.3.9.8; cells. In contrast, incubating the mixture of DCs and

348 Koido et al: Direct Antigen Presentation by DC/Tumor Fusions

Figure 1. Characterization of fused DC/tumor cell preparations. Cell morphology of DCs (A), MC38/MUC1 cells (B), fused DC/MUC1 cell preparations cultured for two days, after initiating the fusion process (C), DC/MUC1 fusion cell preparations cultured for eight days after initiating fusion process (D), and DCs mixed with MC38/MUC1 cells and cultured for eight days (E) were examined under an inverted microscope (left hand panel). Cells were stained with fluorescein isothiocyanate-anti-MUC1 and phycoerythrin-anti-MHC class II mAb. Cell aggregates were gated out (middle panel) at the time of fluorescence-activated cell sorting analysis (right hand panel). Similar results were obtained in repeated experiments. Magnification A-D, ×40; E, ×20.

MC38/MUC1 cells for eight days resulted in a small number Morphology of DC/MUC1 cells. To examine the global and of cells being positive for MUC1 and MHC class II (Figure intracellular morphology, DC, MC38/MUC1, and sorted 1E). These cells may have been DCs that had phagocytosed DC/MUC1 cells were preparad and viewed under TEM. DCs MC38/MUC1 fragments. The data indicate that physical exhibited characteristic morphology, with a lobulated nucleus contact is not sufficient to generate fused DC/tumor cells. and a distinctive pattern of euchromatin and heterochromatin, Interestingly, viewing under the inverted microscope revealed and displayed short cytoplasmic processes (Figure 2A, left morphological changes in DC/MUC1 cells (Figure 1D). panel). In contrast, MC38/MUC1 cells had a large nucleus

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Figure 2. Cell surface and intracellular structure of fused DC/MUC1 cells. Preparations of fused DC/MUC1 cells were stained with fluorescein isothiocyanate-anti-MUC1 and phycoerythrin-anti-MHC class II mAbs and sorted by MoFlo. Intracellular structure of DCs (left hand panel), MC38/MUC1 cells (middle panel), and sorted fused DC/MUC1 cells (right hand panel) were examined by immunoelectron microscopy (A, ×4,800; B, ×72,500). The lower right hand panel shows the integration of DCs and tumor cells (arrow). T-N, tumor nucleus; DC-N, DC nucleus.

with conspicuous chromatin and a relatively smooth cell ability to present TAAs directly through MHC class I and surface (Figure 2A, middle panel). Compared with DCs and class II pathways in the presence of co-stimulatory MC38/MUC1 cells, sorted DC/MUC1 cells had multiple molecules. To test this hypothesis, we used an nuclei derived from DCs and MC38/MUC1. Moreover, immunoelectron microscopic technique that is able to bridge DC/MUC1 cells had short cytoplasmic processes (Figure 2A, the information gap between molecular biology and right panel). Interestingly, the cytoplasm of DCs and ultrastructural studies, placing macromolecular functions MC38/MUC1 cells was integrated in sorted DC/MUC1 cells within a cellular context. DCs, MC38/MUC1, and sorted (Figure 2B, right panel, arrows), whereas their nuclei DC/MUC1 cells were dually-stained with pre-embedded remained separate entities. immunogold-labeled anti-MUC1 mAb (5-nm particles) and anti-MHC class I, class II, or CD86 mAb (10-nm particles), Antigen presentation through MHC class I and class II and examined by immunoelectron microscopy. DC/MUC1 pathways by DC/MUC1 cells. As previously reported (12, cells expressed both MUC1 and MHC class II molecules on 22), both CD4+ and CD8+ T-cells are involved in induction the surface (Figure 3A, right panel). Importantly, MUC1 and of antitumor immunity by fused DC/tumor cells. Therefore, MHC class II molecules were co-localized on the DC/MUC1 we hypothesize that the fused DC/tumor cells have the cell surface, suggesting the physical association of these two

350 Koido et al: Direct Antigen Presentation by DC/Tumor Fusions

Figure 3. Co-localization of MUC1 and MHC class I and class II molecules in fused DC/MUC1 cells. Sorted fused DC/MUC1 cells were prepared, stained with pre-embedded immunogold-labeled anti-MUC1 mAb (gold 5-nm size) and anti-MHC class II mAb (gold 10-nm size) (A), anti-MHC class I mAb (gold 10-nm size) (B), or anti-CD86 mAb (gold 10-nm size) (C) and examined by immunoelectron microscopy (×47,500). The middle and right hand panels are enlarged views of the area shown in the inset of images in the left hand and middle panels, respectively, showing co- localization of MUC1 (narrow arrow) and MHC class II, class I, or CD86 (wide arrow) on the surface of fused DC/MUC1 cells.

molecules (Figure 3A, right panel). MUC1 and MHC class Subcellular co-localization of MUC1 and MHC class II I molecules were also co-localized on the DC/MUC1 cell molecules in fused DC/MUC1 cells. Given the observation surface (Figure 3B, right panel). Although CD86 molecules that co-localization of MUC1 and MHC class II molecules were expressed on the fusion cells with MUC1 antigen, they expression is found on the DC/MUC1 cell sufrace, it is of were not as closely associated as those of MHC class I and interest to determine how this antigen processing and class II with MUC1 (Figure 3C, right panel). These results presentation pathway is preserved in the fused cells. To suggest that the fused DC/MUC1 cells possess the ability to address this issue, sorted DC/MUC1 cells were prepared for present MUC1 through both MHC class I and class II immunoelectron microscopic analysis. The labeling for pathways in the presence of CD86. In contrast, DCs MUC1 (5-nm particles) and MHC class II (10-nm particles) expressed MHC class II, CD86, and not MUC1 on the were co-localized in the subcellular structures of DC/MUC1 surface, while MC38/MUC1 were positive for MUC1 but not cells (Figure 4). The expression of MUC1 and MHC class II MHC class II and CD86 (data not shown). was well-preserved in the fused cells. In contrast, only MHC

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Figure 4. Intracellular distribution of MUC1 and MHC class II molecules in fused DC/MUC1 cells. Ultrathin cryosections were stained with an immunogold-labeled anti-MUC1 (gold 5-nm size) mAb and an anti-MHC class II (gold 10-nm size) mAb and examined by immunoelectron microscopy (×72,500). The right hand panels are an enlarged view of vesicles in fused DC/MUC1 cells showing co-localization of MUC1 (narrow arrow) and MHC class II (wide arrow).

class II expression and MUC1 was detected in the Unlike electrofusion, fusion by PEG treatment is an active subcellular structures of DCs and MC38/MUC1 cells, and evolving process, thus, it is conceivable that a larger respectively (data not shown). This finding suggests that initial contact surface between the DC and tumor cell leads direct antigen presentation through MHC class II molecules the integration of these cells. In this study, short-term culture can be sustained in fused DC/tumor cells. of DC and tumor cells promoted fusion efficiency. The duration of culture of PEG-mediated cells may allow for the Discussion cells undergoing fusion sufficient time to integrate and display TAAs through MHC class I and class II pathways. Although the level of fusion efficiency is closely correlated Indeed, the fusion efficiency was lower immediately after the with antitumor immunity (23), a variation of fusion efficiency fusion process was initiated, however, a short-duration culture has been reported (17, 32). Fusion efficiency, at least in part, resulted in more than a 10-fold increase in fusion efficiency. depends on the cell types used for fusion (13) as well as the Prolonged culture should be avoided since unfused tumor fusion methods used (33). There are some differences in the cells overgrow. Previously, most investigators vaccinated the sensitivity of cells to PEG treatment (data not shown), thus, it fused cell preparations into patients shortly after the fusion is desirable to perform a dose–response test to evaluate the process (34, 35). It is likely that such cell preparations may conditions of PEG treatment for cells and to determine the not at this time be in an optimal form to present TAAs to the optimal exposure time. Moreover, any improper handling of host immune cells. Moreover, it is also useful to activate DCs cells, such as excessive force or deprivation of culture by Toll-like receptor agonists before fusion (23). medium prior to fusion, may also have a detrimental effect There are two different pathways for antigen presentation. on the health of cells and subsequently on fusion efficiency. Endogenously-synthesized are processed and

352 Koido et al: Direct Antigen Presentation by DC/Tumor Fusions presented through the MHC class I-restricted pathway. In References contrast, exogenous proteins are processed and displayed in association with MHC class II molecules through the 1 Steinman RM: The dendritic cell system and its role in endocytic pathway (36). However, little is known whether immunogenicity. Annu Rev Immunol 9: 271-296, 1991. 2 Nestle FO, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R, fused DC/tumor cells can process and present TAAs directly Burg G and Schadendorf D: Vaccination of patients through MHC class I and class II pathways in the presence of with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 4: co-stimulatory molecules. 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RJ, Rosenberg SA and Shu S: Hybrids of co-stimulatory molecules and may result in simultaneous of dendritic cells and tumor cells generated by electrofusion activation of CD4+ and CD8+ T-cells. simultaneously present immunodominant from multiple human tumor-associated antigens in the context of MHC class I Competing Interests and class II molecules. J Immunol 170: 5317-5325, 2003. 13 Salomskaite-Davalgiene S, Cepurniene K, Satkauskas S, Venslauskas MS and Mir LM: Extent of cell electrofusion in The Authors declare that they have no competing interests. vitro and in vivo is cell line dependent. Anticancer Res 29: 3125- 3130, 2009. Acknowledgements 14 Lindner M and Schirrmacher V: Tumour cell dendritic cell fusion for : Comparison of therapeutic This work was supported by National Cancer Institute Grant R01 efficiency of polyethylen-glycol versus electro-fusion protocols. CA87057; by the US Department of Defense Eur J Clin Invest 32: 207-217, 2002. 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