Antitumor effects of vaccination with dendritic cells transfected with modified receptor for hyaluronan-mediated motility mRNA in a mouse glioma model

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Object

The receptor for hyaluronan-mediated motility (RHAMM) is frequently overexpressed in brain tumors and was recently identified as an immunogenic antigen by using serological screening of cDNA expression libraries. In this study, which was conducted using a mouse glioma model, the authors tested the hypothesis that vaccination with dendritic cells transfected with RHAMM mRNA induces strong immunological antitumor effects.

Methods

The authors constructed a plasmid for transduction of the mRNAs transcribed in vitro into dendritic cells, which were then used to transport the intracellular protein RHAMM efficiently into major histocompatibility complex class II compartments by adding a late endosomal–lysosomal sorting signal to the RHAMM gene. The dendritic cells transfected with this RHAMM mRNA were injected intraperitoneally into the mouse glioma model 3 and 10 days after tumor cell implantation. The antitumor effects of the vaccine were estimated by the survival rate, histological analysis, and immunohistochemical findings for immune cells.

Mice in the group treated by vaccination therapy with dendritic cells transfected with RHAMM mRNA survived significantly longer than those in the control groups. Immunohistochemical analysis revealed that greater numbers of T lymphocytes containing T cells activated by CD4+, CD8+, and CD25+ were found in the group vaccinated with dendritic cells transfected with RHAMM mRNA.

Conclusions

These results demonstrate the therapeutic potential of vaccination with dendritic cells transfected with RHAMM mRNA for the treatment of malignant glioma.

Abbreviations used in this paper:MHC = major histocompatibility complex; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; RHAMM = receptor for hyaluronan-mediated motility.

Article Information

Address reprint requests to: Koji Kajiwara, M.D., Department of Neurosurgery, Yamaguchi University School of Medicine, 1-1-1, Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan. email: koji@yamaguchi-u.ac.jp.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Schematic construction of a plasmid cassette for tumor antigen mRNA transcription. The signal sequence fragment (SS) and transmembrane–cytoplasmic domain (TM-Cyto) were amplified from TRP-2 cDNA by using PCR. The PCR products were cloned as a Hind III-Pst I signal sequence fragment and a BamH I-Sma I transmembrane–cytoplasmic domain fragment into the pSP64 vector. The Ampr label denotes the β-lactamase coding region.

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    Schematic construction of the RHAMM mRNA transcription cassette. The mouse RHAMM genes were separately cloned into a mouse TRP-2 cassette plasmid.

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    Graph showing the survival ratio after tumor cell implantation into the mouse brain. The group vaccinated with dendritic cells transfected with modified RHAMM mRNA (squares) survived significantly longer than the mice vaccinated with dendritic cells without transfection (diamonds) or the ones with no treatment (triangles, 20 mice in each group; p < 0.001, log-rank analysis of survival curves in each group). The group vaccinated with dendritic cells transfected with modified RHAMM mRNA also survived significantly longer than the ones vaccinated with chloroquine-treated dendritic cells transfected with modified RHAMM mRNA (circles, 15 mice; p < 0.001, log-rank analysis of survival curves).

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    Photomicrographs of coronal sections of brain obtained in mice killed at 20 days after implantation of tumor cells; the sections were stained for histological investigation. The tumor volume of the sections obtained in mice vaccinated with dendritic cells transfected with modified RHAMM mRNA was smaller than that of the other groups. A: Brain tumor implanted after the animal received vaccination with dendritic cells transfected with modified RHAMM mRNA. B: Brain tumor in a mouse that received no treatment. H & E, original magnification × 12.5.

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    Immunocytochemical analysis performed with RHAMM to show its expression in mouse glioma tissues (A) and in the normal brain (B). In the mouse glioma models in which KR158B tumors were implanted, RHAMM was consistently overexpressed homogeneously. In contrast, RHAMM was not detected in the normal brain tissues. Bars = 50 μm.

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    Photomicrographs obtained for immunocytochemical analysis, in which immune cell markers were used to stain the mouse brains collected 20 days after the implantation of tumor cells. The CD45+ leukocytes were detected within and around the tumors in all groups (A, no treatment; B, vaccination with dendritic cells transfected with modified RHAMM mRNA). In the brains implanted with KR158B glioma cells in the group that received no treatment, the infiltrating cells were mainly macrophages (C), and only a few CD3+ lymphocytes were present in the tumor or surrounding tissue (D). In contrast, in the group vaccinated with dendritic cells transfected with modified RHAMM mRNA, a greater number of CD3+ lymphocytes (E) infiltrated in and around the tumor, and these lymphocytes contained not only CD8+ (F) but also CD4+ (G) T cells, which contained T cells activated by CD25+ (H). Bar = 50 μm.

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