J Korean Med Sci.  2011 Oct;26(10):1270-1276. 10.3346/jkms.2011.26.10.1270.

Intratumoral Administration of Secondary Lymphoid Chemokine and Unmethylated Cytosine-phosphorothioate-guanine Oligodeoxynucleotide Synergistically Inhibits Tumor Growth in Vivo

• Oh SM
• Oh K
• Lee DS

• KMID: 1785982
• DOI: http://doi.org/10.3346/jkms.2011.26.10.1270

Abstract

Secondary lymphoid tissue chemokine (SLC), which is expressed in T cell zones of secondary lymphoid organs, including the spleen and lymph nodes, strongly recruits both T lymphocytes and mature dendritic cells. As appropriate interaction of tumor-specific T cells and mature dendritic cells, equipped with tumor antigens, is a prerequisite for effective T cell immunity against established tumors, we mobilized lymphocytes and dendritic cells to tumor sites by intratumoral injection of secondary lymphoid tissue chemokine-Fc (SLC-Fc) fusion protein using the B16F10 murine melanoma model. Activation of dendritic cells, another prerequisite for the effective activation of naive tumor-specific T cells, was achieved by the addition of immunostimulatory cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG-ODN) into the tumor site. Intratumoral administration of SLC-Fc or CpG-ODN revealed antitumor effects against B16F10 murine melanoma grown in the subcutaneous space. Co-treatment of SLC-Fc and CpG-ODN displayed synergistic effects in reducing the tumor size. The synergistic antitumor effect in co-treatment group was correlated with the synergistic/additive increase in the infiltration of CD4+ T cells and CD11c+ dendritic cells in the tumor mass compared to the single treatment groups. These results suggest that the combined use of chemokines and adjuvant molecules may be a possible strategy in clinical tumor immunotherapy.

Keyword

SLC; CpG-ODN; Melanoma; Tumor Immunotherapy

MeSH Terms

Animals
Antigens, CD11c/immunology
CD4-Positive T-Lymphocytes/immunology
Cell Line, Tumor
Cell Proliferation/drug effects
Chemokine CCL21/*administration & dosage/pharmacology
Chemotaxis, Leukocyte
Dendritic Cells/immunology/metabolism
Immunotherapy
Injections, Intralesional
Melanoma, Experimental/*immunology/*therapy
Mice
Mice, Inbred C57BL
Oligodeoxyribonucleotides/*administration & dosage/pharmacology
T-Lymphocytes/immunology/metabolism

Figure

• 

  Fig. 1 SLC-Fc is chemotactic for CD4+ and CD8+ T cells in vitro. A total of 1 × 106 lymphocytes from peripheral LNs of B6 male mice were incubated in the upper chamber of the Transwell. After 4 hr incubation, migrated cells in the lower chamber were stained with anti-CD4, anti-CD8, and anti-B220 antibodies and analyzed by FACSCalibur. (A) Flow cytometric analysis of migrated cells in the lower chamber with culture media, 300 ng SLC-Fc, or 200 ng recombinant SLC (rSLC). Data shown are representative of two independent experiments. (B, C) Chemotaxis assay performed with 0.2 µg recombinant SLC or 3, 0.3, or 0.03 µg purified SLC-Fc in the lower chamber. The chemotaxis index was calculated as described in Materials & Methods. Data represent means ± SD, based on three independent determinations using samples in triplicate. *P ≤ 0.05; †P ≤ 0.005.

• 

  Fig. 2 Intratumoral injection of CpG-ODN induces the rejection of established tumors. Ten- to 12-week-old B6 male mice were inoculated subcutaneously with 2 × 105 B16F10 melanoma cells in the shaved left flank. Tumor size was recorded every 2 days. Intratumoral injection was performed when the tumor volume reached approximately 150 µL. (A) SLC-Fc treatment (3 µg), two or four times. (B) CpG-ODN treatment (1, 5, or 10 µg) once. (C) CpG-ODN treatment (1, 5, or 10 µg), two times. Data represent means ± SD of two independent determinations with n = 6 mice/group. *P < 0.05; †P < 0.01; ‡P < 0.001.

• 

  Fig. 3 Synergistic antitumor effect of SLC-Fc and CpG-ODN. Ten- to 12-week-old B6 male mice were inoculated subcutaneously with 2 × 105 B16F10 melanoma cells in the shaved left flank. Tumor size was recorded every 2 days. Intratumoral injection was performed when the tumor volume reached approximately 150 µL. Data represent means ± SD of two independent determinations with n = 6 mice/group.

• 

  Fig. 4 Recruitment of T cells and DCs inside the tumor. B16F10 melanoma cells (2 × 105) were inoculated in the shaved left flank of B6 mice. When the tumor volume reached approximately 150 µL, mice were received with PBS, 10 µg CpG-ODN (twice), 3 µg SLC-Fc (twice), or 10 µg CpG-ODN plus 3 µg SLC-Fc (twice). Eleven days after intratumoral administration of CpG-ODN and/or SLC-Fc, tumors were removed. Infiltrating cells in the tumor mass were obtained by treatment with collagenase D (100 µg/ea) for 25 min at 37℃ and stained with anti-CD4, anti-CD8, anti-CD11c, anti-CD86, and anti-I-Ab (Y3P) antibodies. (A) Populations of the infiltrating cells in the tumor mass were analyzed by flow cytometry. (B) Total numbers of the infiltrating cells in the tumor mass were counted. (C) Each population of the infiltrating cells in the tumor mass was calculated from data based on flow cytometry analysis. Data represent means±SD of two independent determinations with n = 4 mice/group.

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