Transfer of the IL-37b gene elicits anti-tumor responses in mice bearing 4T1 breast cancer

Abstract

Aim: IL-37b has shown anti-cancer activities in addition to its anti-inflammatory properties. In this study, we investigated the effects of IL-37b on breast carcinoma growth in mice and to determine the involvement of T cell activation in the effects.

Methods: IL-37b gene was transferred into mouse breast carcinoma cell line 4T1 (4T1-IL37b cells), the expression of secretory IL-37b by the cells was detected, and the effects of IL-37b expression on the cell proliferation in vitro was evaluated. After injection of 4T1 cells or 4T1-IL37b cells into immunocompetent BALB/c mice, immunodeficient BALB/c nude mice and NOD-SCID mice, the tumor growth and survival rate were measured. The proliferation of T cells in vitro was also detected.

Results: IL-37b was detected in the supernatants of 4T1-IL37b cells with a concentration of 12.02 ± 0.875 ng/mL. IL-37b expression did not affect 4T1 cell proliferation in vitro. BALB/c mice inoculated with 4T1-IL37b cells showed significant retardation of tumor growth. BALB/c mice inoculated with both 4T1 cells and mitomycin C-treated 4T1-IL37b cells also showed significant retardation of tumor growth. But the anti-cancer activity of IL-37b was abrogated in BALB/c nude mice and NOD-SCID mice inoculated with 4T1-IL37b cells. Recombinant IL-37b slightly promoted CD4(+) T cell proliferation without affecting CD8(+) T cell proliferation.

Conclusion: IL-37b exerts anti-4T1 breast carcinoma effects in vivo by modulating the tumor microenvironment and influencing T cell activation.

Figure 1

Expression of IL-37b did not affect 4T1 breast carcinoma cell growth in vitro. (A) A total of 2×10⁵ 4T1 cells were transduced with Ad-IL37b in each well of 12-well plates. At 48 and 72 h after virus transduction, the amounts of IL-37b in the culture supernatants were detected by ELISA. (B) Cells (1×10⁴) from each subline were seeded in 48-well plates, and cell numbers were counted in triplicate at 24, 36, 48, 60, and 72 h. Mean±SEM. n=3.

Figure 2

4T1-IL37b showed retarded tumor growth in immunocompetent BALB/c mice. (A) Groups of five BALB/c mice were challenged with 4T1, 4T1-eGFP and 4T1-IL37b. Mice were injected with 1×10⁵ cells into the intramammary gland fat pad in the right flank on d 0. Tumor size was measured every seven days starting on d 7 after tumor cell injection. (B–D) Groups of four BALB/c tumor models of 4T1-eGFP and 4T1-IL37b were euthanized on d 14, and tumors were excised. Then, the tumor volumes and weights were measured. Mean±SEM. n=3. ^cP<0.01.

Figure 3

M-4T1-IL37b suppressed 4T1 tumor growth in immunocompetent BALB/c mice. Groups of five BALB/c mice were challenged with 1×10⁵ 4T1, 1×10⁵ 4T1+1×10⁵ M-4T1-eGFP, or 1×10⁵ 4T1+1×10⁵ M-4T1-IL37b. Tumor cell injections and tumor volume measurements were conducted as described previously. Mean±SEM. n=3. ^bP<0.05, ^cP<0.01.

Figure 4

Recombinant IL-37b promoted CD4⁺ but not CD8⁺ T cell proliferation in vitro. Isolated CD4⁺ or CD8⁺ T cells from lymphocytes were stimulated with an anti-CD3 antibody for 72 h, with or without the addition of recombinant IL-37b. Cell size measured by FSC, the CFSE proliferation profile, and the expression of CD25 and CD69 were analyzed by flow cytometry. The data are representative of four independent experiments.

Figure 5

4T1-IL37b did not show retarded tumor growth in BALB/c nude and NOD/SCID mice. (A) Because BALB/c nude mice do not have mammary glands, they (seven in each group) were injected subcutaneously with tumor cells (1×10⁵) into the right flank on d 0. (B) Groups of seven NOD/SCID mice were injected with tumor cells as described previously. Tumor volume was measured on d 12. Mean±SEM. n=2.