Low frequency magnetic fields enhance antitumor immune response against mouse H22 hepatocellular carcinoma

Abstract

Objective: Many studies have shown that magnetic fields (MF) inhibit tumor growth and influence the function of immune system. However, the effect of MF on mechanism of immunological function in tumor-bearing mice is still unclear.

Methods: In this study, tumor-bearing mice were prepared by subcutaneously inoculating Balb/c mice with hepatocarcinoma cell line H22. The mice were then exposed to a low frequency MF (0.4 T, 7.5 Hz) for 30 days. Survival rate, tumor growth and the innate and adaptive immune parameters were measured.

Results: MF treatment could prolong survival time (n = 28, p<0.05) and inhibit tumor growth (n = 9, p<0.01) in tumor-bearing mice. Moreover, this MF suppressed tumor-induced production of cytokines including interleukin-6 (IL-6), granulocyte colony- stimulating factor (G-CSF) and keratinocyte-derived chemokine (KC) (n = 9-10, p<0.05 or 0.01). Furthermore, MF exposure was associated with activation of macrophages and dendritic cells, enhanced profiles of CD4(+) T and CD8(+) T lymphocytes, the balance of Th17/Treg and reduced inhibitory function of Treg cells (n = 9-10, p<0.05 or 0.01) in the mice model.

Conclusion: The inhibitory effect of MF on tumor growth was related to the improvement of immune function in the tumor-bearing mice.

Figure 1. Magnetic field exposure system.

Figure 2. Magnetic fields raise survival rate and inhibit tumor growth in tumor-bearing mice.

(A) Statistical analyses of survival after subcutaneous challenge of Balb/c mice with H22 cells and treated with magnetic fields using log-rank test. Mice were treated by magnetic fields (MF) for 30 days, 2 h per day, and cared without magnetic fields for another 40 days. (B) The mean mass and volume of tumor for each group after treatment with magnetic fields 30 days. ***P<0.001, Mann-Whitney U test. The Hematoxylin and eosin staining (C, 10×) and immunohistochemical staining of Ki67 (D) in tumors.

Figure 3. Magnetic fields reverse levels of cytokines/chemokines in plasma of tumor-bearing mice.

Bio-Plex was used to analyze cytokine/chemokine changes in mice plasma, (A) IL-6, (B) granulocyte colony-stimulating factor (G-CSF), (C) IL-12p40, (D) Keratinocyte-derived Chemokine (KC), (E) IL-1α, (F) IL-1β, (G) IL-2, (H) IL-3, (I)IL-4, (J)Rantes, and (K) TNF-α (±s.e.m.). *P<0.05, **P<0.01, or ns = No significant difference.

Figure 4. Magnetic fields transform the development of macrophage subsets.

(A) Proportion of F4/80⁺ macrophage cells in PBMC was detected by flow cytometry. (B) The mean proportion of F4/80⁺ macrophage cells in PBMC for each group (Mean ± s.e.m.). **P<0.01.

Figure 5. Magnetic fields enhances the expression of CD40 in Dendritic Cell.

Proportion of CD11c⁺ DC (A) and CD11c⁺CD40⁺ DC (B) in PBMC was detected by flow cytometry. (C) The mean proportion of CD11c⁺CD40⁺ DC in PBMC for each group (Mean ± s.e.m.). (D) The mean proportion of CD11c⁺CD40⁺ DC in tumor infiltrating lymphocytes for each group (Mean ± s.e.m.). *P<0.05, **P<0.01 or ns = No significant difference.

Figure 6. Magnetic fields promotes the development of CD4⁺ and CD8⁺ T cells polarization in PBMC.

(A) Proportion of CD4⁺ T cells in PBMC was detected by flow cytometry. (B) The mean proportion of CD4⁺ T cells in PBMC for each group (Mean ± s.e.m.). (C) Proportion of CD8⁺ T cells in PBMC was detected by flow cytometry. (D) The mean proportion of CD8⁺ T cells in PBMC for each group (Mean ± s.e.m.). *P<0.05.

Figure 7. Magnetic fields regulate Th17/Treg balance and down-regulate the inhibitory function of Treg cells in spleen.

(A) Flow cytometry analysis of Th1, Th2, Th17 and Treg cell populations in spleen. (B) Summarized data of Th1, Th2, Th17 and Treg cell populations in spleen. (C) mRNA expression of T-bet, GATA3, RORγ and Foxp3 in splenic lymphocytes were detected by qPCR. (D) Inhibitory function of Treg cells in each group were detected by mixed lymphocyte reaction. (E) The mean mass of spleen for each group (Mean ± s.e.m.). *P<0.05, **P<0.01, ***P<0.001, or ns = No significant difference.