The cryo-thermal therapy eradicated melanoma in mice by eliciting CD4+ T-cell-mediated antitumor memory immune response

Abstract

Tumor metastasis is a major concern in tumor therapy. In our previous studies, a novel tumor therapeutic modality of the cryo-thermal therapy has been presented, highlighting its effect on the suppression of distal metastasis and leading to long-term survival in 4T1 murine mammary carcinoma model. To demonstrate the therapeutic efficacy in other aggressive tumor models and further investigate the mechanism of long-term survival induced, in this study, spontaneous metastatic murine B16F10 melanoma model was used. The cryo-thermal therapy induced regression of implanted melanoma and prolonged long-term survival while inhibiting lung metastasis. It also promoted the activation of CD4⁺ CD25^- conventional T cells, while reduced the percentage of CD4⁺ CD25⁺ regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in the spleen, lung and blood. Furthermore, the cryo-thermal therapy enhanced the cytolytic function of CD8⁺ T cells and induced differentiation of CD8⁺ T cells into memory stem T cell (T_SCM), and differentiation of CD4⁺ T cells into dominant CD4-CTL, Th1 and Tfh subsets in the spleen for 90 days after the treatment. It was found that good therapeutic effect was mainly dependent on CD4⁺ T cells providing a durable memory antitumor immune response. At the same time, significant increase of serum IFN-γ was also observed to provide an ideal microenvironment of antitumor immunity. Further study showed that the rejection of re-challenge of B16F10 but not GL261 tumor in the treated mice in 45 or 60 days after the treatment, implied a strong systemic and melanoma-specific memory antitumor immunity induced by the treatment. Thus the cryo-thermal therapy would be considered as a new therapeutic strategy to prevent tumor recurrence and metastasis with potential clinical applications in the near future.

Figure 1

The cryo-thermal therapy eradicated B16F10 melanoma established and prolonged survival by inhibiting lung metastasis. (a) Kaplan–Meier survival curves in five independent trials. Kaplan–Meier survival curves were compared using log-rank tests. Survival rate in each trial was indicated; n=37 for control and the cryo-thermal group, respectively. (b)Tumor regression in the treated mice in week (I) 0, (II) 1, (III) 2, (IV) 3 and (V) 4 after the treatment. (c) Histological section of the lungs of the untreated mice on day 12 (I) and 26 (II), and of mice treated with the cryo-thermal therapy on day 26 (III)and 102 (IV) following tumor inoculation (red arrows indicated the metastasis lesions); × 20, scale bar, 500 μm. n=3 per group

Figure 2

The cryo-thermal therapy shifted an immunosuppressive to immunostimulatory state. The phenotype of immune cells collected from the spleen of the treated mice on day 0, 1, 3, 5, 7, 14, 21, 28 were analyzed by flow cytometry. (a) The percentages of MDSCs. (b) The percentages of CD4⁺ T cells. (c) The percentages of CD4⁺CD25⁺. (d) The percentages of CD4⁺CD25⁻. (e) The percentages of CD8⁺. The percentages of T_SCM (f), T_CM (g) and T_EM (h) subsets in total CD8⁺ populations of spleen after the treatment. The percentages of MDSCs (i), CD4⁺ (j) and CD8⁺ (k) T cells within spleen 3 and 6 months after the cryo-thermal therapy. Data were shown as mean±S.D., n=3 per group. Data for bar graphs were calculated using two-way ANOVA. *P<0.05; **P<0.01 compared with baseline values. ^&P<0.05; ^&&P<0.01 compared with control group on day 14 after the treatment. ^#P<0.05; ^##P<0.01; ^###P<0.001 compared with the control group on day 5 after the treatment

Figure 3

The cryo-thermal therapy enhanced memory CD8⁺ T-cell response. mRNA expression of IFN-γ, perforin, granzyme B and Sca-1 in splenic CD8⁺ T cells from the treated mice on day 14, 21, 28 and 90 after the treatment. The expression of IFN-γ, perforin, granzyme B and Sca-1 were examined by real-time PCR. The expression of IFN-γ (a), perforin (b), granzyme B (c) and Sca-1(d) in splenic CD8⁺ T cells from the treated mice. Data were shown as mean±S.D. Data for bar graphs were calculated using unpaired Student's t-test. *P<0.05; **P<0.01; ***P<0.001 in comparisons with the control group at different time points

Figure 4

mRNA expression of marker profiles in splenic CD4⁺ T cells from the treated mice. The expression of IFN-γ, T-bet, IL-2, TNF-α (for Th1 cells); IL-5, IL-4, IL-13, GATA3 (for Th2 cells); TGF-β, IL-10, FoxP3 (for Treg cells); IL-17A, RORγt, CCL20 (for Th17 cells); perforin, GzmB, IFN-γ, Eomes (for CD4-CTL cells); Bcl-6 and IL-21 (for Tfh cells) in splenic CD4⁺ T cells from the treated mice were examined by real-time PCR. The expression of marker profiles in splenic CD4⁺ T cells on day 14 (a), 21 (b), 28 (c) and 90 (d) after the treatment. Data were shown as mean±S.D. Data for bar graphs were calculated using two-way ANOVA. **P<0.01; ***P<0.001 in comparison with the control group at different time points

Figure 5

Long-term survival was mainly CD4⁺ T-cell-dependent. The treated mice on day 45 after the treatment were depleted with anti-CD4 or anti-CD8 monoclonal antibodies, respectively, then the treated mice after CD4⁺ or CD8⁺ lymphocyte depletion were re-challenged with 1 × 10⁵ B16F10 cells. (a) The depletion potential of each Ab was confirmed and percentages of CD3⁺ CD4⁺ and CD3⁺ CD8⁺ T cells were analyzed by flow cytometry. (b) Mean percentages of CD3⁺ CD4⁺ T cells and CD3⁺ CD8⁺ T cells in each group were shown (data for bar graphs were calculated using one-way ANOVA, ***P<0.001). (c) Tumor growth curves of individual mice in each group. Data were shown as mean±S.D., n=6 per group

Figure 6

The cryo-thermal therapy protected mice from tumor re-challenge. (a) Schematic of experimental design. The long-term survivors were re-challenged with a subcutaneous flank injection of B16F10 cells on day 45 after the treatment. (b) The long-term survivors did not develop tumor after the re-challenge (left). Growth kinetics of secondary tumors were shown (right). Data were shown as mean±S.D. Tumor growth curves were analyzed using two-way ANOVA. ***P<0.001, n=3 per group. B16F10 melanoma cells were intravenously infused to generate lung metastasis on day 45 after the treatment, and lung metastasis were enumerated. (c) Schematic of experimental design. (d) Photographic images of lungs from long-term survivors and control mice after tumor re-challenge. (e) B16F10 lung metastatic tumor foci were quantified. Data were shown as mean±S.D. Data for bar graphs were calculated using Student's t-test. *P<0.05, n=3 per group

Figure 7

The cryo-thermal therapy resulted in a melanoma-specific memory response. (a) Experimental designed to determine whether the systemic antitumor memory response was specific for B16F10 melanoma tumor on day 45 after the treatment. A total 1 × 10⁶ GL261 cells were implanted subcutaneously into the left flank and 1 × 10⁵ B16F10 cells were implanted subcutaneously into the right flank. The age-matched naive mice (n=6) were inoculated with B16F10 cells as control (tumor-bearing) group. Tumor volumes were calculated in three dimensions. (b) Photographic images of tumor growth in control mice and long-term survivors (red circle indicated the palpable nodules). Control mice had progressive B16F10 (c) and GL261 (d) flank tumor growth. Long-term survivors developed a protective memory response and rejected B16F10 melanoma tumor growth (e), and did not affect the tumor growth of GL261 (f). Data were shown as mean±S.D., n=6 per group. (g) Schematic representation of the cryo-thermal therapy induced long-term tumor-free survival associated with antitumor immune memory response; 14 d, 21 d, 28 d and 90 d indicated different time points following the treatment