Cryo-thermal therapy induces macrophage polarization for durable anti-tumor immunity

Abstract

Many cancer therapies are being developed for the induction of durable anti-tumor immunity, especially for malignant tumors. The activation of antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), can bridge innate and adaptive immune responses against tumors. However, APCs have an immunosuppressive phenotype and reversing it for effective tumor-specific antigen presenting is critical in developing new cancer treatment strategies. We previously developed a novel cryo-thermal therapy to treat malignant melanoma in a mouse model; long-term survival and durable anti-tumor immunity were achieved, but the mechanism involved was unclear. This study revealed cryo-thermal therapy-induced macrophage polarization to the M1 phenotype and modulated the phenotypic and functional maturation of DCs with high expression of co-stimulatory molecules, increased pro-inflammatory cytokine production, and downregulated immuno-inhibitory molecule expression. Further, we observed CD4⁺ T-cell differentiation into Th1 and cytotoxic T-cell sub-lineages and generation of cytotoxic CD8⁺ T cells, in which M1 macrophage polarization had a direct, important role. The results indicated that cryo-thermal-induced macrophage polarization to the M1 phenotype was essential to mediate durable anti-tumor immunity, leading to long-term survival. Thus, cryo-thermal therapy is a promising strategy to reshape host immunosuppression, trigger persistent memory immunity for tumor eradication, and inhibit metastasis in the long term.

Fig. 1. Cryo-thermal therapy protected mice from pulmonary metastatic B16F10 tumors.

Photographic images of lungs from cryo-thermal-treated and B16F10 tumor-bearing mice, respectively. Upper: schematic of experimental design. Lower: photographic images of lungs

Fig. 2. Cryo-thermal therapy induced phenotypically maturation of splenic DCs.

The phenotype of immune cells collected from the spleen in the treated mice by cryo-thermal therapy and untreated tumor-bearing mice on day 5 and 14 were analyzed by flow cytometry. a–c Percentage of CD11c⁺CD86⁺MHC II⁺ DCs on day 5 and 14 were analyzed by flow cytometry; all data was shown as mean ± SD. n = 6 per group. Data for bar graphs were calculated using Student’s t-test. *p < 0.05. Total RNA was isolated from splenic CD11c⁺ DCs (purified from splenocytes using micro-bead kit) in cryo-thermal-treated mice on day 5 and 14. The mRNA in splenic CD11c⁺ DCs from tumor-bearing mice on day 17 and 26 after tumor inoculation was used as control. The expression level of CXCL10 (d), IL-1β (e), IL-6 (f), IL-7(g), IL-15(h), IL-12p40(i), TNF-α (j), and IL-10 (k). Data were shown as mean ± SD. Data for bar graphs were calculated using two-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001. IL-12 refer to IL-12p40 in the figure

Fig. 3. Cryo-thermal therapy re-educated immunosuppressive macrophages phenotypically.

The phenotype of immune cells collected from the spleen in cryo-thermal-treated mice and untreated tumor-bearing mice on day 5 and 14 were analyzed by flow cytometry. a–b Percentage of CD11b⁺F4/80⁺CD86⁺MHC II⁺ macrophages on day 5 and 14 were analyzed by flow cytometry. All data was shown as mean ± SD. n = 6 per group. Data for bar graphs were calculated using Student’s t-test. **p < 0.01. Total RNA was isolated from splenic CD68⁺ macrophages (purified from splenocytes using micro-bead kit) in cryo-thermal-treated mice on day 5 and 14. The mRNA in splenic CD68⁺ macrophages from tumor-bearing mice on day 17 and 26 after tumor inoculation was used as control. The mRNA level of CD86 (c), MHC II (d), CXCL10 (e), IL-12p40 (F), IL-6 (g), TNF-α (h), iNOS (i), CD206 (j), Arg-1 (k), CCL2 (l), and IL-10 (m). Data were shown as mean ± SD. Data for bar graphs was calculated using two-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001. IL-12 refer to IL-12p40 in the figure

Fig. 4. Cryo-thermal-re-educated macrophages remodeled host immune environment.

a Schematic of experimental design. b, c The efficiency of macrophage depletion was assessed by flow cytometry assay. Data were shown as mean ± SD. n = 6 per group. ***p < 0.001. Data for bar graphs were calculated using one-way ANOVA. d–g The splenocytes were collected from the mice in cryo-thermal + PBS-lip, cryo-thermal + Clod-lip, and control groups, then the population of CD11c⁺ cells and percentage of CD86⁺MHC II⁺ DCs (gated on CD11c⁺ cells shown in d) on day 5 and 14 were analyzed by flow cytometry. All data were shown as mean ± SD. n = 6 per group. *p < 0.05 or **p < 0.01. Data for bar graphs were calculated using two-way ANOVA

Fig. 5. Cryo-thermal-re-educated macrophages remodeled host immune environment (continued).

a, b The splenocytes were collected from the mice in cryo-thermal + PBS-lip, cryo-thermal + Clod-lip, and control groups, then the percentage of CD3⁺CD4⁺ T cells on day 5 and 14 were analyzed by flow cytometry. All data were shown as mean ± SD. n = 6 per group. **p < 0.01 or ***p < 0.001. Data for bar graphs were calculated using two-way ANOVA. c The level of thPOK (for CD4-CTL cells), IFN-γ (for Th1 cells), IL-4 (for Th2 cells), IL-17 (for Th17 cells), Bcl-6 (for Tfh cells), and FoxP3 (for Treg cells) in splenic CD4⁺ T cells were examined by flow cytometry. Data were shown as mean ± SD. Data for bar graphs were calculated using two-way ANOVA. *p < 0.05 or **p < 0.01, or ***p < 0.001

Fig. 6. Cryo-thermal-re-educated macrophages remodeled host immune environment (continued).

a, b The splenocytes were collected from the mice in cryo-thermal + PBS-lip, cryo-thermal + Clod-lip, and control groups, then the percentage of CD3⁺CD8⁺ T cells on day 5 and 14 were analyzed by flow cytometry. All data were shown as mean ± SD. n = 6 per group. ***p < 0.001, cryo-thermal + Clod-lip group compared with other groups. Data for bar graphs were calculated using two-way ANOVA. c The level of IFN-γ, perforin, granzyme-B in splenic CD8⁺ T cells were examined by flow cytometry. Data were shown as mean ± SD. Data for bar graphs were calculated using two-way ANOVA. *p < 0.05 or **p < 0.01, or ***p < 0.001

Fig. 7. Cryo-thermal-induced macrophage polarization to the M1 phenotype was crucial for triggering anti-tumor memory immunity.

a Schematic of experimental design. Macrophage depletion was achieved by intraperitoneal injection of Clod-lip (red arrows indicated Clod-lip injection) on day 1 before cryo-thermal therapy and on day 5, 10 after cryo-thermal therapy, respectively. The tumor growth was tracked on day 50 after tumor inoculation (38 days after the cryo-thermal therapy). b Photographic images of tumor growth in cryo-thermal + PBS-lip, cryo-thermal + Clod-lip, and control groups

Fig. 8

Schematic representation of cryo-thermal therapy drove macrophages polarization toward M1 phenotype that remodeled host immune environment triggering the durable anti-tumor memory immunity. Cryo-thermal therapy triggered the macrophage polarization towards M1 phenotype. Cryo-thermal therapy also induced the phenotype and functional maturation of DCs. Moreover, cryo-thermal-induced M1 polarization macrophage could trigger DC maturation, CD4⁺ T cell differentiation into Th1 and CTL sublineages, and the generation of cytotoxic CD8⁺ T cells, which lead to long-lasting adaptive immune-mediated inhibition of distant lung metastases