Figure 1.
Tumor Growth and tumor related factors of implanted tumor in non-irradiated or pre-irradiated tumor beds with 5 Gy.
(A) Growth rate of HCa-1 in various dose irradiated tumor beds. 1×106 murine hepatocarcinoma cells (HCa-1) were injected intramuscularly in the right thigh of the mice on day 1 after the implantation site had been irradiated. (n = 5 mice per group) *p<0.05, pre 5 Gy/D1 vs control (ANOVA) (B) Growth rate of HCa-I and (C) MIH-2 tumors (n = 5 mice per group), (D) expression of CD31 and (E) p-stat3 in implanted tumor. Approximately 1×106 murine hepatocarcinoma cells (HCa-1 and MIH-2) were injected intramuscularly in the right thigh of the mice on day 1 and 3 after the implantation site had been irradiated. Error bars denote ± SEM. *p<0.05, pre 5Gy/D3 vs control (ANOVA). The tumors were resected for histologic examination when the tumor volume reached 800 to 1200 mm3 and CD31 and p-stat3 were stained.
Figure 2.
(A) TGF-β expression and (B) mRNA level in both tumors implanted into irradiated tumor beds or non-irradiated tumor beds at day 3 after irradiation. (C) TGF-β expression and (D) TGF-β mRNA level of tumor beds with or without 5 Gy-irradiation. p<0.05 (*) and p<0.01 (**) (t-test).
Figure 3.
TIL frequency and IL-6 expression in implanted tumors and irradiated beds.
TILs were isolated from the tumor and stained with CD4+ and CD8+ antibodies which was analyzed by FACS. (A) frequency of infiltrated CD4+ and CD8+ T cells and (B) Summary of TIL frequency in HCa-I and MIH-2 tumors. (C) IL-6 expression in both tumors. Irradiated beds were analyzed at day 3 after 5 Gy irradiation. (D) Representative immunofluorescence staining for IL-6, (E) western blotting for IL-6, (F) IL-6 mRNA level and (G) histology in 5 Gy irradiated beds. p<0.05 (*) and p<0.01 (**) (t-test).
Figure 4.
IL-6 expression in the tumor-draining lymph node of tumor bearing mice.
(A and B) IL-6 expression in the tumor draining lymph node from HCa-I and MIH-2 bearing mice. p<0.05 (*) and p<0.01 (**) (t-test).
Figure 5.
IL-17A expression in the tumor-draining lymph node of tumor bearing mice and Th17 differentiation by irradiation in vitro.
(A) Expressions of IL-17A and (B) Th17 cells (CD4+ and Ror-γt double positive cells) in both tumors. (C) IL-17A mRNA level and protein expression in the tumor draining lymph node of HCa-I bearing mice. (D) IL-17A mRNA level and protein expression in the tumor-draining lymph node of MIH-2 bearing mice. (E) Th17 differentiation by irradiation in vitro. Murine fibroblasts were cultured for 3 days after irradiating cells with 5 Gy. Naïve CD4+ T cells were isolated from the spleen, which were then cultured for 4 days in conditioned medium derived from irradiated fibroblasts or non-irradiated fibroblasts. Secreted IL-17A from Th17 cells was analyzed by ELISA. IL-17A mRNA level was evaluated using quantitative real time PCR. rmIL-6 (10 ng/ml), α-IFN-γ (2 µg/ml), and α-IL-4 (2 µg/ml) were added to the culture media. p<0.05 (*) and p<0.01 (**) (t-test).
Figure 6.
The effect of treatment with α-IL-17A neutralizing antibody on tumor growth.
(A and C) Growth delay and (B and D) IL-17A expression of HCa-I and MIH-2 tumors implanted in 5 Gy irradiated beds or non-irradiated beds by α-IL-17A treatment, respectively (n = 5 mice per group). Error bars denote ± SEM. *p<0.05, pre 5 Gy/D3/α-IL-17 vs pre 5 Gy/D3/IgG2a (ANOVA).