Figure 3. Increase of tumor cell–derived... | Rockefeller University Press

$Increase of tumor cell–derived IL-15 is responsible for the enhanced antitumor immunity after radiation by Piezo2 deficiency. (A and B) WT and Piezo2−/− MC38 cells were irradiated at a single fraction of 30 Gy. At 60 h after radiation, total RNA was extracted and further analyzed. (A) RNA-seq analysis of indicated cytokine mRNA expression. Bar plots from RNA-seq reveal the genes coding T cell–related cytokines. (B)Il-15 mRNA expression in WT and Piezo2−/− MC38 or WT and Piezo2−/− B16F1 with or without radiation was shown from two independent experiments. (C) C57BL/6 were transplanted subcutaneously with 2 × 106 WT MC38-tdTomato and Piezo2−/− MC38-tdTomato cells. Tumors were treated locally with one fraction of 18-Gy IR. On days 7 and 14 after radiation, IL-15 expression in MC38-tdTomato+ cells was represented from three independent experiments (n = 5–9 mice per group). (D) C57BL/6 were transplanted subcutaneously with 2 × 106 WT MC38 and Piezo2−/− MC38 cells. Tumors were treated locally with one fraction of 18-Gy IR. Anti-IL-15 was administered intratumor at 100 µg per mouse to mice every 2 days for a total of four times from the day receiving radiotherapy. Tumor growth was measured twice a week. The tumor growth curve was represented from two independent experiments (n = 6 mice per group). (E) Representative data and quantification of the percentage of IFN-γ+ in CD8+ T cells from irradiated WT and Piezo2−/− MC38 tumors with or without anti-IL-15 treatment on day 11 after IR were shown from two independent experiments (n = 4–5 mice per group). (F and G) Representative data and quantification of the percentage of TOX+ (F) and TCF-1+ (G) in PD-1+CD44+TIM3lowCD8+ T cells from tumors with or without anti-IL-15 treatment on day 11 after IR were shown from two independent experiments (n = 4–6 mice per group). (H) Representative data and quantification of the percentage of Ki-67+ in CD8+ T cells from irradiated WT and Piezo2−/− MC38 tumors with or without anti-IL-15 treatment on day 11 after IR were shown from two independent experiments (n = 4–5 mice per group). (I) C57BL/6 were transplanted subcutaneously with 2 × 106 WT, IL-15−/−, Piezo2−/−, and Piezo2−/−IL-15−/− MC38 cells. Tumors were treated locally with one fraction of 18-Gy IR. The tumor growth curve was represented from two independent experiments (n = 4–6 mice per group). (J) Representative data and quantification of the frequency of IFN-γ+TNF-α+CD8+ T cells from irradiated WT, IL-15–/–, Piezo2−/−, and Piezo2–/–IL-15−/− MC38 tumors on day 16 after IR were shown from two independent experiments (n = 4–6 mice per group). Data were represented as means ± SEM. B, C, E–H, and J were calculated by one-way ANOVA with multiple comparison tests. D and I were analyzed by two-way ANOVA with multiple comparison tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significant difference.$

Figure 3.

Increase of tumor cell–derived IL-15 is responsible for the enhanced antitumor immunity after radiation by Piezo2 deficiency. (A and B) WT and Piezo2^−/− MC38 cells were irradiated at a single fraction of 30 Gy. At 60 h after radiation, total RNA was extracted and further analyzed. (A) RNA-seq analysis of indicated cytokine mRNA expression. Bar plots from RNA-seq reveal the genes coding T cell–related cytokines. (B)Il-15 mRNA expression in WT and Piezo2^−/− MC38 or WT and Piezo2^−/− B16F1 with or without radiation was shown from two independent experiments. (C) C57BL/6 were transplanted subcutaneously with 2 × 10⁶ WT MC38-tdTomato and Piezo2^−/− MC38-tdTomato cells. Tumors were treated locally with one fraction of 18-Gy IR. On days 7 and 14 after radiation, IL-15 expression in MC38-tdTomato⁺ cells was represented from three independent experiments (n = 5–9 mice per group). (D) C57BL/6 were transplanted subcutaneously with 2 × 10⁶ WT MC38 and Piezo2^−/− MC38 cells. Tumors were treated locally with one fraction of 18-Gy IR. Anti-IL-15 was administered intratumor at 100 µg per mouse to mice every 2 days for a total of four times from the day receiving radiotherapy. Tumor growth was measured twice a week. The tumor growth curve was represented from two independent experiments (n = 6 mice per group). (E) Representative data and quantification of the percentage of IFN-γ⁺ in CD8⁺ T cells from irradiated WT and Piezo2^−/− MC38 tumors with or without anti-IL-15 treatment on day 11 after IR were shown from two independent experiments (n = 4–5 mice per group). (F and G) Representative data and quantification of the percentage of TOX⁺ (F) and TCF-1⁺ (G) in PD-1⁺CD44⁺TIM3^lowCD8⁺ T cells from tumors with or without anti-IL-15 treatment on day 11 after IR were shown from two independent experiments (n = 4–6 mice per group). (H) Representative data and quantification of the percentage of Ki-67⁺ in CD8⁺ T cells from irradiated WT and Piezo2^−/− MC38 tumors with or without anti-IL-15 treatment on day 11 after IR were shown from two independent experiments (n = 4–5 mice per group). (I) C57BL/6 were transplanted subcutaneously with 2 × 10⁶ WT, IL-15^−/−, Piezo2^−/−, and Piezo2^−/−IL-15^−/− MC38 cells. Tumors were treated locally with one fraction of 18-Gy IR. The tumor growth curve was represented from two independent experiments (n = 4–6 mice per group). (J) Representative data and quantification of the frequency of IFN-γ⁺TNF-α⁺CD8⁺ T cells from irradiated WT, IL-15^–/–, Piezo2^−/−, and Piezo2^–/–IL-15^−/− MC38 tumors on day 16 after IR were shown from two independent experiments (n = 4–6 mice per group). Data were represented as means ± SEM. B, C, E–H, and J were calculated by one-way ANOVA with multiple comparison tests. D and I were analyzed by two-way ANOVA with multiple comparison tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significant difference.

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