Radiotherapy-induced anti-tumor immunity contributes to the therapeutic efficacy of irradiation and can be augmented by CTLA-4 blockade in a mouse model

Abstract

Purpose: There is growing evidence that tumor-specific immune responses play an important role in anti-cancer therapy, including radiotherapy. Using mouse tumor models we demonstrate that irradiation-induced anti-tumor immunity is essential for the therapeutic efficacy of irradiation and can be augmented by modulation of cytotoxic T lymphocyte (CTL) activity.

Methods and materials: C57BL/6 mice, syngeneic EL4 lymphoma cells, and Lewis lung carcinoma (LL/C) cells were used. Cells were injected into the right femurs of mice. Ten days after inoculation, tumors were treated with 30 Gy of local X-ray irradiation and their growth was subsequently measured. The effect of irradiation on tumor growth delay (TGD) was defined as the time (in days) for tumors to grow to 500 mm3 in the treated group minus that of the untreated group. Cytokine production and serum antibodies were measured by ELISA and flow cytometry.

Results: In the EL4 tumor model, tumors were locally controlled by X-ray irradiation and re-introduced EL4 cells were completely rejected. Mouse EL4-specific systemic immunity was confirmed by splenocyte cytokine production and detection of tumor-specific IgG1 antibodies. In the LL/C tumor model, X-ray irradiation also significantly delayed tumor growth (TGD: 15.4 days) and prolonged median survival time (MST) to 59 days (versus 28 days in the non-irradiated group). CD8(+) cell depletion using an anti-CD8 antibody significantly decreased the therapeutic efficacy of irradiation (TGD, 8.7 days; MST, 49 days). Next, we examined whether T cell modulation affected the efficacy of radiotherapy. An anti-CTLA-4 antibody significantly increased the anti-tumor activity of radiotherapy (TGD was prolonged from 13.1 to 19.5 days), while anti-FR4 and anti-GITR antibodies did not affect efficacy.

Conclusions: Our results indicate that tumor-specific immune responses play an important role in the therapeutic efficacy of irradiation. Immunomodulation, including CTLA-4 blockade, may be a promising treatment in combination with radiotherapy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Therapeutic effects of X-ray irradiation on EL4 tumors in C57BL/6 mice.

(A) Growth curves of EL4 tumors in the non-treated group (open circles, n = 5) and irradiated group (closed triangles, n = 5). X-ray irradiation was performed on Day 10; bars, SD. (B) Survival curves for EL4-inoculated C57BL/6 mice, the non-treated group (dotted line), and the irradiated group (solid line). (C) Lymph node metastasis (arrows) in a representative mouse 20 days after tumor inoculation. (D) Growth curve of EL4 tumors (second inoculation). EL4 cells were re-inoculated into the left hind limbs of mice 24 days after the first challenge with EL4 cells (closed triangles, n = 5) or B16 cells (open circles, n = 5); bars, S.D.

Figure 2. Effect of X-ray irradiation on EL4 tumors in BALB/c-nu/nu mice.

(A) Growth curves of EL4 tumors in the irradiated group (n = 5). X-ray irradiation was performed on Day 10; bars, SD. (B) Survival curves for EL4-inoculated BALB/c-nu/nu mice. (C) Systemic metastases (arrows) in the internal organs of a representative mouse 20 days after tumor inoculation.

Figure 3. Immune reaction in irradiated EL4 tumor-bearing mice.

(A) IFN-γ (left) and TNF-α (right) concentrations in C57BL/6 and BALB-c nu/nu mouse splenocyte culture supernatants as measured by ELISA. Mice were sacrificed 24 days after tumor inoculation. Control (cont) splenocytes from non-tumor-bearing irradiated mice; Ra, splenocytes from irradiated EL4 tumor-bearing mice. (B) Mice were sacrificed on the indicated day and splenocytes were cultured for ELISA. Irradiation was performed on Day 10. Day 0, splenocytes from non-tumor-bearing non-irradiated mice; Day 10, tumor-bearing non-irradiated mice; Days 11, 14, 17, 20, tumor-bearing irradiated mice. (C) Antibodies specific for EL4 cells produced by local irradiation of EL4 tumors in C57/BL6 (left) and BALB/c-nu/nu (right) mice.

Figure 4. Irradiation of EL4 tumors in C57BL/6 mice results in the abscopal effect.

(A) Timing of primary EL4 cell inoculation and tumor irradiation in control and synchronous models. (B) Volume of irradiated tumors in the right hind limbs of synchronous model mice (closed circles, n = 5) and of non-irradiated tumors in the left hind limbs of synchronous model mice (open circles) and the control tumors in control model mice (closed triangles, n = 5); bars, S.D.

Figure 5. X-ray irradiation delays LL/C tumor growth, and CTL depletion reduces TGD, in C57BL/6 mice.

(A) Growth curves of LL/C tumors in the non-treated group (open circles, n = 7), irradiated group (open triangles, n = 7), anti-CD8 antibody-treated group (closed circles, n = 3), and irradiation plus anti-CD8 antibody-treated group (closed triangles, n = 7). X-ray irradiation was performed when tumors reached 100 mm3 in volume (Day 0); bars, SD. (B) Survival curves for LL/C-inoculated C57BL/6 mice, non-treated group (thin solid line), irradiated group (thick solid line), anti-CD8 antibody-treated group (thin dotted line), and irradiation plus anti-CD8 antibody-treated group (thick dotted line).

Figure 6. X-ray irradiation delays LL/C tumor growth and antibody-mediated immunomodulation increases TGD in C57BL/6 mice.

(A) Growth curves of LL/C tumors in non-irradiated (solid line, n = 5/each group) and irradiated (dashed line, n = 5/each group) groups. Effects of the anti-CTLA-4 (closed triangles), anti-FR4 (open squares), anti-GITR (open diamonds) immunomodulatory antibodies, and the saline control (for the non-treated group, closed circles) were examined. X-ray irradiation was performed when tumors reached 100 mm3 in volume (Day 0); bars, SD. (B) Survival curves for each group of LL/C-inoculated C57BL/6 mice; non-treated (thin solid line), irradiated (thick solid line), anti-CTLA-4 antibody-treated (thin dotted line), irradiation plus anti-CTLA-4 antibody-treated (thick dotted line), anti-FR4 antibody-treated (thick dashed line), irradiation plus anti-FR4 antibody-treated (thin dashed line), anti-GITR antibody-treated (thick dash-dotted line), and irradiation plus anti-GITR antibody-treated (thin dash-dotted line).

Figure 7. X-ray irradiation induces HMGB1 protein in vitro.

Cells were exposed to the indicated doses of X-ray irradiation and then cultured for 48 h. HMGB1 concentrations in culture supernatants were measured by ELISA.