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. 2024 Feb 25;15(8):2245-2259.
doi: 10.7150/jca.91559. eCollection 2024.

Carbon ion irradiation combined with PD-1 inhibitor trigger abscopal effect in Lewis lung cancer via a threshold dose

Affiliations

Carbon ion irradiation combined with PD-1 inhibitor trigger abscopal effect in Lewis lung cancer via a threshold dose

Ruifeng Liu et al. J Cancer. .

Abstract

Background and goal: Carbon ion beam is radio-biologically more efficient than photons and is beneficial for treating radio-resistant tumors. Several animal experiments with tumor-bearing suggest that carbon ion beam irradiation in combination with immunotherapy yields better results, especially in controlling distant metastases. This implies that carbon ion induces a different anti-tumor immune response than photon beam. More complex molecular mechanisms need to be uncovered. This in vivo and in vitro experiment was carried out in order to examine the radio-immune effects and the mechanism of action of carbon ion beam versus X-ray in combination with PD-1 inhibitors. Methods and Materials: Lewis lung adenocarcinoma cells and C57BL/6 mice were used to create a tumor-bearing mouse model, with the non-irradiated tumor growing on the right hind leg and the irradiated tumor on the left rear. 10Gy carbon ion beam or X-ray radiation, either alone or in combination with PD-1 inhibitor, were used to treat the left back tumor. The expression of molecules linked to immunogenicity and the infiltration of CD8+ T lymphocytes into tumor tissues were both identified using immunohistochemistry. IFN-β in mouse serum was measured using an ELISA, while CD8+ T cells in mouse peripheral blood were measured using flow cytometry. Lewis cells were exposed to different dose of X-ray and carbon ion. TREX1, PD-L1, and IFN-β alterations in mRNA and protein levels were identified using Western blot or RT-PCR, respectively. TREX1 knockdown was created by siRNA transfection and exposed to various radiations. Using the CCK8 test, EdU assay, and flow cytometry, changes in cell viability, proliferation, and apoptosis rate were discovered. Results: Bilateral tumors were significantly inhibited by the use of carbon ion or X-ray in combination with PD-1, particularly to non-irradiated tumor(p<0.05). The percentage of infiltrating CD8+ T cells and the level of IFN-β expression were both raised by 10Gy carbon ion irradiation in the irradiated side tumor, although PD-L1 and TREX1 expression levels were also elevated. Lewis cell in vitro experiment further demonstrated that both X-ray and carbon ion irradiation can up-regulate the expression levels of PD-L1 and TREX1 with dose-dependent in tumors, particularly the trend of up-regulation TREX1 is more apparent at a higher dose in carbon ion, i.e. 8 or 10Gy, while the level of IFN-β is decreased. IFN-β levels were considerably raised under hypofractionated doses of carbon ion radiation by gene silencing TREX1. Conclusions: By enhancing tumor immunogenicity and increasing CD8+T infiltration in TME through a threshold dosage, X-ray or carbon ion radiation and PD-1 inhibitors improve anti-tumor activity and cause abscopal effect in Lewis lung adenocarcinoma-bearing mice. TREX1 is a possible therapeutic target and prognostic marker.

Keywords: Abscopal effect; Carbon ion; In vitro experiment; Irradiation; PD-1 inhibitor.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
A was C57BL/6 mice with bilateral tumor bearing models, the left back was an irradiated area (indicated by the red arrow) and the right hind limb was a non-irradiated area (indicated by the green arrow). B were homemade irradiation-shielded lead boxes for mice.
Figure 2
Figure 2
Schematic diagram of tumor irradiation and aPD-1 intervention in tumor-bearing mice.
Figure 3
Figure 3
Strategies for sorting CD3+T and CD3+CD8+T subsets in peripheral blood of mice.
Figure 4
Figure 4
Changes in body weight, tumor weight and volume of mice in different intervention groups (A. Changes in average body weight of 6 mice. B. Changes of average tumor weight in irradiated area. C. Changes of average tumor weight in non-irradiated area. D. Changes of average tumor volume in irradiated area. E. Changes of average tumor volume in non-irradiated area. **p < 0.01, ***p < 0.001, ns p > 0.05).
Figure 5
Figure 5
Effects of carbon ion or X-ray combined with PD-1 inhibitors on the expression of immune-related molecules in irradiated tumor tissues. A is the representative figure of hematoxylin and immunohistochemical staining (PD-L1, TREX1, IFN-β, CD8+T) on the irradiated side tumor (Multiple 400X). B were tumor immunohistochemical staining score (PD-L1, TREX1, IFN-β) and CD8+T cell count (*p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05).
Figure 6
Figure 6
Effects of carbon ion or X-ray combined with PD-1 inhibitors on the expression of immune-related molecules in non-irradiated tumor tissues. A is the representative figure of hematoxylin and immunohistochemical staining (PD-L1, TREX1, IFN-β, CD8+T) on the non-irradiated side tumor (Multiple 400X). B were tumor immunohistochemical staining score (PD-L1, TREX1, IFN-β) and CD8+T cell count (*p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05).
Figure 7
Figure 7
Changes of T lymphocyte subsets and IFN-β in peripheral blood of mice in each group (A. CD3+T lymphocyte, B. D3+CD8+T lymphocyte, C. IFN-β, * p < 0.05).
Figure 8
Figure 8
Effects of X-ray and carbon ion irradiation on mRNA and protein levels of PD-L1, IFN-βand TREX1 molecules (A. Effects of X-ray on PD-L1, IFN-βand TREX1 mRNA level in Lewis cells; B. Effects of carbon ion on PD-L1, IFN-β and TREX1 mRNA level in Lewis cells; C&E. Effects of X-ray on the expression of PD-L1, IFN-β and TREX1 proteins in Lewis cells; D&F. The effects of carbon ion rays on the expression of PD-L1, IFN-β and TREX1 proteins in Lewis cells; * p < 0.05, **p < 0.01).
Figure 9
Figure 9
Effects of irradiation on proliferation and apoptosis of Lewis cells after TREX1 gene silencing (A. Agarose gel electrophoresis after TREX1 gene silencing; B. The transfection efficiency of siTREX1-Lewis was verified by Western blot; C. Protein immunoblotting of TREX1; D-F. The changes of cell proliferation and viability over time in each group, D was Edu representative image, E was Edu cell proliferation rate, F was CCK8 cell viability; G-H. The changes of cell apoptosis rate in each group; *p < 0.05, **p < 0.01, ***p < 0.001).
Figure 10
Figure 10
Changes of IFN-β and TREX1 in siTREX1 Lewis cells with 10 Gy carbon ion irradiation (A. The statistical results of qRT-PCR; B. Protein immunoblotting; *p<0.05).

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