Carbon ion combined photon radiotherapy induces ferroptosis via NCOA4-mediated ferritinophagy in glioblastoma

Abstract

Glioblastoma (GBM), the most prevalent and lethal primary malignancy of the central nervous system, remains refractory to conventional photon radiotherapy due to inherent limitations in dose distribution. Although carbon ion radiotherapy offers distinct advantages, including its characteristic Bragg peak deposition and superior relative biological effectiveness, its clinical application is constrained by high costs and increased toxicity. This study explores the radiobiological interactions underlying a mixed carbon ion-photon irradiation regimen, a promising strategy in advanced particle therapy. Our findings demonstrate that combined irradiation exerts synergistic cytotoxic effects in GBM models. Mechanistic analysis reveals that this combination induces clustered DNA double-strand breaks, leading to the cytoplasmic accumulation of double-stranded DNA (dsDNA) fragments. This, in turn, activates the cGAS-STING-mediated cytosolic DNA sensing pathway, which facilitates NCOA4-FTH1 axis-driven ferritinophagy and ultimately triggering iron-dependent ferroptosis. These findings offer a new mechanistic perspective on optimizing combined particle therapy regimens for GBM treatment, with significant implications for translational applications in clinical radiation oncology.

Keywords: Carbon ion radiotherapy; Ferritinophagy; Ferroptosis; Glioblastoma; Photon radiotherapy.

Carbon ion combined photon radiotherapy enhances cell lethality in glioblastoma by inducing ferroptosis through activation of STING and triggering the NCOA4-mediated ferritinophagy pathway.

Fig. 1

Carbon ion combined with photon radiotherapy effectively inhibits the proliferation of glioblastoma cells. (A) Schematic diagram of carbon ion irradiation of cells. (B) Schematic diagram of photon irradiation of cells. (C) Colony formation results of GBM cell lines after different irradiation treatments. (D) Immunofluorescence assay detecting the expression of γ-H2AX in each group post-irradiation. (E) Flow cytometry analysis of γ-H2AX expression in each group post-irradiation. (F) Western blot analysis of phosphorylated γ-H2AX protein expression in each group post-irradiation. Scale bar: 20 μm. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 2

Carbon ion combined with photon radiotherapy induces glioblastoma cell death by promoting ferroptosis. (A) GSVA heatmap of RNA-seq. (B) Flow cytometry to detect the level of lipid peroxidation in cells. (C) MDA assay to measure the level of lipid peroxidation in cells. (D) Flow cytometry to detect the level of Fe²⁺ in cells. (E) PI staining to assess cell death. (F) Transmission electron microscopy (TEM) analysis of changes in mitochondria and autophagosomes in each group of cells. Red arrows indicate mitochondria, and yellow arrows indicate autolysosomes. Scale bar (up): 2 μm, Scale bar (down): 500 nm. (G) Western blot analysis of ACSL4 and SLC7A11 expression in GBM cell lines. (H) Cell immunofluorescence to detect the level of unstable iron ions in cells. (I–J) Flow cytometry to detect the level of Fe²⁺ in cells. (K–N) Flow cytometry to detect the level of lipid peroxidation in cells. Ferr-1: 5 μM; DFO: 50 μM. Ferroptosis inhibitors were applied immediately after irradiation and maintained for 48 h. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 3

Carbon ion combined with photon radiotherapy inhibits glioblastoma tumor growth in vivo by promoting ferroptosis. (A) Schematic diagram of mouse immobilization for irradiation. (B) Illustration of carbon ion irradiation in mice. (C) Illustration of photon irradiation in mice. (D) In vivo imaging of intracranial tumors in mice (n = 7). Irradiation was performed on day 10 after orthotopic tumor implantation, followed by daily intraperitoneal injection of Lipro-1 (10 mg/kg). The control group received an equivalent volume of normal saline. (E) Quantification of in vivo imaging on day 31 after orthotopic tumor implantation. (F) HE staining of mouse brain tumors. (G) 4HNE and Iba-1 staining of mouse brain tumors. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 4

Carbon ion combined photon radiotherapy promotes ferroptosis in glioblastoma cells by enhancing ferritinophagy. (A) Heatmap of the expression of ferroptosis-driving and inhibiting genes in each group. Three biological replicates per group. (B,C) RT-qPCR to detect the expression of NCOA4 and FTH1. (D) Western blot to detect the expression of NCOA4, FTH1, and LC3B. (E, F) Flow cytometry to detect the autophagy level in each group. (G, H) Flow cytometry to detect the lysosomal level in each group. (I) Cell immunofluorescence to detect the expression and colocalization of Fe²⁺ and lysosomes. Scar bar: 20 μm. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 5

Carbon ion combined photon radiotherapy promotes ferroptosis in glioblastoma cell lines by enhancing NCOA4-mediated ferritinophagy. (A) Kaplan-Meier survival curve of NCOA4 expression in glioblastoma patients. (B) Box plots of NCOA4 expression associated with various clinical pathologies of glioblastoma (WHO grade, IDH mutation status, 1p/19q co-deletion). (C) Western blot to detect the expression of NCOA4 and FTH1 in GBM cells after NCOA4 knockdown. (D) Western blot analysis of NCOA4 and FTH1 protein expression in NCOA4-knockdown cells. (E) Flow cytometry to detect the level of lipid peroxidation in NCOA4-knockdown cells. (F) Flow cytometry to detect the level of Fe²⁺ in NCOA4-knockdown cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 6

Carbon ion combined photon radiotherapy promotes ferroptosis in glioblastoma cells by activating STING. (A) Immunofluorescence detection of dsDNA expression in each group after irradiation 48h. (B) ELISA detection of 8-OHdG expression in each group after irradiation 48h. (C) Western blot of cGAS-STING pathway-related proteins in glioblastoma cells after different radiation treatments. (D) Flow cytometry to detect the level of lipid peroxidation in sting-knockdown cells. (E) Flow cytometry to detect the level of Fe²⁺ in sting-knockdown cells. (F) Flow cytometry to detect the level of lipid peroxidation in cells treated with the STING inhibitor C-176 (1 μM), with treatment immediately after irradiation and lasting for 48 h. (G). Flow cytometry to detect the level of Fe²⁺ in C-176 treated cells. (H, I) Western blot analysis of NCOA4 and FTH1 protein expression in sting-knockdown cells. (J, K) Western blot analysis of NCOA4 and FTH1 protein expression in C-176 treated cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 7

Carbon ion combined photon radiotherapy enhances cell lethality in glioblastoma by inducing ferroptosis through activation of STING and triggering the NCOA4-mediated ferritinophagy pathway.