Low dose radiotherapy combined with immune checkpoint inhibitors induces ferroptosis in lung cancer via the Nrf2/HO-1/GPX4 axis

Abstract

Background: Immune checkpoint inhibitors (ICI) have revolutionized the therapeutic direction for lung cancer, yet their response rates remain unsatisfactory. Recently, the combination of ICI and low dose radiotherapy (LDR), a novel approach that effectively mobilizes innate and adaptive immunity, has gained interest among scientists. However, the underlying molecular mechanisms are not clearly elucidated.

Methods: The in vivo anti-tumor effects of LDR and ICI were measured in murine tumor models. The immune response and alterations in the tumor microenvironment were measured using flow cytometry and enzyme-linked immunosorbent assay (ELISA). Cell viability and death were assessed using CCK-8 assays. Fluorescent probes and ELISA were used to assess ferroptosis induced by the combination therapy in vitro and in vivo. Western blotting and qPCR were performed to detect alterations in the Nrf2/HO-1/GPX4 pathway. Furthermore, a phase 1 clinical trial with a combined regimen of LDR and anti-PD-1 antibodies in patients with lung cancer was conducted.

Results: The combined LDR and ICI regimen exhibited considerable anti-tumor effects in murine tumor models, promoting immune response and increasing the IFN-γ levels. In vitro data showed that LDR plus ICI induced ferroptosis in cancer cells by increasing reactive oxygen species and MDA levels, promoting Fe2⁺ accumulation, and suppressing GSH. Furthermore, ferroptosis induced by combination therapy was associated with suppression of the Nrf2/HO-1/GPX4 antioxidant axis. Importantly, a phase 1 clinical trial of the combination therapy showed promising efficacy in patients with lung cancer with chemoimmunotherapy resistance.

Conclusion: This study demonstrated that LDR plus ICI induces ferroptosis through the Nrf2/HO-1/GPX4 pathway, resulting in a significant anti-tumor effect and providing a combinatorial strategy to overcome lung cancer. However, this combined strategy merits further clinical investigation.

Keywords: Nrf2/HO-1/GPX4; chemoimmunotherapy-resistant; ferroptosis; immune checkpoint inhibitor; low dose radiotherapy.

Figure 1

LDR combined with ICI had a significant anti-tumor effect in the LLC lung cancer model. (A) Workflow for the LLC lung cancer model and treatment with LDR combined with ICI. (B) Tumor volume, (C) body weight changes, (D) representative images of tumors, (E) tumor weights of mice bearing LLC tumors treated with LDR combined with ICI, as well as the combination therapy compared with control treatment (n = 4 mice per group). (F) Tumor growth curves of individual mice in different groups. LDR, low dose radiotherapy; αPD-1, anti-PD-1; i.p., intraperitoneal injection; ICI, immune checkpoint inhibitor. Data are presented as means ± SD. ***p < 0.01. Unpaired two-tailed Student’s t-test.

Figure 2

LDR combined with ICI increased IFN-γ levels in both the TME and blood. Quantification of tumor-infiltrating (A) CD8⁺ T cells, including IFNγ⁺ CD8⁺, TNFα⁺ CD8⁺, Perforin⁺ CD8⁺, and GranzymeB⁺ CD8⁺ TILs from mice across different treatment groups; (B) CD4⁺ T cells, including Tregs from mice across different treatment groups; (C) DC; (D) TAM; and (E) NK cells from mice across the different treatment groups. (F) ELISA of IFN-γ, TNF-α and important interleukins in the serum of mice in the different treatment groups. LDR, low dose radiotherapy; ICI, immune checkpoint inhibitor; DC, dendritic cell; TAM, tumor-associated macrophages; Treg, regulatory T cells; NK, natural killer; IL, interleukin. Data are presented as means ± SD. *p < 0.05; ***p < 0.01; ns, not significant. Unpaired two-tailed Student’s t-test.

Figure 3

LDR combined with ICI induced cell death. (A) LDR combined with ICI induced cell death in LLC lung cancer and ID8 ovarian cancer cells. (B) Cell death induced by LDR combined with ICI in the absence and presence of ferrostatin-1 (1 μM), Z-VAD-FMK (10 μM), and necrosulfonamide (1 μM). LDR, low dose radiotherapy; ICI, immune checkpoint inhibitor; Fer1, ferrostatin-1; Z-VAD, Z-VAD-FMK; Nec1, necrosulfonamide. Data are presented as means ± SD. ***p < 0.01. Unpaired two-tailed Student’s t-test.

Figure 4

LDR combined with ICI induced ferroptosis and suppressed the Nrf2/HO-1/GPX4 pathway. Representative images of FerroOrange fluorescent (A) and fluorescent intensity (B) reflecting Fe2⁺ concentrations in LLC lung cancer cells in different treatment groups. (C) The GSH and MDA (F) levels of LLC lung cancer cells in different treatment groups. (D) Representative images of DCF fluorescent and fluorescent intensity (E) reflecting ROS levels of LLC lung cancer cells in different treatment groups. (G) Representative images of C11-BODIPY probe in LLC lung cancer cells across different treatment groups. Green fluorescence indicates oxidized lipid and red fluorescence indicates non-oxidized lipid. NC, normal control; LDR, low dose radiotherapy; ICI, immune checkpoint inhibitor; ROS, reactive oxygen species; gMFI, geometric mean fluorescence intensity. Data are presented as mean ± SD. *p < 0.05; ***p < 0.01. Unpaired two-tailed Student’s t-test.

Figure 5

ROS levels and Nrf2/HO-1/GPX4 pathway analysis in mice across different treatment groups. (A) Western blot of Nrf2/HO-1/GPX4 activation in LLC lung cancer cells. (B) ROS levels in tumor tissues from mice. (C-F) Nrf2/HO-1/GPX4 pathway activation in tumor tissues from mice. LDR, low dose radiotherapy; ICI, immune checkpoint inhibitor; ROS, reactive oxygen species. Data are presented as mean ± SD. *p < 0.05; ***p < 0.01. Unpaired two-tailed Student’s t-test.

Figure 6

The combination of LDR and anti-PD-1 has an anti-tumor effect in patients with chemoimmunotherapy-resistant lung cancer. (A) CT images of a representative patient with NSCLC who underwent four cycles of chemoimmunotherapy and experienced PD. The patient received LDR plus αPD-1 therapy; the dose distribution plan is shown in (B). Following LDR and PD-1 treatment, the patient showed PR. The representative CT images are shown in (C). (D) The best of response of the combination therapy from the base line. (E) Serum IFN-γ levels of the six patients before and after the combination therapy. NSCLC, non-small cell lung cancer; LDR, low dose radiotherapy; PD, progression disease; SD, stable disease; PR, partial response. Data are presented as mean ± SD. ***p < 0.01. Unpaired two-tailed Student’s t-test.

Figure 7

An overview of the present study.