Radiotherapy-activated NBTXR3 nanoparticles promote ferroptosis through induction of lysosomal membrane permeabilization

Abstract

Purpose: Radiotherapy-activated NBTXR3 (NBTXR3 + RT) has demonstrated superior efficacy in cancer cell destruction and tumor growth control, compared to radiotherapy (RT), in preclinical and clinical settings. Previous studies highlighted the immunomodulatory properties of NBTXR3 + RT, such as modification of tumor cell immunogenicity/adjuvanticity, producing an effective local tumor control and abscopal effect, related to an enhanced antitumor immune response. Furthermore, NBTXR3 + RT has shown potential in restoring anti-PD1 efficacy in a refractory tumor model. However, the early events leading to these results, such as NBTXR3 endocytosis, intracellular trafficking and primary biological responses induced by NBTXR3 + RT remain poorly understood.

Methods: We analyzed by transmission electron microscopy endocytosis and intracellular localization of NBTXR3 nanoparticles after endocytosis in various cell lines, in vitro and in vivo. A kinetic of NBTXR3 endocytosis and its impact on lysosomes was conducted using LysoTracker staining, and a RNAseq analysis was performed. We investigated the ability of NBTXR3 + RT to induce lysosomal membrane permeabilization (LMP) and ferroptosis by analyzing lipid peroxidation. Additionally, we evaluated the recapture by cancer cells of NBTXR3 released from dead cells.

Results: NBTXR3 nanoparticles were rapidly internalized by cells mainly through macropinocytosis and in a less extend by clathrin-dependent endocytosis. NBTXR3-containing endosomes were then fused with lysosomes. The day following NBTXR3 addition, we measured a significant increase in LysoTracker lysosome labeling intensity, in vitro as in vivo. Following RT, a significant lysosomal membrane permeabilization (LMP) was measured exclusively in cells treated with NBTXR3 + RT, while RT had no effect. The day post-irradiation, a significant increase in lipid peroxidation, a biomarker of ferroptosis, was measured with NBTXR3 + RT compared to RT. Moreover, we demonstrated that NBTXR3 nanoparticles released from dead cells can be recaptured by cancer cells.

Conclusions: Our findings provide novel insights into the early and specific biological effects induced by NBTXR3 + RT, especially LMP, not induced by RT in our models. The subsequent significant increase in lipid peroxidation partially explains the enhanced cancer cell killing capacity of NBTXR3 + RT compared to RT, potentially by promoting ferroptosis. This study improves our understanding of the cellular mechanisms underlying NBTXR3 + RT and highlights its potential as an agnostic therapeutic strategy for solid cancers treatment.

Keywords: Ferroptosis; Lipid peroxidation; Lysosome membrane permeabilization; NBTXR3; Nanoparticle; Radiotherapy.

Fig. 1

NBTXR3 nanoparticles are internalized by different endocytosis pathways. Transmission electronic microscopy (TEM) representative images of NBTXR3 nanoparticles uptake by macropinocytosis and clathrin-mediated endocytosis in CT26.WT (upper panel), HT1080 (middle panel) and 42-MG-BA (lower panel) at 1 h, 3 h and 24 h after addition of 400 µM NBTXR3. The membrane invagination and lamellipodia observed indicate membrane perturbations typical of macropinocytosis. White arrow: membrane ruffles enclosing a small number of NBTXR3 nanoparticles; black arrow: internalized NBTXR3 nanoparticles cluster in early endosomes. Scale bar, 300 nm. Abbreviations: C, Clathrin; CTL, control cells (no NBTXR3); Cyt, cytoplasm; Nuc, nucleus

Fig. 2

Endocytosis of NBTXR3 nanoparticles by cancer cells increases constantly over time. Following addition of 400 µM NBTXR3, the evolution of the cellular granularity at the indicated time-points was carried out by flow cytometry for the CT26.WT, HT1080, 42-MG-BA, and HCT116 cells. A Representative flow cytometry analysis of the evolution of the cellular granularity over time. B Kinetic of cellular granularity profile evolution for the CT26.WT (n = 4), HT1080 (n = 5), 42-MG-BA (n = 8), and HCT116 (n = 3) cells. Data of independent experiments are represented as the relative granularity to CTL ± SEM. Statistical test: One-way ANOVA. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001. C Baseline granularity of the tested cell lines. Data of independent experiments are represented as the mean granularity (SSC-A) ± SEM. D Comparison of granularity profile evolution for tested cell lines. Data of independent experiments are represented as the relative granularity to CTL ± SEM. E Evolution of the cellular granularity profile for 42-MG-BA cells (n = 4) according to NBTXR3 concentrations

Fig. 3

Endosomes containing NBTXR3 clusters are fused with lysosomes. Immuno-TEM images obtained 24 h after treatment of 42-MG-BA cells, showing that A NBTXR3 and B NBTXR3^RED clusters colocalize with LAMP-1, a specific marker for lysosomes. Black arrows indicate the location of AuNPs allowing identification of LAMP-1, surrounding the clusters of NBTXR3 and NBTXR3^RED. Scale bar, 300 nm. C Representative confocal microscopy images taken 24 h after treatment of CT26.WT, HT1080, 42-MG-BA and HCT116 cells with NBTXR3.^RED ., LysoTracker-stained lysosomes ;, NBTXR3 nanoparticle labelled with utilized Dextran Tetramethylrhodamine (NBTXR3^RED); , actin filaments; , nucleus. Scale bar, 10µm

Fig. 4

Endocytosis of NBTXR3 nanoparticles by cancer cells increases LysoTracker signal over the time. Following addition of 400 µM NBTXR3, the evolution LysoTracker signal at the indicated time-points was carried out by flow cytometry for the CT26.WT, HT1080, 42-MG-BA, and HCT116 cells. Representative evolution of LysoTracker signal over time analyzed by A flow cytometry and B fluorescent microscopy. C Kinetic of modification of LysoTracker profile for the CT26.WT (n = 4), HT1080 (n = 5), 42-MG-BA (n = 8), and HCT116 (n = 3) cells. Data of independent experiments are represented as the relative LysoTracker MFI to CTL ± SEM. Statistical test: One-way ANOVA or Friedman test. *, p < 0.05; **, p < 0.01; ***, p < 0.001, p < 0.0001; ****. D Baseline LysoTracker signal of the tested cell lines. Data of independent experiments are represented as the MFI ± SEM. E Comparison of MFI LysoTracker profile evolution for tested cell lines. Data of independent experiments are represented as the relative MFI to CTL ± SEM. F Evolution of the MFI LysoTracker profile for 42-MG-BA cells (n = 4) according to NBTXR3 concentrations

Fig. 5

Endocytosis of NBTXR3 nanoparticles induces upregulation of genes linked to lysosomes. RNAseq analysis of CT26.WT cells treated for 24 h with NBTXR3 (n = 3). A Heatmap of lysosome-associated genes (GO:0005764). B Volcano plot of lysosome-associated genes (GO:0005764) showing significantly differentially expressed genes (p < 0.05) of NBTXR3-treated (NBTXR3) vs. untreated (CTL) CT26.WT cells. Heatmap of expression of C primary lysosome (GO:0005766) D secondary lysosome (GO:0005767) and E late endosome to lysosome transport (GO:1,902,774). For all presented heatmaps, each column stands for the mean value of three independent experiments. Expression values are shown as the Z-score mean of the log₂ transformed normalized counts. Red and green are assigned to higher and lower expression, respectively, according to the color key in the right

Fig. 6

Intratumoral injection of NBTXR3 lead to significant increase of both granularity and LysoTracker signal, and endocytosis by cancer cells. A Schematic representation of the experiment workflow. Box and whisker plots representation of cancer cells B granularity and C LysoTracker signal measured 24 h after intratumoral injection of NBTXR3 or 5% Glc (vehicle, CTL). Presented data were obtained from 4–5 mice per group (each dot represents one value) from two independent experiments. Statistical test: Unpaired t-test. *, p < 0.05. D TEM representative images of NBTXR3 nanoparticles uptake by CT26.WT cells 24 h after intratumoral injection of NBTXR3 from five mice (CTL, mouse #1; NBTXR3-treated mice: mouse #2, pictures 1–2; mouse #3, picture 3; mouse #4, picture 4; mouse #5, pictures 6–8). White arrow: membrane ruffles enclosing a small NBTXR3 nanoparticle aggregate; black arrow: internalized NBTXR3 nanoparticles cluster. Scale bars: CTL, 2µm; 1–8, 0.5µm. Abbreviations: CTL, control cells (no NBTXR3); Cyt, cytoplasm; Nuc, nucleus

Fig. 7

NBTXR3 nanoparticles released from cancer cells killed by RT can be again endocytosed by live cancer cells. Analysis of recaptures NBTXR3^RED released from 42-MG-BA dead cells. A Schematic representation of the experiment workflow. B Fluorescent microscopy representative images of NBTXR3^RED nanoparticles recapture experiment 48h after co-culture of untreated (CTL, upper panel) or RT-treated (RT, lower panel) conditions. White arrows indicate the presence of NBTXR3^RED nanoparticles in the cytoplasm of 42-MG-BA-ACTB-GFP cells. C Box and whisker plots representation of flow cytometry analysis of NBTXR3^RED MFI in 42-MG-BA-ACTB-GFP cells according to the tested condition (CTL, untreated; RT, radiotherapy-treated), from three independent experiments. Statistical test: Paired t-test. *, p < 0.05. D Representative TEM images of NBTXR3^RED nanoparticles uptake by 42-MG-BA-ACTB-GFP cells 48h after co-culture with dead 42-MG-BA NBTXR3^RED cells. Black arrow: internalized NBTXR3^RED nanoparticles cluster. Scale bar: 0.5µm. Abbreviations: Cyt, cytoplasm; MVB, multivesicular body. E FACS gating strategy employed to isolate NBTXR3^RED-containing 42-MG-BA-ACTB-GFP cells from RT condition for TEM analysis

Fig. 8

NBTXR3 + RT promotes ferroptosis cell death through lysosomal membrane permeabilization and lipid peroxidation enhancement. A Clonogenic assay on CT26.WT (n = 5), HT1080 (n = 4), 42-MG-BA (n = 3) and HCT116 (n = 5) cells. Data of independent experiments are represented as the surviving fraction ± SEM. Statistical test: One way ANOVA. B Relative LysoTracker MFI signal measured by flow cytometry 1 h post-RT in CT26.WT (n = 4), HT1080 (n = 3), 42-MG-BA (n = 3) and HCT116 (n = 4) cells to evaluate lysosomal membrane permeabilization. Data of independent experiments are represented as the MFI ± SEM. Statistical test: One way ANOVA. C Fluorescent microscopy representative images of Cathepsin D signal in 42-MG-BA cells 6h post-RT, at the indicated condition. Scale bar, 10µm. D Scatter dot plot of Cathepsin D signal intensity measured in 42-MG-BA cells at the indicated condition, from three independents experiments. Each dot represents one cell intensity. “n” on figures indicates number of cells analyzed. Statistical test: Kruskal–Wallis test. Bar, median. E Relative BODIPY MFI signal measured by flow cytometry 24 h post-RT in CT26.WT, HT1080, 42-MG-BA and HCT116 cells measured, to evaluate lipid peroxidation. Data of independent experiments (n = 3) are represented as MFI ± SEM. Statistical test: One way ANOVA. For all Figures: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, non-significant

Fig. 9

Model presenting the biological responses induced by NBTXR3 alone or activated by radiotherapy. The dotted lines indicate the potential biological pathways impacted by NBTXR3 + RT to be identified or for which a connection between two measured events previously published remains to be established