NeuroD4 converts glioblastoma cells into neuron-like cells through the SLC7A11-GSH-GPX4 antioxidant axis

Abstract

Cell fate and proliferation ability can be transformed through reprogramming technology. Reprogramming glioblastoma cells into neuron-like cells holds great promise for glioblastoma treatment, as it induces their terminal differentiation. NeuroD4 (Neuronal Differentiation 4) is a crucial transcription factor in neuronal development and has the potential to convert astrocytes into functional neurons. In this study, we exclusively employed NeuroD4 to reprogram glioblastoma cells into neuron-like cells. In vivo, the reprogrammed glioblastoma cells demonstrated terminal differentiation, inhibited proliferation, and exited the cell cycle. Additionally, NeuroD4 virus-infected xenografts exhibited smaller sizes compared to the GFP group, and tumor-bearing mice in the GFP+NeuroD4 group experienced prolonged survival. Mechanistically, NeuroD4 overexpression significantly reduced the expression of SLC7A11 and Glutathione peroxidase 4 (GPX4). The ferroptosis inhibitor ferrostatin-1 effectively blocked the NeuroD4-mediated process of neuron reprogramming in glioblastoma. To summarize, our study demonstrates that NeuroD4 overexpression can reprogram glioblastoma cells into neuron-like cells through the SLC7A11-GSH-GPX4 signaling pathway, thus offering a potential novel therapeutic approach for glioblastoma.

Fig. 1. Morphological changes of human glioblastoma cells induced by NeuroD4.

A Schematic representation of the cell reprogramming process. B, C Rapid morphological changes of U251 and KNS89 cells were observed upon ectopic expression of NeuroD4. The control group was infected with GFP-expressing virus. Higher magnification views of the boxed regions are also depicted. Dpi: days after infection; GM: medium for glioblastoma cells; NM: medium for neuronal induction. Scale bar: 200 µm.

Fig. 2. Time-course analysis of NeuroD4-mediated neuronal reprogramming of human glioblastoma cells.

A, C, E, G Immunostaining of neuronal markers TUJ1 and MAP2 in NeuroD4 transformed U251 and KNS89 cells at 14 dpi. B, D, F, H Quantifying neuronal marker expression in NeuroD4-infected U251 and KNS89 cells during the indicated course (9 random fields from triplicate samples were captured for quantification). Dpi: days post infection. ND not detected. Scale: 100 µm.

Fig. 3. NeuroD4-induced neuronal reprogramming suppresses the proliferative marker EdU and Ki67 in glioblastoma cells.

A The experimental design for labeling EdU in glioblastoma cells. B–E EdU detection of U251 and KNS89 cells at 14 dpi and quantitative analysis of EdU+ (9 random fields from triplicate samples were captured for quantification). F–I Immunocytochemical analysis of the Ki67 marker in U251 and KNS89 cells at 14 dpi and quantitative analysis of Ki67+ (9 random fields from triplicate samples were captured for quantification). J, K Time-course cell counting at 0, 1, 3, 5, 7, 14, and 21 dpi. The data are presented as mean ± SD. ***P < 0.001. Dpi: days post infection; GM glioblastoma cell medium, NM neuronal induction medium. Scale: 100 µm.

Fig. 4. Overexpression of NeuroD4 resulted in cell cycle exit.

A Time-course flow cytometric analysis of cell cycle displaying the cellular DNA content of NeuroD4 overexpressed cells in U251, KNS89, and U87 cells. B Quantitative analysis of DNA content (n = 3). C, D The expression of various cell cycle-related mRNA in U251 and KNS89 cells was detected by qRT-PCR. The data are presented as mean ± SD. **P < 0.01, ***P < 0.001 by student’s t-test and One-way analysis of variance (ANOVA). Dpi days post infection. NS no significance.

Fig. 5. The xenografts derived from NeuroD4 overexpressing U87 cells shrink significantly.

A The experimental protocol for orthotopic cell transplantation. B, C In vivo bioluminescent images and the quantification of U87-derived xenografts (n = 5). D Post-imaging Kaplan-Meier survival analysis of transplanted mice (n = 10, P < 0.001 using log-rank test). E, F Immunohisto fluorescence analysis of tumor size in mice implanted with GFP and GFP+NeuroD4 virus infected U87 cells 28 days after transplantation (n = 6). Tumor mass (outlined by dashed lines) was quantified based on the area occupying the ipsilateral brain. G–I Ki67 and TUJ1 markers of NeuroD4 overexpressing U87 cells in xenografts 28 days after implantation. Forty-five random fields from a series of every tenth coronal brain section of six nude mice were collected for quantification in Immunohisto fluorescence analysis. The data are presented as mean ± SD. ***P < 0.001 by student’s t-test and One-way analysis of variance (ANOVA). NS: no significance. ND not detected. GM glioblastoma cell medium. Scale: 100 µm.

Fig. 6. Screening of drugs for inhibiting cell death and analysis of RNA sequencing.

A, B TUJ1 detection at 7 dpi revealed the reprogramming efficiency of GFP+NeuroD4 lentivirus infected U251 cells treated with four cell death inhibitors and quantitative analysis (9 random fields from triplicate samples were captured for quantification). C A heatmap shows the expression of 200 distinct genes in GFP and GFP+NeuroD4 U251 cells (n = 3). D, E The volcano map and pathway enrichment analysis of distinct expression genes between GFP and GFP+ NeuroD4 U251 cells. F The log10 (FPKM) value of neuronal markers in glioblastoma cell reprogramming (n = 3). G The FPKM of GPX4 between GFP and GFP+ NeuroD4 U251 cells (n = 3). The data are presented as mean ± SD. ***P < 0.001 by student’s t-test. Dpi days post infection. NS no significance. ND not detected. Scale: 100 µm.

Fig. 7. SLC7A11-GSH-GPX4 antioxidant axis is a key in NeuroD4-mediated neuronal reprogramming.

A, B The fluorescence intensity of Reactive oxygen species (ROS) was detected by flow cytometry at 5 dpi. C–E Western blot analysis of SLC7A11 and GPX4 protein in U251 cells infected with GFP and GFP+NeuroD4 virus at 3 and 7 dpi. (n = 3). F–H Ki67 and TUJ1 staining revealed the reprogramming efficiency of particular lentiviruses-infected U251 cells at 14 dpi (9 random fields from triplicate samples were captured for quantification). I Pathways involved in NeuroD4-induced reprogramming. GSR glutathione-disulfide reductase. GSSG glutathione oxidized. The data are presented as mean ± SD. *P < 0.01, **P < 0.005, ***P < 0.001 by student’s t-test. Dpi days post infection. NS no significance. ND not detected. Scale: 100 µm.