EGFP Reporter Cell Line - GL261

The progression of gliomas is closely linked to neuronal activity. Glutamatergic neurons form functional synapses with glioma cells (neuron-glioma synapses, NGS), directly promoting tumor growth via electrophysiological signaling. Here, researchers investigated the critical role of the free 19S proteasome within NGSs and elucidated its underlying molecular mechanisms. By integrating in situ mouse glioma models with in vitro NGS models, the study confirmed a specific enrichment of free 19S proteasomes within tumor-infiltrating regions. These proteasomes maintain the stability of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) through their deubiquitinating activity, thereby enhancing synaptic transmission and driving tumor progression. Functionally, inhibiting the deubiquitinating activity of the free 19S proteasome significantly attenuates postsynaptic potentials and suppresses both tumor proliferation and invasion. Notably, radiotherapy was found to further activate the free 19S-AMPAR axis, suggesting that targeting this pathway to enhance radiosensitivity could represent a highly promising therapeutic strategy. In therapeutic experiments utilizing mouse models, the combined inhibition of the 19S-AMPAR axis with radiotherapy resulted in a significant suppression of glioma growth. These findings elucidate a novel mechanism of interaction between gliomas and neurons mediated by proteasome-dependent synaptic modulation, laying a solid foundation for the development of combination therapies targeting synaptic pathways.

To assess cellular proliferation within neuron-glioma synapses (NGS), researchers co-cultured GL261-EGFP glioma cells with HT22 neurons for 72 hours to establish an NGS model. Immunofluorescence analysis revealed that, compared to the monoculture group, glioma cells co-cultured with neurons exhibited a significantly higher rate of Ki67 positivity. Notably, this increase was significantly attenuated following treatment with bAP15 (Figure 1A–B). Quantitative analysis of fluorescently labeled cells after 5 days of co-culture confirmed a significant increase in glioma cell numbers compared to the monoculture control group. This co-culture-induced proliferative effect was once again significantly inhibited by bAP15 treatment (Figure 1C). Importantly, both the specific AMPAR inhibitor CNQX and AMPAR knockdown produced anti-proliferative effects similar to those of bAP15, suggesting that the 19S proteasome primarily regulates tumor proliferation via the AMPAR pathway (Figure 1A-C). In invasion assays, Transwell analysis demonstrated that, compared to the monoculture control group, the number of glioma cells penetrating the Matrigel matrix was significantly higher in the co-culture group comprising GL261-EGFP glioma cells and HT22 neurons. Treatment with bAP15 significantly suppressed this enhanced invasive capacity (Figure 1D–E). Consistent with the results of the proliferation assays, CNQX treatment and AMPAR knockdown similarly inhibited glioma cell invasion, further highlighting the pivotal role of the 19S-AMPAR signaling axis in driving the malignant progression of gliomas (Figure 1D-E).

Figure 1. The free 19S-AMPAR Axis Promotes Glioma Proliferation and Invasion. (Lu Y, et al., 2026)