Low-LET X-ray Radiation Enhances the Cellular Connectivity Through Tunneling Nanotubes in Glioblastoma Cells

Developing therapy resistance and exhibiting high invasiveness are significant challenges in treating aggressive cancers, such as glioblastoma, where intercellular communication plays a crucial role in cellular organization, survival, and resistance to treatment. Tunneling nanotubes (TNTs), nanometer-sized membranous channels that connect distant cells, have emerged as an efficient form of intercellular communication that may enable cancer cells to evade therapeutic interventions.

In this study, we investigated the responses of TNT networks to low linear energy transfer (low-LET) X-ray irradiation in two established glioblastoma cell lines, U87 MG and LN229. Initially, we assessed radiosensitivity using colony formation assays to measure cell survival. Then, we used confocal live-cell microscopy to monitor TNT network dynamics over a 24-hour period following irradiation and performed co-staining experiments to identify cargoes transported through TNTs.

We observed a significant increase in TNT-mediated cellular connectivity 6 to 10 hours after 1.8 Gy X-ray irradiation in both cell lines. In contrast, cells treated with a higher radiation dose (3.9 Gy) exhibited reduced TNT connectivity; however, it remained slightly elevated compared to sham-irradiated controls. The co-staining experiments revealed the presence of calcium and mitochondria within TNTs. These cargoes are known to facilitate cancer cell migration and survival, potentially contributing to treatment resistance.

Taken together, these results strongly suggest that TNT-mediated intercellular communication may be a critical mechanism that supports glioblastoma resistance to radiotherapy.