Irradiation of Glioblastoma Cells Increases CD147 Levels in their Extracellular Vesicles: Contribution to Increased MMP9 Activity in the Tumor Microenvironment

Abstract

Glioblastoma is a locally invasive primary cancer of the central nervous system. Current treatment modalities are unable to adequately limit this invasiveness, leading to a 95% recurrence rate. Moreover, although radiotherapy has produced the largest improvement in survival for these patients, it is also associated with increased tumor invasiveness, perhaps contributing to the high recurrence rate. This study found that CD147, a protein which is commonly overexpressed in glioblastoma, may contribute to this invasion, in particular the increased invasion after radiation exposure. Interestingly, CD147 works to promote invasion in cancer cells via intercellular communication: tumors secrete CD147 in extracellular vesicles (EVs) to induce neighboring cells to produce matrix metalloproteinases (MMPs), which can degrade the extracellular matrix. We irradiated T98G human glioblastoma cells with 8Gy of 137Cs γ-rays, and collected EVs from the medium at 24h using serial ultracentrifugation. The EVs were characterized by cryo-electron microscopy, NanoSight, and immunoblots. They were either collected for biochemical analyses or added at a 3x concentration to SVG human astrocytes maintained in culture for 24 h. One-way ANOVA with p<0.05 was considered significant, and the Holm test was used to control for multiple comparisons. Mass spectrometry analysis had CD147 in the top 1% of increased proteins in EVs from irradiated glioblastoma cells (IR-EVs) relative to EVs from control, non-irradiated glioblastoma cells (C-EVs). This increase was validated by immunoblot. Notably, EVs were enriched in the active (highly glycosylated) form of the protein. In contrast, CD147 protein levels in the glioblastoma cells were not increased, and PCR demonstrated the mRNA levels had not changed – suggesting irradiated cells mobilize existing CD147 into EVs. We then examined the effects of glioblastoma cells on the microenvironment, reporting fold change in astrocytes receiving glioblastoma cell EVs relative to those receiving no EVs. All results have an n=6 with p<0.01. Astrocytes receiving C-EVs or IR-EVs had 3-fold higher MMP2 activity as determined by zymography. The activity of MMP9 secreted by astrocytes increased 10-fold in response to receiving C-EVs, but increased 14-fold in response to IR-EVs – a significant difference. Knockdown of CD147 by shRNA in the glioblastoma cells rendered its levels in the EVs undetectable by immunoblot. The C-EVs from these CD147-knockdown glioblastoma cells could still produce a 3-fold increase in MMP2 and an 8-fold increase in MMP9 activity, but IR-EVs could no longer produce an increase in MMP9 activity, i.e. MMP9 activity was reduced from 14-fold to 7-fold of the control astrocytes’. Glioblastoma cells can increase CD147 levels in their EVs in response to irradiation, contributing to increased MMP9 activity, but not necessarily MMP2 activity, through intercellular communication with astrocytes.