Extracellular vesicles originating from glioblastoma cells increase metalloproteinase release by astrocytes: the role of CD147 (EMMPRIN) and ionizing radiation

Abstract

Background: Glioblastoma multiforme is an aggressive primary brain tumor that is characterized by local invasive growth and resistance to therapy. The role of the microenvironment in glioblastoma invasiveness remains unclear. While carcinomas release CD147, a protein that signals for increased matrix metalloproteinase (MMP) release by fibroblasts, glioblastoma does not have a significant fibroblast component. We hypothesized that astrocytes release MMPs in response to CD147 contained in glioblastoma-derived extracellular vesicles (EVs) and that ionizing radiation, part of the standard treatment for glioblastoma, enhances this release. Methods: Astrocytes were incubated with EVs released by irradiated or non-irradiated human glioblastoma cells wild-type, knockdown, or knockout for CD147. Levels of CD147 in glioblastoma EVs and MMPs secreted by astrocytes were quantified. Levels of proteins in the mitogen activated protein kinase (MAPK) pathway, which can be regulated by CD147, were measured in astrocytes incubated with EVs from glioblastoma cells wild-type or knockdown for CD147. Immunofluorescence was performed on the glioblastoma cells to identify changes in CD147 localization in response to irradiation, and to confirm uptake of the EVs by astrocytes. Results: Immunoblotting and mass spectrometry analyses showed that CD147 levels in EVs were transiently increased when the EVs were from glioblastoma cells that were irradiated with γ rays. Specifically, the highlyglycosylated 45 kDa form of CD147 was preferentially present in the EVs relative to the cells themselves. Immunofluorescence demonstrated that astrocytes incorporate glioblastoma EVs and subsequently increase their secretion of active MMP9. The increase was greater if the EVs were from irradiated glioblastoma cells. Testing MAPK pathway activation, which also regulates MMP expression, showed that JNK signaling, but not ERK1/2 or p38, was increased in astrocytes incubated with EVs from irradiated compared to non-irradiated glioblastoma cells. Knockout of CD147 in glioblastoma cells blocked the increased JNK signaling and the rise in secreted active MMP9 levels. Conclusions:The results support a tumor microenvironment-mediated role of CD147 in glioblastoma invasiveness, and reveal a prominent role for ionizing radiation in enhancing the effect. They provide an improved understanding of glioblastoma intercellular signaling in the context of radiotherapy, and identify pathways that can be targeted to reduce tumor invasiveness.

Function of exosomes in neurological disorders and brain tumors

“…Astrocytes cultured with GBM EVs show enhanced secretion of immunosuppressive cytokines such as CSF2 and interleukins 4, 10 and 13, thus providing a tumor supportive microenvironment. EVs secreted by irradiated GBM cells have enhanced presence of CD147, which in turn stimulates increased MMP9 release from recipient astrocytes, suggesting the contribution of astrocyte signaling in promoting GBM invasiveness, particularly in response to ionizing radiation [ 229 ] . A recent study reported that GSC EVs induce metabolic reprogramming of pre-transformed astrocytes to enhance proliferation, self-renewal and tumor growth in a mouse allograft model [ 230 ] .…”

Extracellular Vesicle-Mediated Bilateral Communication between Glioblastoma and Astrocytes

“…In response to IR, glioblastoma cells increase the levels of CD147 shed in EVs. This protein, involved in regulation of MMPs, enhances MMP9 release from astrocytes exposed to these EVs by increasing JNK signaling, a process that was blocked by knocking out CD147 in glioma cells [69]. Since MMPs break down ECM, this result supports a tumor microenvironment-mediated role of CD147 in glioblastoma invasiveness, and reveals a prominent role for IR, used in treatment for glioblastoma, in enhancing this effect [69].…”

“…This protein, involved in regulation of MMPs, enhances MMP9 release from astrocytes exposed to these EVs by increasing JNK signaling, a process that was blocked by knocking out CD147 in glioma cells [69]. Since MMPs break down ECM, this result supports a tumor microenvironment-mediated role of CD147 in glioblastoma invasiveness, and reveals a prominent role for IR, used in treatment for glioblastoma, in enhancing this effect [69]. In addition, astrocytes exposed to glioblastoma-derived EVs were tested in an 'invadopodia' assay to determine the effects on astrocyte migration and ECM degradation [65].…”

Advances in research on glioma microenvironment and immunotherapeutic targets

“…A study showed that a distinct astrocytic phenotype is caused by the coexistence of microglia and astrocytes in the tumor environment, which leads to a large release of antiinflammatory cytokines such as TGF-β, IL-10 and G-CSF [83]. RAs increase the expression levels of MMP9, which promotes the tumor invasiveness by breaking down the ECM [84]. After radiation, RAs will increase the secretion of transglutaminase 2 (TG2), which triggers the MES trans-differentiation of GSCs [85,86].…”