The Impact of Glioblastoma Differentiation on Blood-Brain Barrier Permeability

Background Introduction

Glioblastoma Multiforme (GBM) is a highly aggressive brain tumor, and its treatment is extremely challenging due to its complex biological characteristics and the presence of the Blood-Brain Barrier (BBB). The BBB is a complex structure composed of Tight Junctions (TJs) and ATP-binding Cassette (ABC) transporters, which prevent most drugs from entering the brain. Although the BBB is disrupted in the core region of GBM tumors, it remains intact in the Brain-Adjacent-to-Tumor (BAT) areas, making it difficult for drugs to reach these regions and leading to tumor recurrence.

The heterogeneity of GBM is one of the main reasons for treatment failure. The tumor contains both highly differentiated cells and Cancer Stem Cells (CSCs), with the latter playing a key role in tumor initiation, development, and recurrence despite their smaller numbers. CSCs exhibit multidrug resistance, primarily due to the high expression of ABC transporters such as P-glycoprotein (Pgp). Additionally, CSCs can spread to adjacent brain tissues, causing tumor recurrence.

Although it is known that GBM cells release soluble factors that affect BBB permeability, the specific mechanisms remain unclear. This study aims to explore how the differentiation degree of GBM cells influences BBB permeability and to reveal the underlying molecular mechanisms.

Source of the Paper

This paper was co-authored by Sabrina Di Giovanni, Martina Lorenzati, Olga Teresa Bianciotto, and others, from multiple research institutions including the University of Torino in Italy and the Institute Cochin in France. The paper was published in 2024 in the journal Fluids and Barriers of the CNS, titled “Blood-brain barrier permeability increases with the differentiation of glioblastoma cells in vitro.”

Research Process and Results

1. Research Design

This study conducted in vitro experiments by constructing a human BBB model and co-culturing it with GBM cells derived from three patients. The GBM cells were divided into two types: Stem Cell/Neurosphere (SC/NS) and Differentiated/Adherent Cell (AC). The study also included experiments using GBM cell-conditioned medium to investigate the effects of soluble factors on the BBB.

2. Experimental Process

a) Construction of the BBB Model

The researchers used human brain microvascular endothelial cells (HCMEC/D3) as the main component of the BBB, cultured in Transwell inserts. To simulate a more physiologically relevant BBB, the study also constructed a triple-culture model, including endothelial cells, human astrocytes (HAS), and human pericytes (HPEs).

b) Co-culture of GBM Cells with the BBB

GBM cells (AC and NS) were co-cultured with the BBB model for 72 hours. The functional changes in the BBB were assessed by measuring Transendothelial Electrical Resistance (TEER) and the permeability of drugs such as doxorubicin, mitoxantrone, and dextran-70.

c) Conditioned Medium Experiments

The researchers also used GBM cell-conditioned medium, with or without IL-6 (Interleukin-6), to verify the role of IL-6 in regulating BBB permeability.

d) Molecular Mechanism Studies

Through immunoblotting and quantitative real-time PCR (qRT-PCR), the researchers detected changes in the expression of ABC transporters and tight junction proteins in BBB cells. Additionally, they used the STAT3 (Signal Transducer and Activator of Transcription 3) inhibitor STA-21 and the PROTAC (Proteolysis Targeting Chimera) SD-36 targeting STAT3 to further validate the role of the IL-6/STAT3 signaling pathway.

3. Main Results

a) GBM Cells Increase BBB Permeability

The study found that co-culture with GBM cells significantly increased the permeability of the BBB to doxorubicin, mitoxantrone, and dextran-70, while TEER values decreased. These effects were more pronounced in the AC co-culture group, indicating that more differentiated GBM cells have a greater impact on the BBB.

b) Effects of Conditioned Medium

The conditioned medium from GBM cells also increased BBB permeability, with the AC-conditioned medium showing stronger effects than the NS-conditioned medium. ELISA detection revealed that the level of IL-6 in the AC-conditioned medium was significantly higher than in the NS-conditioned medium.

c) Key Role of IL-6

By adding or removing IL-6, the researchers identified IL-6 as a key factor in regulating BBB permeability. IL-6 activates the STAT3 signaling pathway, downregulating the expression of ABC transporters and tight junction proteins, thereby increasing BBB permeability.

d) Role of the STAT3 Signaling Pathway

Treatment with the STAT3 inhibitor STA-21 or the PROTAC SD-36 significantly reduced BBB permeability and restored the expression of ABC transporters and tight junction proteins. This indicates that the IL-6/STAT3 axis plays a central role in regulating BBB permeability.

4. Conclusion

This study is the first to reveal the impact of GBM cell differentiation on BBB permeability. More differentiated GBM cells release IL-6, activating the STAT3 signaling pathway in BBB cells, downregulating the expression of ABC transporters and tight junction proteins, and thereby increasing BBB permeability. This discovery provides new insights for improving drug delivery in GBM treatment, particularly in the tumor margin regions where the BBB remains intact.

Research Highlights

1. First to Reveal the Impact of Differentiation on BBB Permeability: This study is the first to demonstrate the relationship between GBM cell differentiation and BBB permeability, filling a gap in this field of research.

2. Key Role of the IL-6/STAT3 Axis: The study clarifies the molecular mechanism by which IL-6 regulates BBB permeability through the STAT3 signaling pathway, offering a potential target for future drug development.

3. Construction of Multiple Experimental Models: The study not only used a single BBB model but also constructed a more physiologically relevant triple-culture model, enhancing the reliability of the experimental results.

4. Potential Therapeutic Applications: By modulating the IL-6/STAT3 axis, new therapeutic strategies may be developed to improve the delivery efficiency of chemotherapeutic drugs in GBM treatment.

Research Significance

The scientific value of this study lies in revealing the association between GBM cell differentiation and BBB permeability and identifying the central role of the IL-6/STAT3 axis in this process. This discovery not only deepens our understanding of GBM biology but also provides new ideas for improving GBM treatment. By targeting the IL-6/STAT3 axis, more effective drug delivery strategies may be developed in the future, enhancing treatment outcomes for GBM patients.

Furthermore, this study offers new research perspectives for other neurological diseases involving the BBB, such as Alzheimer’s disease and Parkinson’s disease, with broad application prospects.