Department of Oncology, University of Torino, piazza Nizza 44, Torino, 10126, Italy.
Molecular Biotechnology Center "Guido Tarone", University of Torino, piazza Nizza 44, Torino, 10126, Italy.
Fluids Barriers CNS. 2024 Nov 1;21(1):89. doi: 10.1186/s12987-024-00590-0.
Glioblastoma multiforme (GBM) is an aggressive tumor, difficult to treat pharmacologically because of the blood-brain barrier (BBB), which is rich in ATP-binding cassette (ABC) transporters and tight junction (TJ) proteins. The BBB is disrupted within GBM bulk, but it is competent in brain-adjacent-to-tumor areas, where eventual GBM foci can trigger tumor relapse. How GBM cells influence the permeability of BBB is poorly investigated.
To clarify this point, we co-cultured human BBB models with 3 patient-derived GBM cells, after separating from each tumor the stem cell/neurosphere (SC/NS) and the differentiated/adherent cell (AC) components. Also, we set up cultures of BBB cells with the conditioned medium of NS or AC, enriched or depleted of IL-6. Extracellular cytokines were measured by protein arrays and ELISA. The intracellular signaling in BBB cells was measured by immunoblotting, in the presence of STAT3 pharmacological inhibitor or specific PROTAC. The competence of BBB was evaluated by permeability assays and TEER measurement.
The presence of GBM cells or their conditioned medium increased the permeability to doxorubicin, mitoxantrone and dextran-70, decreased TEER, down-regulated ABC transporters and TJ proteins at the transcriptional level. These effects were higher with AC or their medium than with NS. The secretome analysis identified IL-6 as significantly more produced by AC than by NS. Notably, AC-conditioned medium treated with an IL-6 neutralizing antibody reduced the BBB permeability to NS levels, while NS-conditioned medium enriched with IL-6 increased BBB permeability to AC levels. Mechanistically, IL-6 released by AC GBM cells activated STAT3 in BBB cells. In turn, STAT3 down-regulated ABC transporter and TJ expression, increased permeability and decreased TEER. The same effects were obtained in BBB cells treated with STA-21, a pharmacological inhibitor of STAT3, or with a PROTAC targeting STAT3.
Our work demonstrates for the first time that the degree of GBM differentiation influences BBB permeability. The crosstalk between GBM cells that release IL-6 and BBB cells that respond by activating STAT3, controls the expression of ABC transporters and TJ proteins on BBB. These results may pave the way for novel therapeutic tools to tune BBB permeability and improve drug delivery to GBM.
多形性胶质母细胞瘤（GBM）是一种侵袭性肿瘤，由于血脑屏障（BBB）富含三磷酸腺苷结合盒（ABC）转运体和紧密连接（TJ）蛋白，因此在药理学上难以治疗。GBM 实质内的 BBB 被破坏，但在肿瘤邻近脑区仍具有功能，最终的 GBM 病灶可能引发肿瘤复发。GBM 细胞如何影响 BBB 的通透性还不清楚。
为了阐明这一点，我们将 3 例患者来源的 GBM 细胞与体外 BBB 模型共培养，从每个肿瘤中分离出干细胞/神经球（SC/NS）和分化/贴壁细胞（AC）成分。此外，我们还建立了含有 NS 或 AC 条件培养基的 BBB 细胞培养物，其中 NS 或 AC 条件培养基中富含或缺乏白细胞介素 6（IL-6）。通过蛋白质芯片和 ELISA 测量细胞外细胞因子。在存在 STAT3 药理学抑制剂或特异性 PROTAC 的情况下，通过免疫印迹测量 BBB 细胞内的信号转导。通过通透性测定和 TEER 测量评估 BBB 的功能。
GBM 细胞或其条件培养基的存在增加了阿霉素、米托蒽醌和右旋糖酐-70 的通透性，降低了 TEER，在转录水平下调了 ABC 转运体和 TJ 蛋白。与 NS 相比，AC 或其培养基的作用更高。分泌组分析表明，AC 比 NS 产生更多的白细胞介素 6（IL-6）。值得注意的是，用抗白细胞介素 6 中和抗体处理的 AC 条件培养基将 NS 水平的 BBB 通透性降低至 NS 水平，而富含白细胞介素 6 的 NS 条件培养基将 BBB 通透性增加至 AC 水平。从机制上讲，AC GBM 细胞释放的白细胞介素 6 激活了 BBB 细胞中的 STAT3。反过来，STAT3 下调了 ABC 转运体和 TJ 的表达，增加了通透性并降低了 TEER。在使用 STAT3 药理学抑制剂 STA-21 或针对 STAT3 的 PROTAC 处理 BBB 细胞时，也获得了相同的效果。
我们的工作首次证明，GBM 分化程度会影响 BBB 的通透性。释放白细胞介素 6 的 GBM 细胞与通过激活 STAT3 做出反应的 BBB 细胞之间的串扰，控制 BBB 上 ABC 转运体和 TJ 蛋白的表达。这些结果可能为调节 BBB 通透性和改善 GBM 药物递送的新型治疗工具铺平道路。
