Cell-specific crosstalk proteomics reveals cathepsin B signaling as a driver of glioblastoma malignancy near the subventricular zone

Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially due to subventricular zone (SVZ) contact. Despite this, crosstalk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. Additionally, GBM brain tumor initiating cells (BTICs) increase expression of CTSB upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Finally, we show LV-proximal CTSB upregulation in patients, showing the relevance of this crosstalk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM. To investigate how CTSB contributes to gene expression in glioblastoma brain tumor initiating cells, we performed RNA sequencing on three technical repliactes of primary line GBM1A transduced with empty vector (EV) or shRNA against CTSB (KD 1). We then performed gene expression comparisons to identify differentially expressed genes.

胶质母细胞瘤（Glioblastoma, GBM）是最常见且恶性程度最高的原发性颅内恶性肿瘤。紧邻侧脑室（Lateral Ventricles, LVs）的胶质母细胞瘤侵袭性更强，这可能与其接触室下区（Subventricular Zone, SVZ）有关。尽管如此，学界对胶质母细胞瘤与神经干细胞/前体细胞（Neural Stem/Progenitor Cells, NSC/NPCs）之间的串扰机制仍知之甚少。
本研究通过细胞特异性蛋白质组学技术发现，紧邻侧脑室的胶质母细胞瘤可通过诱导衰老过程阻断神经干细胞/前体细胞的神经元成熟程序。此外，胶质母细胞瘤脑肿瘤起始细胞（Brain Tumor Initiating Cells, BTICs）在与神经前体细胞相互作用后，会上调组织蛋白酶B（Cathepsin B, CTSB）的表达。
慢病毒敲降与重组蛋白实验证实，细胞内源性及可溶性组织蛋白酶B均可促进脑肿瘤起始细胞出现恶性相关表型。可溶性组织蛋白酶B不仅会阻滞神经前体细胞的神经元成熟过程，还可诱导其衰老，这为侧脑室-肿瘤近距离接触与神经发生紊乱之间的关联提供了分子机制解释。
最后，本研究在患者样本中验证了侧脑室旁胶质母细胞瘤组织中组织蛋白酶B的表达上调，证实了该串扰机制在人体胶质母细胞瘤生物学过程中的临床相关性。上述研究结果彰显了蛋白质组学分析在肿瘤微环境研究中的应用价值，并为胶质母细胞瘤的新型治疗策略提供了研究方向。
为探究组织蛋白酶B对胶质母细胞瘤脑肿瘤起始细胞基因表达的调控作用，本研究对转染空载体（Empty Vector, EV）或靶向组织蛋白酶B的短发夹RNA（shRNA，KD1组）的原代细胞系GBM1A的3次技术重复样本进行了RNA测序，并通过基因表达差异分析筛选得到差异表达基因。