Multi-inlet spheroid generator for modeling immune microenvironment of glioblastoma (GBM)

Using the multi-inlet spheroid generator (MSG) platform, we generated stable GBM spheroids and observed their interactions with various immune cells, including microglia and natural killer (NK) cells, at different time points. HMO6 and NK-92 cells were used for microglia and NK cells, respectively. We examined the interactions between tumor-immune cells as well as between different immune cells. The generation of tri-cultured spheroids successfully recapitulated the immunosuppressive microenvironment of the GBM tumor. We performed gene expression profiling analysis of bulk RNA-seq data obtained from patient-derived GBM spheroids at various time points. Total RNA was isolated from these spheroids using the QIAzol lysis reagent (Qiagen,79306 )following the manufacturer's protocol. Libraries for bulk RNA-seq were prepared using the TruSeq Stranded mRNA Library Prep Kit. GBM spheroids were cultured and maintained in Dulbecco’s modified Eagle’s high glucose medium (SH30243.01, Hyclone) supplemented with 10% heat-inactivated fetal bovine serum (2357664RP, Gibco) and 1% antibiotic-antimycotic (15240062, Gibco). Spheroids were maintained at 37°C in a humidified atmosphere containing 5% CO2 and tested regularly for mycoplasma contamination by mycoplasma detection kit (rep-mys-20, InvivoGen).

本研究采用多入口球状体生成平台（multi-inlet spheroid generator, MSG）构建了稳定的胶质母细胞瘤（Glioblastoma Multiforme, GBM）球状体，并在不同时间点观测其与多种免疫细胞的相互作用，包括小胶质细胞与自然杀伤（natural killer, NK）细胞。我们分别选用HMO6细胞与NK-92细胞作为小胶质细胞与NK细胞的实验模型。本研究同时检测了肿瘤-免疫细胞间以及不同免疫细胞间的相互作用。三细胞共培养球状体的成功构建，复现了胶质母细胞瘤的免疫抑制微环境。我们对不同时间点从患者来源的胶质母细胞瘤球状体中获取的批量RNA测序（bulk RNA-seq）数据开展了基因表达谱分析。采用QIAzol裂解液（Qiagen，货号79306）并严格遵循制造商的实验规程，从上述球状体中提取总RNA。批量RNA测序文库的制备采用TruSeq Stranded mRNA Library Prep Kit试剂盒。胶质母细胞瘤球状体的培养与维持采用添加了10%热灭活胎牛血清（Gibco，货号2357664RP）与1%抗生素-抗真菌混合液（Gibco，货号15240062）的高糖达尔伯克改良伊格尔培养基（DMEM高糖培养基，SH30243.01，Hyclone）。球状体于37℃、含5% CO₂的饱和湿度培养箱中维持培养，并定期采用支原体检测试剂盒（InvivoGen，货号rep-mys-20）开展支原体污染检测。