CpG on carbon nanotubes inhibits migration of brain tumor cells while activating immune cells. Mus musculus

Even when treated with aggressive current therapies, patients with glioblastoma usually survive less than two years and exhibit a high rate of recurrence. CpG is an oligonucleotide that activates the innate immune system via TLR9 activation. Injection of CpG into glioblastoma tumors showed promise as an immunotherapy in mouse models but proved disappointing in human trials. One aspect of glioma that is not addressed by CpG therapy alone is the highly invasive nature of glioma cells, which is associated with resistance to radiation and chemotherapy. Here, we demonstrate that single-walled carbon nanotubes non-covalently functionalized with CpG (SWNT/CpG) not only retain the immunostimulatory property of the CpG, but interestingly also inhibit the migration of glioma cells without affecting cell viability or proliferation. SWNT/CpG activated macrophages by induction of the TLR9/NF-κB pathway, but actually decreased NF-κB activation in glioma cells. By using RNA-seq to compare the effect of SWNT/CpG treatment on glioma cells and macrophages, we confirmed that SWNT/CpG treatment has cell type-specific effects on gene expression. Through RNA-seq analysis, we identified a number of differentially affected pathways which may be involved in glioma cell migration inhibition and observed reduced gene expression in DNA repair pathways for SWNT/CpG-treated glioma cells. The migration inhibition of glioma cells was correlated with reduced intracellular levels of reactive oxygen species (ROS), suggesting that an antioxidant-based mechanism mediates the observed effects. To our knowledge, SWNT/CpG is the first therapy that inhibits the migration of cancer cells while stimulating the immune system. Overall design: K-Luc glioma or RAW-Blue macrophage cells were plated in a scratch assay (ibidi 2-well insert) and treated with either 1) water (untreated control), 2) CpG-only control, 3) SWNT/LP-only control (SWNTs coated with a control material), 4) CpG + SWNT/LP co-treatment control, or 5) SWNT/CpG treatment (SWNTs coated with CpG DNA). At the conclusion of the migration assay, each treatment group was compared to the untreated control for that cell line to determine if there was any observable migration inhibition. After this observation was noted, total RNA was purified for each replicate.

即使采用当前的强化治疗方案，胶质母细胞瘤（glioblastoma）患者的总生存期通常仍不足两年，且复发率居高不下。CpG是一类通过激活TLR9通路激活先天免疫系统的寡核苷酸。将CpG直接注射至胶质母细胞瘤瘤体内，在小鼠模型中展现出了免疫治疗的应用前景，但在人体临床试验中却未能取得理想效果。仅依靠CpG治疗无法解决胶质瘤细胞极强的侵袭性这一关键问题——该侵袭性与肿瘤对放疗和化疗的耐药性密切相关。本研究证实，经CpG非共价功能化修饰的单壁碳纳米管（single-walled carbon nanotubes, SWNT），即SWNT/CpG，不仅保留了CpG的免疫刺激活性，还能在不影响细胞活力与增殖能力的前提下，显著抑制胶质瘤细胞的迁移。SWNT/CpG可通过诱导TLR9/NF-κB通路激活巨噬细胞，但在胶质瘤细胞中却会下调NF-κB的激活水平。通过RNA测序（RNA-seq）对比SWNT/CpG处理对胶质瘤细胞与巨噬细胞的影响，我们确认该复合物对基因表达的调控具有细胞类型特异性。通过RNA-seq分析，我们鉴定出多个可能参与胶质瘤细胞迁移抑制的差异调控通路，并观察到经SWNT/CpG处理的胶质瘤细胞中，DNA修复通路的基因表达水平显著降低。胶质瘤细胞的迁移抑制效应与细胞内活性氧（reactive oxygen species, ROS）水平降低呈正相关，这提示其作用机制可能依赖于抗氧化通路。据我们所知，SWNT/CpG是首款兼具癌细胞迁移抑制与免疫系统激活双重功效的治疗方案。总体实验设计：将K-Luc胶质瘤细胞或RAW-Blue巨噬细胞接种于ibidi 2孔插入式培养板的划痕实验体系中，分别接受以下5种处理：1）纯水（未处理对照组）；2）单纯CpG对照组；3）单纯SWNT/LP对照组（包被对照材料的单壁碳纳米管）；4）CpG与SWNT/LP联合处理对照组；5）SWNT/CpG处理组（包被CpG DNA的单壁碳纳米管）。迁移实验结束后，将每个处理组与其对应细胞系的未处理对照组进行比对，以评估是否存在可观测的迁移抑制效应。确认该效应后，对每个重复样本的总RNA进行纯化提取。