PDF(Pubmed)
Abstract：
Enhancing immune cell functions in tumors remains a major challenge in cancer immunotherapy. Natural killer cells (NK) are major innate effector cells with broad cytotoxicity against tumors. Accordingly, NK cells are ideal candidates for cancer immunotherapy, including glioblastoma (GBM). Hypoxia is a common feature of solid tumors, and tumor cells and normal cells adapt to the tumor microenvironment by upregulating the transcription factor hypoxia-inducible factor (HIF)-1α, which can be detrimental to anti-tumor effector immune cell function, including that of NK cells. We knocked out HIF-1α in human primary NK cells using clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9). Then, cellular characterizations were conducted in normoxic and hypoxic conditions. Electroporating two HIF-1α-targeting guide RNA-Cas9 protein complexes inhibited HIF-1α expression in expanded NK cells. HIF-1α knockout human NK cells, including populations in hypoxic conditions, enhanced the growth inhibition of allogeneic GBM cells and induced apoptosis in GBM-cell-derived spheroids. RNA-sequencing revealed that the cytotoxicity of HIF-1α knockout NK cells could be related to increased perforin and TNF expression. The results demonstrated that HIF-1α knockout human NK cells, including populations, enhanced cytotoxicity in an environment mimicking the hypoxic conditions of GBM. CRISPR-Cas9-mediated HIF-1α knockout NK cells, including populations, could be a promising immunotherapeutic alternative in patients with GBM.
摘要：
增强肿瘤中的免疫细胞功能仍然是癌症免疫治疗的主要挑战。自然杀伤细胞（NK）是主要的先天效应细胞，对肿瘤具有广泛的细胞毒性。因此，NK细胞是癌症免疫治疗的理想候选者。包括胶质母细胞瘤（GBM）。缺氧是实体瘤的共同特征,肿瘤细胞和正常细胞通过上调转录因子缺氧诱导因子(HIF)-1α适应肿瘤微环境,这可能不利于抗肿瘤效应免疫细胞的功能，包括NK细胞。我们使用成簇的规则间隔短回文重复序列（CRISPR）相关蛋白9（Cas9）在人原代NK细胞中敲除HIF-1α。然后，在常氧和低氧条件下进行细胞表征。电穿孔两种HIF-1α靶向指导RNA-Cas9蛋白复合物抑制扩增NK细胞中的HIF-1α表达。HIF-1α敲除人NK细胞,包括处于缺氧状态的人群，增强了同种异体GBM细胞的生长抑制作用，并诱导了GBM细胞衍生球体的凋亡。RNA测序显示，HIF-1α敲除NK细胞的细胞毒性可能与穿孔素和TNF表达增加有关。结果表明HIF-1α敲除人NK细胞,包括人口,在模拟GBM缺氧条件的环境中增强的细胞毒性。CRISPR-Cas9介导的HIF-1α基因敲除NK细胞,包括人口,可能是GBM患者的一种有前途的免疫治疗替代方案。
