Pathania Anup S, Prathipati Philip, Olwenyi Omalla A, Chava Srinivas, Smith Oghenetejiri V, Gupta Subash C, Chaturvedi Nagendra K, Byrareddy Siddappa N, Coulter Don W, Challagundla Kishore B
Department of Biochemistry and Molecular Biology & The Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Laboratory of Bioinformatics, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki City, Osaka 567-0085, Japan.
Mol Ther Oncolytics. 2022 Mar 31;25:308-329. doi: 10.1016/j.omto.2022.03.010. eCollection 2022 Jun 16.
Neuroblastoma (NB) is an enigmatic and deadliest pediatric cancer to treat. The major obstacles to the effective immunotherapy treatments in NB are defective immune cells and the immune evasion tactics deployed by the tumor cells and the stromal microenvironment. Nervous system development during embryonic and pediatric stages is critically mediated by non-coding RNAs such as micro RNAs (miR). Hence, we explored the role of miRs in immune response via a range of data-driven workflows and & experiments. Using the TARGET, NB patient dataset (n=249), we applied the robust bioinformatic workflows incorporating differential expression, co-expression, survival, heatmaps, and box plots. We initially demonstrated the role of miR-15a-5p (miR-15a) and miR-15b-5p (miR-15b) as tumor suppressors, followed by their negative association with stromal cell percentages and a statistically significant negative regulation of T and natural killer (NK) cell signature genes, especially (PD-L1) in stromal-low patient subsets. The NB phase-specific expression of the miR-15a/miR-15b-PD-L1 axis was further corroborated using the PDX (n=24) dataset. We demonstrated miR-15a/miR-15b mediated degradation of PD-L1 mRNA through its interaction with the 3'-untranslated region and the RNA-induced silencing complex using sequence-specific luciferase activity and Ago2 RNA immunoprecipitation assays. In addition, we established miR-15a/miR-15b induced CD8T and NK cell activation and cytotoxicity against NB . Moreover, injection of murine cells expressing miR-15a reduced tumor size, tumor vasculature and enhanced the activation and infiltration of CD8T and NK cells into the tumors . We further established that blocking the surface PD-L1 using an anti-PD-L1 antibody rescued miR-15a/miR-15b induced CD8T and NK cell-mediated anti-tumor responses. These findings demonstrate that miR-15a and miR-15b induce an anti-tumor immune response by targeting PD-L1 in NB.
神经母细胞瘤（NB）是一种难以治疗且最为致命的儿科癌症。NB有效免疫治疗的主要障碍包括免疫细胞缺陷以及肿瘤细胞和基质微环境所采用的免疫逃逸策略。胚胎期和儿童期的神经系统发育主要由非编码RNA（如微小RNA，miR）介导。因此，我们通过一系列数据驱动的工作流程和实验，探索了miR在免疫反应中的作用。利用TARGET数据库中的NB患者数据集（n = 249），我们应用了强大的生物信息学工作流程，包括差异表达、共表达、生存分析、热图和箱线图。我们首先证明了miR-15a-5p（miR-15a）和miR-15b-5p（miR-15b）作为肿瘤抑制因子的作用，随后发现它们与基质细胞百分比呈负相关，并且对T细胞和自然杀伤（NK）细胞特征基因具有统计学上显著的负调控作用，尤其是在基质含量低的患者亚组中对程序性死亡配体1（PD-L1）的调控。使用患者来源的异种移植（PDX，n = 24）数据集进一步证实了miR-15a/miR-15b-PD-L1轴在NB中的阶段特异性表达。我们通过序列特异性荧光素酶活性和AGO2 RNA免疫沉淀试验证明，miR-15a/miR-15b通过与3'非翻译区相互作用以及RNA诱导沉默复合体介导PD-L1 mRNA的降解。此外，我们证实miR-15a/miR-15b可诱导CD8 + T细胞和NK细胞活化，并对NB具有细胞毒性。此外，注射表达miR-15a的鼠细胞可减小肿瘤大小、减少肿瘤血管生成，并增强CD8 + T细胞和NK细胞向肿瘤内的活化和浸润。我们进一步证实，使用抗PD-L1抗体阻断表面PD-L1可挽救miR-15a/miR-15b诱导的CD8 + T细胞和NK细胞介导的抗肿瘤反应。这些发现表明，miR-15a和miR-15b通过靶向NB中的PD-L1诱导抗肿瘤免疫反应。
