NanoString PanCancer Immune Profiling of 4′‐bromo‐resveratrol treated BrafV600E/PtenNULL melanoma mice. NanoString PanCancer Immune Profiling of 4′‐bromo‐resveratrol treated BrafV600E/PtenNULL melanoma mice

We performed differential gene expression analysis using high throughput multiplex analysis via NanoString nCounter PanCancer Immune Profiling panel, which analyzes 770 genes related to cancer-immune pathways (cancer progression, chemokines and cytokines and their receptors, and innate and adaptive immune response). nSolver software was used to analyze the data and a heat map was generated to show differential expression of 770 genes. Further, using a fold cut-off of ≥2 and p-value <0.05, we observed that 4'-BR significantly modulated expression of multiple cancer immune genes. Overall design: Tumors were induced by topical application of 4-hydroxy-tamoxifen on shaved backs of 10-week-old mice, and the effects of 4′‐BR (30 mg/kg b.wt.; intraperitoneally; 3d/week for 5 weeks) were assessed on melanoma development and progression. RNA was extracted from tumors obtained from control and 30 mg/kg 4′‐BR treated mice (n = 6), followed by determination of expression of 770 genes (730 immune genes and 40 housekeeping genes) via the Mouse PanCancer Immune Profiling Panel using the nCounter platform in the NanoString Technologies .

本研究采用NanoString nCounter泛癌免疫分析检测面板（NanoString nCounter PanCancer Immune Profiling panel）开展高通量多重差异基因表达分析，该检测面板可靶向覆盖770个与癌症免疫通路相关的基因，涵盖癌症进展、趋化因子、细胞因子及其受体，以及先天与适应性免疫应答相关的基因。实验采用nSolver软件对数据进行分析，并绘制热图以展示770个基因的差异表达谱。进一步以倍数变化阈值≥2、p值<0.05作为筛选标准，本研究发现4'-BR可显著调控多个癌症免疫相关基因的表达。

实验整体设计如下：对10周龄小鼠剃毛的背部皮肤局部涂抹4-羟基他莫昔芬以诱导肿瘤生成，随后给予4'-BR处理（剂量为30 mg/kg体重，腹腔注射，每周3次，持续5周），评估其对黑色素瘤发生与进展的影响。从对照组与30 mg/kg 4'-BR处理组的小鼠肿瘤组织中提取RNA（每组样本量n=6），随后依托NanoString Technologies的nCounter检测平台，采用小鼠泛癌免疫分析检测面板（Mouse PanCancer Immune Profiling Panel）对770个基因（包含730个免疫相关基因与40个持家基因）的表达水平进行检测。