RNAseq of tumor samples from CT26 syngeneic mouse treated with a PI3Ka/d inhibitor (AZD8835). RNAseq of tumor samples from CT26 syngeneic mouse treated with a PI3Ka/d inhibitor (AZD8835)

PI3K inhibitors with differential selectivity to distinct PI3K isoforms have been tested extensively in clinical trials, largely to target tumor epithelial cells. PI3K signaling also regulates the immune system and inhibition of PI3Kdelta modulate the tumor immune microenvironment of pre-clinical mouse tumor models by relieving T-regs-mediated immunosuppression. While PI3K inhibitors as a class and PI3Ka/d specifically are associated with immune-related side effects. However, the impact of mixed PI3K inhibitors in tumor immunology is under-explored. Here we examine the differential effects of AZD8835 a dual PI3Ka/d inhibitor specifically on the tumor immune microenvironment using syngeneic CT26 mice. CT-26 (5x10^6 cells/mouse) tumor cells were implanted subcutaneously (s.c.) in the left flank of female Balb/c and C57/Bl6 mice, respectively. AZD8835 was dosed at 50mg/kg twice daily in at 2days on/ 5 days off schedule for times indicated in figures or 25mg/kg BID daily. At end of study tumor tissues were then transferred into the gentleMACS C Tube containing RPMI. Tumor samples were processed using the mouse tumor dissociation kit from Miltenyi Biotec. Cells were liberated from tumors for downstream application using a mouse tumor dissociation kit and octodissociator (Miltenyi) according to manufacturer’s instructions. For RNA sequencing, total RNA was extracted using the RNeasy 96 Qiacube HT Kit (Qiagen), quality validated using nanodrop and Quantit RNA Assay Kit (Thermo Fisher), and submitted for TrueSeq Stranded mRNA library preparation, following the manufacturer’s instructions (Illumina). Resulting libraries were sequenced on the HiSeq4000 System.

对不同PI3K同工型（PI3K isoforms）具有差异化选择性的PI3K抑制剂已在临床试验中得到广泛研究，其主要靶向肿瘤上皮细胞。PI3K信号通路亦可调控免疫系统，而PI3Kδ（PI3Kdelta）的抑制可通过缓解调节性T细胞（T-regs）介导的免疫抑制，改善临床前小鼠肿瘤模型的肿瘤免疫微环境。尽管作为一类药物的PI3K抑制剂，尤其是PI3Kα/δ亚型抑制剂，常与免疫相关不良反应相关，但多靶点PI3K抑制剂在肿瘤免疫学中的作用仍有待深入探索。本研究利用同源移植（syngeneic）CT26小鼠模型，探究双重PI3Kα/δ抑制剂AZD8835对肿瘤免疫微环境的差异化影响。实验中，分别向雌性Balb/c及C57/Bl6小鼠左侧皮下（s.c.）接种5×10^6个CT-26肿瘤细胞。AZD8835采用两种给药方案：其一为按“给药2天、停药5天”的周期，以50mg/kg的剂量每日给药两次；其二为以25mg/kg的剂量每日给药两次（BID），具体给药时长如图中所示。实验结束后，将肿瘤组织转移至含RPMI培养基的gentleMACS C管中。采用美天旎生物技术（Miltenyi Biotec）的小鼠肿瘤组织解离试剂盒处理肿瘤样本，并依照制造商说明书，使用小鼠肿瘤解离试剂盒及OctoDissociator（Miltenyi）从肿瘤中解离出细胞，用于后续实验。对于RNA测序，使用RNeasy 96 Qiacube HT试剂盒（Qiagen）提取总RNA，通过纳米分光光度计（nanodrop）及Quant-iT RNA检测试剂盒（Thermo Fisher Scientific）进行质量验证，随后依照因美纳（Illumina）的说明书进行TrueSeq链特异性mRNA文库构建。最终构建的文库在HiSeq4000测序系统上完成测序。