Identification of immune-related genes prognostic index for predicting survival and immunotherapy in colorectal carcinoma

1.Clinical DataTranscript data and Clinicopathological information was downloaded from The Cancer Genome Atlas (TCGA) database (https://portal.Gdc.cancer.gov/), including 41 cases of para-tumor, 473 cases of CRC tumor and 452 clinical cases. The survival and transcriptional data of 250 CRC cases were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The transcript dataset GSE161158 uploaded in November 2020 by Moffitt Cancer Research Center, University of Miami was used (10). Lists of immune related genes were download from ImmPort (https://www.immport. org/home) and Innate DB (https://www.innatedb.ca/). KEGG (http://www.gsea-msigdb.org/gsea/index.jsp) gene sets and all Gene Ontology (GO) gene sets were used as Gene Symbols. Gene mutation information was downloaded from cBioPortal (http://www.cbioportal.org/). 2.Murine Data2.1 IRGPI Genes Expression in CRC Murine ModelSPF Balb/c male mice, 6 - 8 weeks old, body mass (20 ± 5) g, purchased from Huaxing Experimental Animal Farm of Huiji District (Zhengzhou City, China), experimental animal license NO. is SCXK (Yu) 2019-0002. All animal experiments was approved by the Experimental Mouse Ethics Committee of Nanjing University of Traditional Chinese Medicine (NO. 202010A026).The above mice were randomly divided into: Control group (C), CRC Model group (M)，10 per group. Five Balb/c male mice were taken as tumor-bearing mice, and 1×107 CT26 cells were subcutaneously injected into the left axilla, and sacrificed one week later. The subcutaneous tumor was removed under sterile conditions, placed in sterile PBS, and disintegrated into several 1 mm3 masses. Under sterile conditions, the two groups of mice were dissected to expose the colon, the 1 mm3 tumor mass was fixed to the colon of the CRC Model group with tissue glue, while nothing was fixed in the Control group, and then the abdomen of the two groups of mice was sutured. After 3 days of postoperative recovery, mice were weighed, and Micro-CT scans were performed on the 26th day (under the condition of isoflurane respiratory anesthesia), and on the 27th day, the mice were sacrificed after anesthesia with 2 % sodium pentobarbital. Total RNA was extracted from the colon of the Control group and the tumor tissue of the CRC Model group by FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, China, Cat#RC101-01) , and after reverse transcribed into cDNA using HiScript® Ⅲ RT SuperMix for q PCR (Vazyme, China, Cat#R323-01), Real-Time PCR was used to detect the expression of IRCPI genes in each group using BlastaqTM Green 2× qPCR MasterMix (abm, Canada, Cat#G891). The primer sequences are shown in Supplementary Table S4.2.2 Immune Infiltration in CRC murine model The liver, colon, tumor, and mesentery of paraffin-embedded mice were sectioned, and then stained with hematoxylin-eosin (HE staining), and photographed with an upright white light photographic microscope (Nikon, Japan, Eclipse Ci-L). TIME immune cells were detected by flow cytometry (FCM). PBMCs were extracted by RBC lysate (FcMACS, China, Cat#FMS-RBC500). At least 5×106 cell suspensions(100 μL) were incubated with FC blocker at 4 ℃ for 10 min, then Anti-Human/Mouse CD11b FITC Antibody (PeproTech, USA, Cat#03221-50)、PE-Cy™7 Rat Anti-Mouse CD86 Antibody (BD Pharmingen™, USA, Cat#560582) and Alexa Fluor® 488 Anti-Mouse CD206 Antibody (Biolegend, USA, Cat#141710) were used to marked Macrophages; Alexa Fluor® 488 anti-mouse CD19 Antibody (Invitrogen, USA, REF#11-0193-81) and PE/Cy7 anti-mouse/rat/human CD27 Antibody (Biolegend, USA, Cat#124216) were used to marked B cells; Anti-Mouse CD4 APC-Cyanine7 (PeproTech, USA, Cat#06122-87)、Anti-Mouse CD8a FITC Antibody (PeproTech, USA, Cat#10122-50)、Anti-Mouse CD25 APC Antibody (PeproTech, USA, Cat#07312-80) and Anti-Mouse/Rat FOXP3 PE Antibody (PeproTech, USA, Cat#83422-60) were used to marked T cells, and PBMCs monochromic tubes were made respectively. The cells were detected on the Amnis FlowSight flow cytometer (Merck Millipore, USA), and immunocyte subsets were analyzed using the IDEAS software (Merck Millipore, USA). Supplement Fig.S7 visualized the analysis strategies for IRGPI immunocyte subsets by Flow cytometry.1. 临床数据 转录组数据与临床病理信息下载自癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库（https://portal.gdc.cancer.gov/），包含41例癌旁组织、473例结直肠癌（Colorectal Cancer, CRC）肿瘤组织及452例临床病例。250例CRC病例的生存数据与转录组数据下载自基因表达综合数据库（Gene Expression Omnibus, GEO，https://www.ncbi.nlm.nih.gov/geo/），采用迈阿密大学莫菲特癌症研究中心于2020年11月上传的转录组数据集GSE161158[10]。免疫相关基因列表下载自ImmPort（https://www.immport.org/home）与Innate DB（https://www.innatedb.ca/）。以京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）基因集及所有基因本体（Gene Ontology, GO）基因集作为基因符号集。基因突变信息下载自cBioPortal（http://www.cbioportal.org/）。2. 小鼠数据2.1 CRC小鼠模型中IRGPI基因的表达 实验使用SPF级Balb/c雄性小鼠，鼠龄6~8周，体质量(20±5)g，购自中国郑州市惠济区华兴实验动物养殖场，实验动物许可证号为SCXK(豫)2019-0002。所有动物实验均经南京中医药大学实验动物伦理委员会批准（批准号：202010A026）。将小鼠随机分为对照组（C组）与CRC模型组（M组），每组10只。另取5只Balb/c雄性小鼠构建荷瘤模型：于左侧腋下皮下注射1×10^7个CT26细胞，1周后处死小鼠，无菌条件下取下皮下肿瘤，置于无菌PBS中，剪切成1 mm³大小的组织块。无菌条件下解剖两组小鼠暴露结肠，将1 mm³的肿瘤组织块用组织胶固定于CRC模型组小鼠的结肠壁，对照组小鼠结肠不做任何固定，随后缝合两组小鼠腹部。术后恢复3天后称量小鼠体质量，于第26天在异氟烷吸入麻醉条件下进行Micro-CT扫描，第27天使用2%戊巴比妥钠麻醉后处死小鼠。采用FastPure细胞/组织总RNA提取试剂盒（Vazyme，中国，货号RC101-01）提取对照组小鼠结肠组织与CRC模型组小鼠肿瘤组织的总RNA，使用HiScript® Ⅲ qPCR反转录预混试剂盒（Vazyme，中国，货号R323-01）将总RNA反转录为cDNA，随后采用Blastaq™ Green 2× qPCR预混液（abm，加拿大，货号G891）通过实时荧光定量PCR（Real-Time PCR）检测各组IRGPI基因的表达，引物序列详见补充表S4。2.2 CRC小鼠模型的免疫浸润 对石蜡包埋的小鼠肝脏、结肠、肿瘤及肠系膜组织进行切片，经苏木精-伊红染色（HE染色）后，使用正置白光显微成像系统（Nikon，日本，Eclipse Ci-L）进行图像采集。采用流式细胞术（FCM）检测肿瘤免疫微环境（Tumor Immune Microenvironment, TIME）细胞。使用红细胞裂解液（FcMACS，中国，货号FMS-RBC500）分离外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMCs）。取至少5×10^6个细胞悬液（100 μL），于4℃与Fc封闭剂孵育10 min，随后使用以下抗体标记免疫细胞亚群：抗人/小鼠CD11b FITC抗体（PeproTech，美国，货号03221-50）、PE-Cy™7 大鼠抗小鼠CD86抗体（BD Pharmingen™，美国，货号560582）及Alexa Fluor® 488 抗小鼠CD206抗体（Biolegend，美国，货号141710）用于标记巨噬细胞；Alexa Fluor® 488 抗小鼠CD19抗体（Invitrogen，美国，货号REF#11-0193-81）与PE/Cy7 抗小鼠/大鼠/人CD27抗体（Biolegend，美国，货号124216）用于标记B细胞；抗小鼠CD4 APC-Cyanine7（PeproTech，美国，货号06122-87）、抗小鼠CD8a FITC抗体（PeproTech，美国，货号10122-50）、抗小鼠CD25 APC抗体（PeproTech，美国，货号07312-80）及抗小鼠/大鼠FOXP3 PE抗体（PeproTech，美国，货号83422-60）用于标记T细胞，并分别制备PBMCs单染管。使用Amnis FlowSight流式细胞仪（Merck Millipore，美国）检测细胞，采用IDEAS软件（Merck Millipore，美国）分析免疫细胞亚群。补充图S7可视化展示了流式细胞术分析IRGPI免疫细胞亚群的实验策略。