结肠保真度的丧失可以使多曲目可塑性和转移

　　所有体内实验均根据英国内政部法规（项目许可：PP9016178，PP7510272和PP3908577）进行，并受到爱丁堡大学和格拉斯哥大学动物福利和伦理委员会的本地道德审查。在19至23°C的温度下，将小鼠在12小时的光线周期下饲养。环境湿度为55±10％。随意提供标准饮食和水。在C57BL/6J上进行了工作，查尔斯河实验室的CD1裸露背景。对于肺转移模型，将500,000个AKP对照或AKP ATRXKO细胞重悬于100 µL PBS中，并注入雌性CD1裸鼠的尾静脉中。对于肝转移模型，用异氟烷麻醉C57BL/6J小鼠，并使用剖腹手术实现脾脏访问。将约500,000个细胞注入50 µL PBS中，然后用钉书钉封闭切口。用50,000个AKP对照，AKP ATRXKO或AKP HNF4AKO细胞重悬于100 µL基底膜提取物（BME）中，对女性CD1裸鼠的皮下注射。对雌性CD1裸小鼠的皮下注射，用12,000个流式细胞仪排序的细胞重悬于100 µL BME中。 　　使用Karl Storz Tele Pack Vet X LED内窥镜视频单元进行结肠粘膜粘膜注射。AKP对照，AKP ATRXKO，BPN对照和BPN Atrxko类器官通过机械移液解离，然后用PBS洗涤，然后用PBS洗涤，然后在雌性CD1裸鼠中原位注射。在单个注射中注入了70μlPBS中约500个类器官。O.J.S.实验室产生了BPN类器官线（雄性）。 　　根据相关许可文件中定义的相关许可终点（皮下注射直径15 mm），根据相应的许可证和研究方案对小鼠进行人工安乐死。 　　根据3RS（替换，还原和改进），选择最小的样本量可能会带来显着差异。 　　将6-12周龄的C57BL/6J或CD1裸鼠随机分组以进行移植实验。所有小鼠都接受了不同基因型或表型的相同数量的细胞。通过注射细胞的基因型（例如，AKP对照与AKP ATRXKO）确定实验组。在进行组织学分析和数据收集期间，监测临床体征时，研究者对肿瘤的基因型视而不见。IHC对肿瘤组织学的分析是使用QUPATH软件进行的，研究者对肿瘤基因型视而不见。 　　将组织固定在甲醛4％稳定，缓冲（VWR）中，然后转移至70％乙醇。使用组织TEK VIP浸润处理器（Sakura）进行组织加工，然后嵌入石蜡中。将组织以5 µm的厚度切开，在37°C下干燥，然后将其脱蜡和再水化。对于每个样品，将一个截面用降血石和曙红染色。其他部分用于标准IHC或免疫荧光分析。使用以下主要抗体：KRT5（兔子； ABCAM 52635; 1：200或鸡肉； Biolegend 905903; 1/200）;Ly6d（兔子； Atlas HPA024755; 1：200）;扭曲（鼠标；圣克鲁斯81417，1：200）;EPCAM（兔子； ABCAM 71916; 1：200）;HNF4A（兔子； CST 3113; 1：500）;Atrx（鼠标； Sigma Mabe1798; 1：500）;β-catenin（小鼠； BD 610154; 1:50）;E-辅助蛋白（兔子； CST 3195，1：200）;和CDX2（鼠标； Atlas Amab 91828，1：1,000）。使用Envision+ System-HRP标记聚合物（DAKO）实现IHC二级检测。使用DAB底物（EPREDIA和2B科学）可视化正信号5-10分钟。切片用血久毒素染色。对于免疫荧光研究，使用了以下二级抗体（1：400）：抗兔594（Invitrogen，A21207）；Anti-Chicken-488（Invitrogen，A78948）;抗链霉菌蛋白647（Invitrogen，S32357）;每个污渍显示了抗兔子-488（ABCAM，150073）的代表性图像。使用NDP.View2plus软件使用纳米室数字幻灯片扫描仪（Hamamatsu）进行数字化幻灯片，并使用Qupath（v.0.2.3）进行分析。使用的商业结肠癌人体组织阵列是CO804B（Biomax）（扩展数据图1B – D）。图5和扩展数据中的TMA图10包括患有I – III期CRC的患者，他们在1997年至2013年在格拉斯哥皇家医院进行了潜在的治愈性切除。从侵入式边缘的每个捐赠块中取出三个核心， 捕获少量的肿瘤及其周围的环境。NHS Grore Glasgow和Clyde Biorepository在其NHS研究伦理委员会下授权患者组织的进入，并根据《苏格兰伦理学22/WS/WS/0207》中的Biorepository申请编号845授予了伦理学批准，如赫尔辛基（Helsinki）的宣布所述，苏格兰伦理学22/WS/WS/0207。总体而言，分析了17例在2002年4月至2010年6月在格拉斯哥皇家医院之间进行原发性CRC和CRC肝转移同步切除的患者。NHS Grood Glasgow和Clyde Biorepository在其NHS研究伦理委员会下授权患者组织的进入，并在苏格兰伦理学22/WS/WS/0207的Bioreportor申请编号357中授予了伦理批准，并根据公认的伦理准则，如Helsinki of Helsinki的宣布所述。 　　使用Quth-0.2.3生成HNF4A，ATRX和CDX2肿瘤细胞表达的H评分。LY6D和KRT5表达阳性的肿瘤核心分别指定为LY6D+和KRT5+。与肿瘤核 <2% tumour cells positive for LY6D or KRT5 expression were designated as LY6D– and KRT5–, respectively. Analysis was performed using QuPath-0.2.3. H-scores were generated for CDH1 cell expression using QuPath-0.2.3. 　　Organoids were resuspended in Cultrex reduced growth factor BME type 2 (Bio-Techne), plated in a 10 µl drop on a culture plate and cultured in organoid culture medium containing advanced DMEM–F12 (Gibco) medium supplemented with 100 units ml–1 penicillin, 100 µg ml–1 streptomycin, 2 mM -glutamine, 10 mM HEPES (all from Life Technologies), 1 ml Primocin (Invivogen), N2, B27 (both from Gibco), 50 ng ml–1 EGF (Peprotech) and 1% Noggin conditioned medium. The Noggin-producing cell line was a gift from H. Clevers’ group (Hubrecht Institute). Organoids were passaged by mechanical fragmentation with a p1000 and p200 pipette. All organoids were grown in a humidified incubator at 37 °C supplemented with 5% CO2. Single cells were generated by incubating organoids in TrypLE Express (Life Technologies) for 15 min at 37 °C and passed through a 40 µm strainer. Cell counting was performed using a Countess II automated cell counter (Invitrogen). To study the effect of TGFβ, dissociated cells (10,000 (or 5,000 for the Tgfbr2KO) cells in 10 µl BME) were cultured in organoid culture medium in the presence of 5 ng ml–1 TGFβ (Peprotech). TGFβ treatment in Extended Data Fig. 3 was performed using a different range of concentrations as specified in the figure. On day 3 the medium was replaced with fresh organoid culture medium containing TGFβ. Cells were collected on day 13 for RNA extraction. Alternatively, cells were stained with calcein (Abcam) or phalloidin-568 (VWR) within 14 days of treatment. 　　For ITGA5+ cell sorting and TGFβ treatment, AKP control and AKP AtrxKO organoids were digested as described above, and ITGA5 FACS was carried out as described in the FACS section below. In brief, 10,000 ITGA5+ or ITGA5– cells were plated in 10 µl BME and treated with 5 ng ml–1 TGFβ as described above. On day 13, organoids were fixed with 4% paraformaldehyde, stained with phalloidin-568 and analysed for the presence of spindle-like structures. 　　For the tail-vein injections, organoids were dissociated in TrypLE Express (Life Technologies) supplemented with a Rho kinase inhibitor (Y-27632, Tocris) for 15 min at 37 °C, passed through a 40 µm strainer, counted and resuspended in PBS. For splenic injections, organoids were collected, washed in PBS, mechanically fragmented by vigorous pipetting and then incubated for 7 min at 37 °C in 0.25% trypsin in EDTA–PBS. After quenching of trypsinization by immersion in 10% FBS, cells were passed through a 40 µm strainer, counted using a haemocytometer and resuspended in PBS to achieve a final volume of 1 × 107cells per ml. Cells were routinely tested for mycoplasma contamination. 　　The patient-derived organoids used in this study were generated by F.V.N.D. and M.G.D. MD20043 is an 81-year-old man with stage 4 rectal cancer: T3, N2, M1 (where ‘T’ is tumour, ‘N’ is nodes and ‘M’ is metastases). Ethics approval for human CRC organoid derivation was carried out under NHS Lothian Ethical Approval Scottish Colorectal Cancer Genetic Susceptibility Study 3 (SOCCS3) (REC reference: 11/SS/0109). The patient provided fully informed consent for the use of their tissues. 　　Human carcinoma organoids were cultured in advanced DMEM–F12 (Gibco) medium supplemented with 100 units ml–1 penicillin, 100 µg ml–1 streptomycin, 2 mM -glutamine, 10 mM HEPES (all from Life Technologies), 1 ml Primocin (Invivogen), 1% Noggin conditioned medium (The Noggin-producing cell line was a gift from H. Clevers’ group, Hubrecht Institute), B27 (Gibco), 50 ng ml–1 EGF (Peprotech), 10 nM gastrin (Sigma), 10 nM PGE2 (Tocris), 10 mM nicotinamide (Sigma), 10 µM SB202190 (Sigma), 600 nM A83-01 (Biotechne) and 12.5 mM N-acetylcysteine (Sigma). 　　For transduction conditions, human organoids were pretreated with IntestiCult (Stem Cell Tech) organoid growth medium and 1 mM valproic acid (Merk) for 48 h after 2 days post-split (day 1). On transduction day (day 1), human organoids were digested into a single-cell suspension in TrypLE Express (Life Technologies) with 10 µM Y-27632 (Tocris) for 8 min at 37 °C with mechanical dissociation every 4 min. Single-cell suspensions were combined with viral particles containing a non-targeting or ATRXKO sgRNA (hATRX: 5′-GCTATAAACAGAAAAAGAAA-3′; pLentiV2-Addgene) and placed on a BME layer. On day 2, medium was changed into IntestiCult supplemented with 1 mM valproic acid and 10 µM Y-27632. On day 3, antibiotic selection started with IntestiCult supplemented with 10 µg ml–1 blasticidin (Gibco) for 3 weeks. IntestiCult medium was used exclusively during transduction and the single-cell stage of organoid development for clone selection. Otherwise, the above-described medium was used for maintaining the organoids. 　　Organoids were dissociated in TrypLE Express (Life Technologies) for 15 min at 37 °C, passed through a 40 µm strainer and resuspended in BME. Next, 1,000 single cells in 10 µl BME were plated in 24-well plates and treated with 250 nM JQ1 (Stratech), 0.25 ng ml–1 IFNγ (Thermo Fisher Scientific), 10 nM FK228 (Stratech) or (0.5 ng ml–1) TNF (Peprotech) for 7 days. 　　For Resazurin cell viability assays, Resazurin (R&D systems) was added at a volume equal to 10% of the cell culture medium volume and incubated at 37 °C. Fluorescence was read using 544 nm excitation and 590 nm emission wavelengths. 　　Total RNA was isolated using a RNeasy Mini kit (Qiagen) accordingly to the manufacture’s protocol. RNA was then subjected to DNA-free DNase treatment (Invitrogen). cDNA was generated using 1 µg RNA by reverse transcription using qScript cDNA SuperMix (Quantabio). RT–qPCR was performed using SYBR Select master mix (Applied Biosystems). Ct values were normalized to β-actin or 18S rRNA. The ΔΔCt method was used to calculate relative gene expression values. Oligonucleotides used in this study are listed in Supplementary Table 11. For the RNA-seq experiments, RNA integrity was evaluated using an Agilent 2200 Bioanalyser. Truseq mRNA-seq libraries were prepared from total AKP and AKP ATRXKO RNA, and these were then sequenced using NovaseqS1 Illumina sequencing at Edinburgh Genomics Facility. AKP and AKP Hnf4aKO libraries were prepared from 100 ng of each total RNA sample using a NEBNEXT Ultra II Directional RNA Library Prep kit (NEB 7760) and the Poly-A mRNA magnetic isolation module (NEB E7490) according to the provided protocol. Sequencing was performed on a NextSeq 2000 platform (Illumina, 20038897) using NextSeq 2000 P3 reagents (200 cycles) (20040560). RNA-seq analysis was carried out using the RaNA-seq pipeline with default settings32. 　　Cells were lysed using RIPA buffer (Sigma) supplemented with 1% phosphatase and protease inhibitors (Sigma). Protein concentration was measured using a BCA Protein Assay kit (Pierce). A total of 20 µg protein lysate was resuspended in 4× LDS sample buffer (Invitrogen) supplemented with sample reducing agent (Invitrogen) and denatured at 100 °C for 5 min. Proteins were separated by electrophoresis on NuPAGE 3–8% Tris-acetate protein gels (Invitrogen) using Tris-acetate buffer and blotted onto an activated PVDF or nitrocellulose (Cytiva) membrane at 100 V for 1.15 h. Membranes were incubated in blocking solution (5% milk, 0.1% Tween-20–PBS) for 1 h at room temperature, and then in primary antibody. The following primary antibodies were used: β-actin (Cell Signalling Technology, 1:5,000); ATRX (MABE1798, Sigma; 1:500); and HNF4A (C11F12, Cell Signalling Technology, 1:,1,000). After 3× 10-min washes in 0.1% Tween-20–PBS, the membrane was incubated in HRP-linked secondary antibody for 1 h at room temperature. The following secondary antibodies were used: anti-rabbit or anti-mouse IgG HRP-linked (Cell Signalling Technology, 1:5,000). Following 3× 10-min washes in 0.1% Tween-20–PBS, antibody signals were detected by using ECL Plus Western blotting substrate (Pierce) and visualized using an ImageQuant 800 (GE Healthcare). Full scans are provided in Supplementary Information 1 and 2. 　　sgRNAs (mAtrx: 5′-ACGGCGCATTAAGGTTCAAG-3′; mHnf4a 5′-CGGGCCACCGGCAAACACTA-3′, mTgfbr2: 5′-AAGCCGCATGAAGTCTGCG-3′, non-targeting controls 5′- GCTTTCACGGAGGTTCGACG-3′ or 5′-ATGTTGCAGTTCGGCTCGAT-3′) were cloned individually into lentiCRISPR v.2 plasmids (Addgene) following Addgene’s protocol. Lentiviral particles were generated using HEK293T cells (provided by J. C. Acosta (IGMM, Edinburgh), originally obtained from the American Type Culture Collection): 10 µg gene-specific lentiviral vector was mixed with 7.5 µg lentiviral packaging vector psPAX2 and 2.5 µg envelope-protein-producing vector pCMV-VSV-G (both from Addgene) and transfected into HEK293T cells in a 10 cm2 dish using polyethylenimine as the transfection reagent (Polysciences). After 48 h, the supernatant medium was filtered using a 0.45 µm syringe filter and concentrated using a Lenti-X concentrator (Takara Bio). Lentiviral transduction was carried out as previously described33. In brief, AKP organoids were expanded and cultured in organoid culture medium supplemented with 10 µM Rho kinase inhibitor (Y-27632, Tocris) and 1 mM valproic acid (Sigma) for 48 h. Spheroids were enzymatically dissociated with StemPro Accutase cell dissociation reagent (Gibco) supplemented with 10 µM Y-27632 for 3 min at 37 °C. Dissociated organoids were then washed twice with advanced DMEM–F12 medium. Cells were counted using a Countess II Automated cell counter (Invitrogen). Next, 5 × 105 cells were plated on a 150 µl bed of BME in a 6-well plate in the presence of Y-27632, 1 mM valproic acid and 4 µg ml–1 polybrene (Sigma). Virus was removed 24 h after transduction, and adhered organoids were overlaid with 150 µl BME. To select transduced cells, 2 µg ml–1 of puromycin or 10 µg ml–1 blasticidin (both from Gibco) was added to the organoid culture medium supplemented with 10 µM Y-27632. Multiple deleted clones were generated. Editing of clonal lines was confirmed by genomic sequencing or western blotting. 　　TissueEnrich analysis34 (https://tissueenrich.gdcb.iastate.edu/) was carried out using the web-based tool with the following settings: gene symbol, Homo sapiens, Human Protein Atlas, All. Lists of gene upregulated or downregulated by >AKP与AKP ATRXKO数据集的2倍用于输入。在图2G中，在Scrna-Seq群集4和15集群中上调的基因列表上进行了组织分析（折叠变化> 1.5）。 　　对于ATRX突变富集分析，我们使用了先前指定的CRIS亚型14，并分析了已知CRIS名称的ATRX突变的TCGA突变数据。计算了CRIS-B亚型携带ATRX突变的肿瘤数量，并使用Fishers的精确测试与所有其他亚型进行了比较。为了生存分析，仅使用从TCGA数据门户下载的TCGA生存数据分析了CRIS-B肿瘤。对于ICM，进行了相同的分析，但根据ICMS指定，TCGA数据分开。 　　使用PGCDNSAM-HNF4A-IRES-GFP质粒（addgene）产生HNF4A过表达。简而言之，如上所述，AKP ATRX类器官被转导。为了选择转导的细胞，进行了针对GFP阳性的FACS分析。 　　从诊断为CRC的患者中，从新鲜切除的手术标本中取样正常的结直肠粘膜和肿瘤。伦理批准是根据NHS Lothian伦理批准苏格兰结直肠癌遗传易感性研究进行的3（SOCCS3 REC：11/SS/0109，IRAS：9556）。所有患者均提供了完全知情的同意书来使用其组织。将组织切成小块，然后在补充1 mg ML – 1胶原酶IV（Sigma），0.5 mg ML – 1透明质酸酶（Sigma）和10 µM Y-27632（Tocris）的37°C下在37°C下完全摇动，直到完全摇晃，直到组织为6090分钟，将组织孵育为1 mg Ml – 1胶原酶IV（Sigma），0.5 mg ML – 1透明质酸酶（Sigma）和10 µM Y-27632（Tocris）。然后将消化反应通过70 µm细胞滤网过滤。将过滤的细胞以500克离心5分钟，在晚期DMEM – F12中洗涤两次，在PBS中以0.1％BSA洗涤一次。然后通过FACS分析单细胞悬浮液。 　　将沉淀的类器官重悬于1 ml Tryple Express（Gibco）中，并在37°C下孵育15分钟。通过移液器剧烈分离细胞，重悬于10 mL晚期DMEM – F12中，通过40 µM细胞过滤器，并在4°C下以300g离心5分钟。将单细胞用0.1％BSA在PBS中洗涤，并用以下抗体染色：EPCAM -APC（Biolegend，118213; 1：200）;LY6D – PE（Biolegend，138603; 1：200）或Ly6d – apc（Miltenyi，130-115-313; 1:50）;和itga5 – pe（Biolegend，103805; 1：200）。用EPCAM-APC（Biolegend，324207; 1:50）和Ly6d – Fitc（Cusabio Biotech，CSB-PA613492LC01HU; 1:50）对人类单细胞悬浮液染色（Biolegend，324207; 1:50）。然后将细胞在PBS中的0.1％BSA中洗涤两次，然后受到FACS（BD FACSARIA II/BD LSR-Fortessa X-20）。通过DAPI负染色，单个可持续细胞被门控。补充信息3中提供了门控策略。使用FlowJo（V.10.8）软件进行分析。 　　使用10倍基因组细胞管道（V.7.2.0）处理原始测序读数并与小鼠参考基因组（MM10）对齐。使用R套件Seurat（V.5）分析了从CellRanger获得的基因表达矩阵。带有细胞条形码 <200 unique genes and >去除10％线粒体基因表达。将过滤后的矩阵标准化为每个细胞的总唯一分子标识符计数，并将细胞周期效应归一化。与Harmony进行了数据积分，随后的Louvain聚类导致了18个集群。使用FindAllmarkers函数在Seurat中实现的Wilcoxon Rank-SUM测试获得了簇之间的差异表达基因。 　　根据制造商的说明，使用主动图案商用套件进行ATAC – SEQ。简而言之，使用杵将50,000个细胞用ATAC裂解裂解，并缓慢散发在冰上25杆，然后在37°C下进行TN5标记30分钟。使用Spriselect协议（Beckam）进行尺寸选择。索引和PCR扩增后，将DNA库多路复用，并通过爱丁堡临床研究设施在100循环试剂盒上与Illumina NextSeq 2000进行测序。 　　使用默认管道设置（https://nf-co.re/atacseq/2.1.2)35，将原始测序输出FASTQ文件输入到NF核ATAC – SEQ管道中。评估了ATAC – SEQ库质量的污染线粒体DNA序列的存在。管道输出BigWig文件用于从IGV浏览器（https://www.igv.org/）创建基因座特定的基因组图像。使用DESEQ2分析了ATAC – SEQ峰的样品之间的差异富集（参考文献36）。使用基本r脚本对ATAC-SEQ读取计数（DESEQ2输出）的表进行了主成分分析。随后，将DESEQ2 ATAC – SEQ读数输入到Monalisa R Package37中，以确定样品组之间存在差异富集的TF结合或DNA结合位点基序。 　　用在37°C的Tryple Express消化细胞15分钟。接下来，将1×106个活细胞固定在室温下含有0.1％甲醛的PBS中，持续1分钟。将甲醛用甘氨酸在0.125 m处淬灭，然后在室温下孵育5分钟。用室温洗涤缓冲液（20毫米HEPES，150 mM NaCl，0.5 mM精子（Sigma-Aldrich），1％Triton X-100、0.05％SDS和1×Prot蛋白酶抑制剂鸡尾酒（ROCHE（ROCHE）），将固定的细胞固定并洗涤两次。将细胞沉淀并重悬于100 µL洗涤缓冲液中。根据制造商的说明，将细胞悬浮液以10：1的比例与姜黄素A珠（CST）添加，并在室温下轻轻混合并孵育20分钟。将细胞孔浆液放在磁架上，直到清除为止，将上清液除去并恢复在冷抗体缓冲液中（20毫米HEPES，150毫米NaCl，0.5 mm的精子，0.0025％digitonin（Thermo Fisher）（Thermo Fisher），2毫米EDTA，1％Triton X-bire x-botiator x-botitor and Intiator and Insiator in 0.05％SDS，并以1％的速度添加到1％（Roche））。添加以下抗体并在4°C下孵育过夜：XP同种型对照DA1E（兔子； CST 1:10）或抗Histone H3，乙酰基K27（兔子； Abcam 1：250）。将样品用digitonin缓冲液（20 mM HEPES，150 mM NaCl，0.5 mM精子，0.0025％Digitonin，1％Triton X-100、0.05％SDS和1×蛋白酶抑制剂鸡尾酒（Roche））和恢复在Digititoninin in digititoninin buffer中。加入1×Cutana Pag-MNase（Epicypher），并在室温下孵育10分钟。将样品用Digitonin缓冲液洗涤两次。通过添加2 mM CaCl2裂解染色质，并在4°C下孵育2小时。通过添加停止缓冲液（340 mM NaCl，20 mM EDTA，4 mM EGTA和50 µg ML – 1糖原（Thermofisher））和通过在37°C孵育10分钟的孵育10分钟来停止消化。收集上清液，并将SDS添加到0.09％的最终浓度。蛋白酶K（CST） 添加并在55°C下孵育过夜。根据制造商的说明，使用Maxtract高密度柱（QIAGEN）纯化DNA，并按照制造商的说明与Glycoblue（Thermofisher）一起纯化DNA。DNA在TE Buffer（Thermofisher）中洗脱。使用具有DNA HS试剂盒（Agilent Technologies）的Agilent 2100电泳生物分解器仪器对样品进行定量并评估尺寸分布。使用简单芯片DNA库准备试剂盒（CST）生成库，并使用量子dsDNA HS测定法对荧光测定法进行了量化，并评估了带有DNA HS套件的Agilent Bioanalyser上的尺寸分布。接下来，使用NextSeq 1000/2000 P1试剂（300个周期）套件在NextSeq 2000平台（Illumina）上进行100 bp配对的测序。 　　NF核切割和运行分析管道（https://doi.org/10.5281/zenodo.7715959）用于使用默认参数处理剪切和运行数据。将读数标准化为DNA（CST）中的尖峰，并与MM10参考基因组排列。删除了MM10编码黑名单中的区域38。Macs2（参考文献39）用于峰值呼叫，并将峰值调用标准化为IgG对照。使用具有默认参数的DiffBind40进行差异分析。BedTools41用于识别使用ATAC数据集的H3K27AC切割和运行中有显着差异的重叠区域。 　　从TCGA27的结直肠腺癌患者中，每千倍酶的大量RNA转录本是从基因组数据共享Commons Portal获得的，并已归一化。具有ATAC -SEQ数据（n = 36）的患者被分层为Atrxko和AtrxWT特征的高，中或低。这是通过在R中使用GSVA计算单样本GSEA评分并选择相应类别的第三，第二或第一分位数的样本来执行的。如前所述23，这些患者还被分配给ICMS类。 　　从基因组数据Commons Portal获得了归一化的ATAC – SEQ峰值计数，可为36例结直肠腺癌患者的77个样本提供。可以找到有关标准化的详细信息，请找到原始出版物27。简而言之，使用“ cpm（矩阵，log = true，priar.count = 5）”将峰值计数矩阵进行标准化。然后使用标准化。在R中使用归一化量表。使用两尾t检验来识别来自分类为hisquam的患者（aTrxko高，对于Atrxwt低）的患者之间的平均值明显不同的峰或hicol（atrxko低，而Atrxwt的高）。差异峰的选择是基于FDR< 0.01 and Δlog2counts >1个截止值。使用R中的复合图绘制了所有样品的计数。 　　对TF基序富集分析进行了对通过荷马分类为Hisquam或HICOL的患者差异的一组峰值进行的。首先使用Chipeakanno注释峰，然后格式化为荷马输入样式。用命令FindMotifSgenome.pl，基因组“ HG38”和“ -size 200 -mask”作为选项进行分析。如前所述42提出了TF基序富集。 　　来自CRC患者的样品的归一化基因表达数据是从基因表达综合（登录GSE39582）中获得的，557例具有复发存活信息的患者用于下游分析。通过使用AtrxWT和Atrxko签名基因进行分层聚类来鉴定Hicol，中间和Hisquam组。三个患者组的无复发存活是通过对数秩检验的Kaplan – Meier生存期确定的。 　　为了了解单细胞水平上ATRXKO驱动的基因表达与ICMS分类之间的关联，我们在从Syn26844071下载后使用SCRNA-SEQ数据进行了分析（参考文献23），并在排除细胞数量少的患者后，使用ICMS分类的肿瘤细胞从原发性肿瘤组织样品中使用ICMS分类进行了分析。使用715个ICMS缔解基因进行了降低分析，并使用Harmonony43校正了患者批次。使用每个签名的平均表达水平计算每个单元格的AtrxWT和AtrxKO分数，然后通过对照基因评分集的汇总表达进行减去。使用100个随机选择基因的平均表达计算对照基因评分，重复了10次。 　　使用GraphPad Prism 9进行了统计分析。在图中显示了所使用的统计测试，并且整个过程中显示了精确的P值。 　　有关研究设计的更多信息可在与本文有关的自然投资组合报告摘要中获得。