基于空间ATAC-seq技术的Apcmin/+小鼠结肠肿瘤表观特征分析

doi:10.3969/j.issn.1674-8115.2025.10.001

摘要/Abstract

摘要：

目的·研究Apc^min/+小鼠自发结肠肿瘤的空间表观遗传特征。方法·使用8月龄雄性Apc^min/+小鼠自发结肠肿瘤模型，搭建空间染色质可及性测序（assay for transposase-accessible chromatin with high-throughput sequencing，ATAC-seq）技术平台。摘取一颗小鼠结肠肿瘤，经冷冻包埋后，进行连续切片；一张组织切片使用苏木精-伊红染色观察其组织学特征，另一张相邻的组织切片进行空间ATAC-seq技术处理，得到带有空间位置信息的DNA文库后进行测序，得到该小鼠结肠肿瘤的空间染色质可及性数据。取同一只小鼠的另一颗肿瘤组织，经消化后得到细胞悬液，通过流式细胞术分选有活性的单细胞悬液，进行单细胞转录组测序，与空间染色质可及性数据联合分析此小鼠的结肠肿瘤微环境表观遗传学特征。结果·成功搭建稳定的空间ATAC-seq技术体系，并据此将小鼠的肿瘤分为恶性区域、非恶性区域以及恶性-非恶性边界区域；恶性区域富集到的转录因子包括NK2同源框转录因子5（NK2 homeobox 5，NKX2-5）、转录因子3（transcription factor 3，TCF3）等。在3个区域分别对转录因子进行富集，得到2种不同的表达趋势：一种为表达水平自恶性‒边界‒非恶性区域逐步降低；另一种为在恶性和边界区域表达水平高，在非恶性区域表达水平低。对不同区域的基因分析显示恶性区域的缺氧反应明显上调，转化生长因子（transforming growth factor，TGF）、Kirsten大鼠肉瘤病毒癌基因同源物（Kirsten rat sarcoma viral oncogene homolog，KRAS）信号通路显著上调，与细胞周期相关的功能明显增强。对肿瘤微环境中细胞间的相互作用分析发现不同区域的细胞互作强度具有显著差别，非恶性区域内部细胞的相互作用强，边界区与非恶性区以及恶性与非恶性区域的相互作用中等，恶性区与边界区的相互作用弱。结论·Apc^min/+小鼠结肠肿瘤呈高度空间异质性；肿瘤恶性区域富集了Tcf3等多种转录因子，恶性区域与边界、非恶性区域之间细胞相互作用相对较弱。

关键词: 结直肠癌, 空间染色质可及性测序, 表观遗传, 单细胞转录组测序

Abstract:

Objective ·To investigate the spatial epigenetic characteristics of spontaneous colon tumors in Apc^min/+ mice. Methods ·A spatial assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) technology platform was established using an eight-month-old male Apc^min/+ mouse model with spontaneous colon tumors. One tumor from a mouse was harvested and embedded in OCT compound for serial cryosectioning; one tissue section was stained with hematoxylin-eosin (H-E) to observe its histological characteristics, while an adjacent section was processed using spatial ATAC-seq technology to generate spatially resolved DNA libraries, followed by sequencing to obtain spatial chromatin accessibility data. Another tumor from the same mouse was digested into a single-cell suspension, in which viable single cells were sorted by flow cytometry and processed for single-cell RNA sequencing. The results were integrated with spatial chromatin accessibility data to jointly analyze the epigenetic characteristics of the colon tumor microenvironment. Results ·A stable spatial ATAC-seq platform was successfully established, dividing the tumor into malignant, non-malignant, and malignant-non-malignant boundary regions. Transcription factors enriched in malignant regions included NK2 homeobox 5 (NKX2-5) and transcription factor 3 (TCF3). Analysis of transcription factor enrichment in the 3 regions revealed two distinct expression trends: one showing a gradual decrease from malignant to boundary to non-malignant regions, and the other exhibiting high expression in malignant and boundary regions but low expression in non-malignant regions. Gene analysis across regions revealed significant upregulation of hypoxia response, transforming growth factor (TGF), and Kirsten rat sarcoma viral oncogene homolog (KRAS) signaling pathways in malignant regions, with cell cycle-related functions markedly enhanced. Analysis of cell-cell interactions in the tumor microenvironment revealed significant differences in interaction strength: strong interactions within non-malignant regions, moderate interactions between boundary and non-malignant regions, and weak interactions between malignant and boundary regions as well as between malignant and non-malignant regions. Conclusion ·Colon tumors in Apc^min/+ mice exhibit high spatial heterogeneity; malignant regions were enriched with transcription factors including TCF3, and cell interactions between malignant regions and boundary/non-malignant regions were relatively weak.

Key words: colorectal cancer (CRC), spatial ATAC-seq, epigenetics, single-cell RNA-seq

中图分类号:

R735.3

梁乐斌, 陈慧芳, 赖淑静, 顾靓, 苏冰. 基于空间ATAC-seq技术的Apc^min/+小鼠结肠肿瘤表观特征分析[J]. 上海交通大学学报（医学版）, 2025, 45(10): 1261-1270.

LIANG Lebin, CHEN Huifang, LAI Shujing, GU liang, SU Bing. Analysis of epigenetic characteristics in colonic tumors of Apc^min/+via spatial ATAC-seq technology[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(10): 1261-1270.

图/表 6

表1 Tn5引物序列

Tab 1 Tn5 primer sequences

Name	Sequence (5'→3')
Tn5MErev	/5Phos/CTGTCTCTTATACACATCT-N2H
Tn5ME-A	/5Phos/CATCGGCGTACGACTAGATGTGTATAAGAGACAG
Tn5ME-B	GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG

表2 标签引物序列

Tab 2 Barcode primer sequences

No.	Barcode A sequence	No.	Barcode B sequence
A1	TTTAGG	B1	GTGGTA
A2	ATTCCA	B2	CCTAGA
A3	GCTCAA	B3	GGGTTT
A4	CATCCC	B4	ATGGCG
A5	TTGGAC	B5	TTCATA
A6	CTGTGT	B6	AACGCC
A7	GGACAT	B7	GGCTGC
A8	CAAAGT	B8	GCTGTG
A9	AAGCGG	B9	AGATGG
A10	AATAAA	B10	GTAATG
A11	GAGGAG	B11	AGGGTC
A12	GGTACA	B12	ATCTCT
A13	AGCGAG	B13	GCCTAG
A14	GTCGGT	B14	TCAAAG
A15	ATTTGC	B15	CATGAT
A16	AGGACT	B16	TGTGCG
A17	GCCCTC	B17	GCAGGA
A18	TCGTAA	B18	TCTACC
A19	CCAGAC	B19	AGTCGT
A20	TATGTA	B20	CGTGGC
A21	ACAATA	B21	GCGTCC
A22	ATGCTT	B22	GAACGC
A23	AGTTTA	B23	ACTTAT
A24	CACAAG	B24	TGGATG
A25	ATCAAC	B25	TATTGT
A26	TAGTCG	B26	ACGTTG
A27	TAGAGA	B27	GAATTA
A28	GTCCCG	B28	CCATCT
A29	TACTTC	B29	TGATCA
A30	AAAGTT	B30	CGTATT
A31	TAAGGG	B31	CGGCAG
A32	GTTGCC	B32	GACACT
A33	AAGTAC	B33	TTCCGC
A34	GATCTT	B34	CTCGCA
A35	TTAACT	B35	GTATAC
A36	GCGAAT	B36	TGTCAC
A37	CCGCTA	B37	TGCGGA
A38	TGAAGC	B38	ACGAGC
A39	ATACAG	B39	ACACCC
A40	CTTCTG	B40	CGCTTG
A41	GAGATC	B41	TGCAAT
A42	CCGACG	B42	CAACAA
A43	CTCCAT	B43	CTGAAA
A44	AAAACG	B44	AACCTA
A45	TAGCAT	B45	ACCTGA
A46	TCGGGT	B46	TCACTT
A47	GTGGTA	B47	GGGCGA
A48	CCTAGA	B48	CGCACC
A49	GGGTTT	B49	CGAGTA
A50	ATGGCG	B50	CCTTTC

图1 空间ATAC-seq应用于 Apcmin/+ 小鼠结肠肿瘤的流程及数据质量Note： A. Workflow of spatial ATAC-seq. B. H-E staining of a tissue section (×20). C. Image of a spatial ATAC-seq tissue section. D. TSS enrichment of the data. E. Peak region fragments of the data. F. Percentage of reads in peaks. G. Overall data quality control (QC).

Fig 1 Workflow and data quality of spatial ATAC-seq applied to colon tumors in Apcmin/+ mice

图2 Apcmin/+ 小鼠肿瘤微环境的空间构成Note： A. Uniform manifold approximation and projection (UMAP) of single-cell RNA-seq of tumor. B. Spatial distribution of different tumor regions. C. Proportion of each cell type within different regions of the tumor.

Fig 2 Spatial composition of tumor microenvironment in Apcmin/+ mice

图3 Apcmin/+ 小鼠肿瘤微环境的空间表观遗传特征Note： A. Spatial distribution of motif activity for transcription factor enrichment in different regions. B. Spatial distribution of two motifs. C. Two expression modes of motifs in different regions. D. Gene activity scores across different tumor regions. E. Hallmark enrichment analysis in different tumor regions. F. GO enrichment analysis in different tumor regions.

Fig 3 Spatial epigenetic characteristics of the tumor microenvironment in Apcmin/+ mice

图4 Apcmin/+ 小鼠肿瘤微环境不同区域细胞的相互作用Note：A. Heatmap of interaction strength in different tumor regions. B. Spatial interaction of two signaling pathway networks in different tumor regions. The thickness of the line represents the strength of the interaction.

Fig 4 Spatial interaction in different regions of the tumor microenvironment in Apcmin/+ mice

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