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