In the final stage of this pathway, malignant transformation, we observe increased accessibility in regions containing HNF4A motifs. We identify regulatory elements and transcription factors (TFs) associated with different stages of transformation from normal colon to carcinoma, including early increases in accessibility of regions containing TCF and LEF motifs and loss of accessibility in regions containing KLF motifs. We find that polyps populate an epigenetic and transcriptional continuum from normal colon to CRC characterized by sequential opening and closing of chromatin and upregulation and downregulation of genes associated with the cancer state. We observe a much larger fraction of cells exhibiting a stem-like state (both transcriptionally and epigenetically) within polyps and CRCs. We identify a subpopulation of exhausted T cells present only in CRC tissue. We find large shifts in fibroblast subpopulations that occur along the transition from normal colon to CRC. From these single-cell datasets, we first catalog immune, stromal and epithelial cell types. Many polyps were obtained from patients with FAP who underwent surgical colectomies, allowing both analysis of polyps with diverse sizes and locations of origin, and collection of neighboring unaffected colon tissue. To chart the regulatory and transcriptomic changes that occur on the phenotypic continuum from healthy colon to invasive carcinoma, as part of the Human Tumor Atlas Network 14, we profiled single-nuclei transcriptomes (single-nucleus RNA sequencing (RNA-seq) (snRNA-seq)) and epigenomes (single-cell assay for transposase-accessible chromatin using sequencing (ATAC-seq) (scATAC-seq)) of healthy colon, polyps and CRCs. These patients typically develop hundreds of polyps by early adulthood 12, 13, and therefore an individual patient can provide numerous polyps of varied molecular ages and stages of progression, all arising in the same germline background. Subsequent mutations in other cancer driver genes such as KRAS, TP53 and SMAD4 result in the transformation to carcinoma.īecause APC mutations are almost universally the initiating event for polyps and CRCs, patients with familial adenomatous polyposis (FAP), who have germline mutations in APC, are a suitable population in which to study the natural progression of polyposis. For example, an estimated 80–90% of colorectal tumors are initiated by loss of APC 9, resulting in β-catenin stabilization and increased WNT signaling 10 leading to intestinal hyperplasia 11. A number of the changes associated with these transitions are nearly universal to all CRC malignancies, as typified by the adenoma-to-carcinoma sequence 6, 7, 8. As a result, a detailed understanding of the progression of phenotypic changes that occur during the transition from normal to precancerous to cancerous state, as well as the molecular drivers of this transformation, remain underexplored.ĬRC is an ideal system to study the continuum of phenotypic states along malignant transformation as it follows a stereotyped progression from normal to atypical to carcinoma that includes the formation of precancerous polyps 4, 5, which can subsequently give rise to CRCs. However, most studies have focused on bulk profiling of advanced stage tumors and have largely ignored premalignant lesions. These efforts have cataloged the diversity and commonality of many genetic and transcriptional changes that accompany malignancy in diverse cancer types. The identification of genes and pathways that drive formation of invasive cancers has been the central focus of a number of large-scale genomics efforts 1, 2, 3. DNA methylation changes in sporadic CRC are strongly anti-correlated with accessibility changes along this continuum, further identifying regulatory markers for molecular staging of polyps. In the cancerous state, we observe T cell exhaustion, RUNX1-regulated cancer-associated fibroblasts and increasing accessibility associated with HNF4A motifs in epithelia. Advanced polyps contain increasing numbers of stem-like cells, regulatory T cells and a subtype of pre-cancer-associated fibroblasts. A large fraction of polyp and CRC cells exhibit a stem-like phenotype, and we define a continuum of epigenetic and transcriptional changes occurring in these stem-like cells as they progress from homeostasis to CRC. To chart cell composition and cell state changes that occur during the transformation of healthy colon to precancerous adenomas to colorectal cancer (CRC), we generated single-cell chromatin accessibility profiles and single-cell transcriptomes from 1,000 to 10,000 cells per sample for 48 polyps, 27 normal tissues and 6 CRCs collected from patients with or without germline APC mutations.
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