Highlights
- •A single-cell resolution atlas of human adenomas and serrated polyps
- •Serrated polyps arise from metaplasia as opposed to stem cell expansion
- •Cytotoxic immunity in serrated polyps occurs independently of hypermutation
- •Distinct immune microenvironments track tumor cell-differentiation states
Summary
Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.
Introduction
Classification schemes for human colorectal cancer (CRC) focus largely on intrinsic features of tumor cells, including histopathology, bulk gene expression (Consensus Molecular Subtypes or CMS), chromosomal instability (CIN), hypermethylation (CpG Island Methylator Phenotype or CIMP), and microsatellite-instability (MSI) (Guinney et al., 2015; Ogino and Goel, 2008). The tumor immune microenvironment is also critical to CRC pathogenesis (Pelka et al., 2021). Hypermutated MSI-high (MSI-H) tumors exhibit a neoantigen-triggered cytotoxic immune infiltration that contributes to their responsiveness to immunotherapy (Le et al., 2015; Llosa et al., 2015). However, a significant subset of low mutation burden CRCs appears to exhibit an activated immune microenvironment via ill-defined mechanisms (Mlecnik et al., 2016). We hypothesize that mapping the routes toward tumorigenesis in precursors of MSI-H and MSS CRCs will uncover mechanisms that define the CRC cellular landscape and identify targets with diagnostic or therapeutic utility.Most MSS and MSI-H CRCs develop from pre-cancerous conventional adenomas (ADs) and sessile serrated lesions (SSLs; formerly sessile serrated adenomas/polyps), respectively. As proposed by Vogelstein and co-workers, ADs arise from truncating mutations in APC, which result in activation of the WNT pathway and CIN (Fearon and Vogelstein, 1990). ADs subsequently accumulate gain-of-function mutations in oncogenes (chiefly KRAS) and loss-of-function mutations in tumor suppressor genes such as TP53, ultimately forming MSS CRCs. Conversely, SSLs resemble MSI-H CRCs molecularly and are distinct from ADs in that tumorigenesis is not initiated by genetic disruptions of APC (Crockett and Nagtegaal, 2019; Thorstensen et al., 2005). Instead, they have epigenetic disruptions, including MLH1 hypermethylation and a 40%–75% prevalence of CIMP (Leggett and Whitehall, 2010; Yang et al., 2004). These tumors harbor BRAF mutations in contrast to KRAS mutations commonly present in ADs. Mirroring the relatively lower incidence of MSI-H CRCs and their prevalence in the proximal colon, SSLs represent only 10%–20% of polyps and are also found in the proximal colon more often, unlike the more frequently distal ADs (Crockett and Nagtegaal, 2019; Markowitz and Bertagnolli, 2009).We present a multi-omic human pre-cancer atlas integrating single-cell transcriptomics, genomics, and immunohistopathology describing the two most common pathways toward CRC. We identify and functionally validate distinct origins and molecular processes that establish divergent tumor landscapes. Notably, this clearer understanding of advanced and highly heterogeneous cancers was enabled only by looking at CRCs through the lens of their originating lesions, paving a path to new strategies for precision prevention, surveillance, and therapeutics.
Results
Distinct histopathologic and molecular features define colonic pre-cancer subtypes
Polyps, as well as matching normal biopsies, were collected from COLON MAP study participants. Most polyps were small (median diameter ≤5 mm) and were bisected for multi-assay analysis. Single-cell RNA sequencing (scRNA-seq), multiplex immunofluorescence (MxIF), and multiplex immunohistochemistry (MxIHC) were performed on two independent sets of specimens collected approximately 1 year apart. The discovery (DIS) set consisted of 65 specimens analyzed including 30 tumors. The validation (VAL) set consisted of 63 specimens analyzed including 32 tumors (Figure 1A). Overall, 128 independent scRNA-seq datasets on 62 tumors were generated (Table S1). Specimens were collected from diverse sex, racial, and age groups (Table S2). In addition, we performed bulk RNA-seq and targeted gene sequencing on an orthogonal set of 66 and 281 polyps, respectively (Figure S1A; Table S2). Polyps were histologically categorized by two pathologists into two subtypes: ADs consisting of tubular ADs (TAs) and tubulovillous ADs (TVAs), or serrated polyps (SERs) consisting of hyperplastic polyps (HPs) and SSLs (Figure 1B). While standard histological features were observed for polyps, HPs were further subdivided into goblet cell-rich HPs (GCHPs) and microvesicular HPs (MVHPs), with MVHPs appearing more advanced and may progress to SSLs (Crockett and Nagtegaal, 2019). Epithelial serrations of GCHPs, if present, were subtle and confined to the mucosal surface, while, for MVHPs, serrations extended from the surface to two-thirds down the crypt, sparing the morphologically normal crypt base. In contrast, SSLs showed epithelial serrations that extended to the base of crypts, which were dilated and spread laterally above the muscularis mucosae….
