Biology subjects

Coffey, R. J.

Publications and source records attributed to Coffey, R. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Novel COX-2 Targeted Nanobodies for Molecular Endoscopic Imaging of Colorectal Adenomas

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality in men and women. Timely detection and diagnosis are key to management of CRC, which is under-diagnosed because colorectal aberrant crypt foci, hyperplastic polyps, and microadenomas are often missed with conventional colonoscopy. The enzyme cyclooxygenase-2 (COX-2) is overexpressed in early stages of colorectal carcinogenesis and plays an important regulatory role in the process, suggesting that it could be a valuable target for enhanced imaging of nascent disease. Thus, we have generated an alpaca-derived library of 73 COX-2-specific nanobody clones. Here, we describe one such nanobody, F9-K45Q-K77Q-ROX, in which two native lysine residues have been mutated followed by conjugation to a fluorophore at the N-terminus with retention of COX-2-selective binding. The site of fluorophore conjugation and COX-2 binding affinity of F9-K45Q-K77Q-ROX were determined by proteomic and microscale thermophoretic analyses, respectively. In cell culture studies using 1483 human head and neck squamous cell carcinoma cells, F9-K45Q-K77Q-ROX accumulated inside cells and bound to intracellular COX-2, as visualized by fluorescence microscopy. In vivo pharmacokinetic, and toxicological analyses revealed that F9-K45Q-K77Q-ROX is detectable in circulation with a plasma half-life of 17.9 min and there is no short-term toxicity associated with single injections of 10 mg/kg, 20 mg/kg, or 40 mg/kg doses at 24 h post-administration. Noninvasive in vivo fluorescence endoscopic imaging validated tumor-specific accumulation of F9-K45Q-K77Q-ROX in azoxymethane/dextran sodium sulfate-induced colorectal adenomas in mice. This work demonstrates the first COX-2-targeted nanobodies including a fluorescent derivative that offers significant promise for targeted endoscopic imaging of COX-2-expressing neoplasms. Significance StatementCurrent colorectal cancer screening procedures, such as white-light colonoscopy, chromoendoscopy, and narrow-band imaging aim to detect solid colon tumors and precursor lesions. However, these methods tend to detect only raised solid tumors and mature cancers, whereas precursor lesions, such as aberrant crypt foci, hyperplastic polyps, and small adenomas are frequently missed. To address the need for better visualization of early lesions, we developed a library of alpaca-derived nanobodies targeted to cyclooxygenase-2 (COX-2), an enzyme that is overexpressed in colorectal adenomas. COX-2-targeted nanobodies bearing a fluorescent tag accumulate and are retained in colonic adenomas, facilitating their endoscopic visualization. This novel COX-2-targeted nanobody platform may also be valuable for early detection of other neoplastic diseases in which COX-2 overexpression occurs. (Word counts 119, limit 120)

bioengineering↗

bioRxiv2025

Unbiased recording of clonal potency reveals species-specific regulation of mammalian intestine

The mammalian intestine regenerates rapidly after damage, yet the clonal dynamics and species-specific regulation of different populations remain poorly understood. Here we used synthetic or naturally occurring DNA alterations to reconstruct clonal histories of the mouse and human intestinal epithelium at single-cell resolution. In mice, we uncovered the clonal architecture of different cell types and their roles in regeneration, supporting a hierarchical regenerative response model. We identified a rare embryonic precursor population that persisted in the adult and was crucial for regeneration after irradiation. This population was marked by Tob2, which is required for nuclear transport of Ascl2. A parallel clonal analysis of 65 human colonic biopsies revealed secretory lineage bias and an age-associated decline in clonal diversity in the distal colon. Unlike highly proliferative murine Lgr5+ stem cells, human LGR5+ cells were found largely quiescent, revealing species-specific difference in clonal potency, and suggesting a distinct regulation of intestinal stemness.

genomics↗

bioRxiv2024

Inhibition of EGFR/ErbB does not protect against C. difficile toxin B

Clostridioides difficile is a common cause of diarrhea and mortality, especially in immunosuppressed and hospitalized patients. C. difficile is a toxin-mediated disease, but the host cell receptors for C. difficile toxin B (TcdB) have only recently been revealed. Emerging data suggest TcdB interacts with receptor tyrosine kinases during infection. In particular, TcdB can elicit Epidermal Growth Factor Receptor (EGFR) transactivation in human colonic epithelial cells. The mechanisms for this function are not well understood, and the involvement of other receptors in the EGFR family of Erythroblastic Leukemia Viral Oncogene Homolog (ErbB) receptors remains unclear. Furthermore, in an siRNA-knockdown screen for protective genes involved with TcdB toxin pathogenesis, we show ErbB2 and ErbB3 loss resulted in increased cell viability. We hypothesize TcdB induces the transactivation of EGFR and/or ErbB receptors as a component of its cell-killing mechanism. Here, we show in vivo intrarectal instillation of TcdB in mice leads to phosphorylation of ErbB2 and ErbB3. However, immunohistochemical staining for phosphorylated ErbB2 and ErbB3 indicated no discernible difference between control and TcdB-treated mice for epithelial phospho-ErbB2 and phospho-ErbB3. Human colon cancer cell lines (HT29, Caco-2) exposed to TcdB were not protected by pre-treatment with lapatinib, an EGFR/ErbB2 inhibitor. Similarly, lapatinib pre-treatment failed to protect normal human colonoids from TcdB-induced cell death. Neutralizing antibodies against mouse EGFR failed to protect mice from TcdB intrarectal instillation as measured by edema, inflammatory infiltration, and epithelial injury. Our findings suggest TcdB-induced colonocyte cell death does not require EGFR/ErbB receptor tyrosine kinase activation.

microbiology↗

bioRxiv2024

Scalable single-cell pooled CRISPR screens with conventional knockout vector libraries

Current methods for single-cell RNA profiling of pooled CRISPR screens are limited, either by indirect capture of single guide RNAs (sgRNAs) or by custom modification of plasmid libraries. Here, we present a direct sgRNA capture platform called Native sgRNA Capture and sequencing (NSC-seq) that enables single-cell CRISPR screens using common knockout plasmid libraries, facilitating genotype-phenotype mapping at multiple scales in vitro and in vivo. Additionally, we characterize sgRNA expression in three whole-genome knockout libraries, revealing a substantial subset of truncated (isoform) spacer reads. We provide this dataset as a reference of expressed sgRNA isoforms that may potentially have compromised CRISPR gene editing efficacy and precision.

genomics↗

bioRxiv2024

miR-100 and miR-125b Contribute to Enhanced Growth and Invasiveness

Extracellular communication via the transfer of vesicles and nanoparticles is now recognized to play an important role in tumor microenvironment interactions. Cancer cells upregulate and secrete abundant levels of miR-100 and miR-125b that can alter gene expression by both cell- and non-cell-autonomous mechanisms. We previously showed that these miRNAs activate Wnt signaling in colorectal cancer (CRC) through noncanonical pairing with 5 negative regulators of Wnt signaling. To identify additional targets of miR-100 and miR-125b, we used bioinformatic approaches comparing multiple CRC cell lines, including knockout lines lacking one or both of these miRNAs. From an initial list of 96 potential mRNA targets, we tested 15 targets with 8 showing significant downregulation in the presence of miR-100 and miR-125b. Among these, Cingulin (CGN) and Protein tyrosine phosphatase receptor type-R (PTPRR) are downregulated in multiple cancers, consistent with regulation by increased levels of miR-100 and miR-125b. We also show that increased cellular levels of miR-100 and miR-125b enhance 3D growth and invasiveness in CRC and glioblastoma cell lines. Lastly, we demonstrate that extracellular transfer of miR-100 and miR-125b can silence both reporter and endogenous mRNA targets in recipient cells and also increase the invasiveness of recipient spheroid colonies when grown under 3D conditions in type I collagen.

cancer biology↗

bioRxiv2024

5-Fluorouracil Treatment Represses Pseudouridine-Containing Small RNA Export into Extracellular Vesicles

5-fluorouracil (5-FU) has been used for chemotherapy for colorectal and other cancers for over 50 years. The prevailing view of its mechanism of action is inhibition of thymidine synthase leading to defects in DNA replication and repair. However, 5-FU is also incorporated into RNA causing toxicity due to defects in RNA metabolism, inhibition of pseudouridine modification, and altered ribosome function. Here, we examine the impact of 5-FU on the expression and export of small RNAs (sRNAs) into small extracellular vesicles (sEVs). Moreover, we assess the role of 5-FU in regulation of post-transcriptional sRNA modifications (PTxM) using mass spectrometry approaches. EVs are secreted by all cells and contain a variety of proteins and RNAs that can function in cell-cell communication. PTxMs on cellular and extracellular sRNAs provide yet another layer of gene regulation. We found that treatment of the colorectal cancer (CRC) cell line DLD-1 with 5-FU led to surprising differential export of miRNA snRNA, and snoRNA transcripts. Strikingly, 5-FU treatment significantly decreased the levels of pseudouridine on both cellular and secreted EV sRNAs. In contrast, 5-FU exposure led to increased levels of cellular sRNAs containing a variety of methyl-modified bases. Our results suggest that 5-FU exposure leads to altered expression, base modifications, and mislocalization of EV base-modified sRNAs.

molecular biology↗

bioRxiv2023

Temporal recording of mammalian development and precancer

Key to understanding many biological phenomena is knowing the temporal ordering of cellular events, which often require continuous direct observations [1, 2]. An alternative solution involves the utilization of irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using NSC-seq, a newly designed and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo, while incorporating assigned cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development and identified new intestinal epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas and single-cell multi-omic profiling of human precancers as part of the Human Tumor Atlas Network (HTAN), including 116 scRNA-seq datasets and clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Thus, our multimodal framework augments existing single-cell analyses and lays the foundation for in vivo multimodal recording, enabling the tracking of lineage and temporal events during development and tumorigenesis.

developmental biology↗

bioRxiv2023

Transcriptomic Profiling Reveals Claudin 18.2 as a Diagnostic Biomarker of Menetrier Disease and the Role of Hedgehog Signaling in Pathogenesis

Both Menetriers disease (MD) and juvenile polyposis syndrome (JPS) are rare premalignant conditions that can lead to gastric cancer development. MD is an acquired disease without known causative mutations. MD patients are characterized by an increased expression of EGF receptor (EGFR) ligand and transforming growth factor alpha (TGF-) in the stomach. JPS is inherited in an autosomal dominant pattern and is caused by BMPR1A or SMAD4 mutations. It is characterized by multiple polyps throughout the gastrointestinal tract along with certain SMAD4 mutations that can result in gastric polyposis. Although there are many distinct clinico- endoscopic and histopathologic features that differ between the two diseases, they also share similar features that often lead to misdiagnosis. This study aimed to identify markers that can help distinguish MD from JPS and to better understand the pathogenesis of MD by comparing differential gene expression patterns. Upon examination of MD and JPS microscopically, we found almost all cases have patchy areas mimicking each other, making it difficult to make a correct diagnosis with histopathologic examination alone. Comparative analysis between MD and JPS using ingenuity pathway analysis (IPA) revealed both common and differential gene signatures. Common gene signatures included estrogen receptor signaling, integrin signaling, mTOR signaling, and others, which may be responsible for histopathologic similarities. Among differential gene signatures, we found that claudin 18 (CLDN18) is upregulated in MD and confirmed that CLDN18.2 (isoform of CLDN18) protein expression is higher in MD than JPS by immunohistochemistry. Comparative analysis between MD and normal control revealed the hedgehog (Hh) signaling pathway is upregulated in MD. Treatment with a hedgehog pathway inhibitor partially rescued the histopathologic phenotypes in a MD mouse model. The current study provides valuable insight into the potential underlying mechanism of why MD and JPS show similar clinico-pathologic features. We also identified a diagnostic marker CLDN18.2 that can help distinguish MD from JPS, genetically. Furthermore, it also shows that Hh signaling plays an important role in the pathogenesis of MD and can function as a potential therapeutic target.

pathology↗

bioRxiv2023

Detection of EGFR and its Activity State in Plasma CD63-EVs from Glioblastoma Patients: Rapid Profiling using an Anion Exchange Membrane Sensor

We present a novel quantitative immunoassay for CD63 EVs (extracellular vesicles) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane and a charged silica nanoparticle reporter. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins and fluorophore degradation, thus enabling direct plasma analysis. With a limit of detection of 30 EVs/L and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. Glioblastoma necessitates improved non-invasive diagnostic approaches for early detection and monitoring. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. This approach offers direct glioblastoma detection from untreated human patient samples. Analysis of glioblastoma clinical samples yielded an area-under-the-curve (AUC) value of 0.99 and low p-value of 0.000033, significantly surpassing the performance of existing assays and markers.

cancer biology↗

bioRxiv2023

GammaGateR: semi-automated marker gating for single-cell multiplexed imaging

MotivationMultiplexed immunofluorescence (mIF) is an emerging assay for multichannel protein imaging that can decipher cell-level spatial features in tissues. However, existing automated cell phenotyping methods, such as clustering, face challenges in achieving consistency across experiments and often require subjective evaluation. As a result, mIF analyses often revert to marker gating based on manual thresholding of raw imaging data. ResultsTo address the need for an evaluable semi-automated algorithm, we developed GammaGateR, an R package for interactive marker gating designed specifically for segmented cell-level data from mIF images. Based on a novel closed-form gamma mixture model, GammaGateR provides estimates of marker-positive cell proportions and soft clustering of marker-positive cells. The model incorporates user-specified constraints that provide a consistent but slide-specific model fit. We compared GammaGateR against the newest unsupervised approach for annotating mIF data, employing two colon datasets and one ovarian cancer dataset for the evaluation. We showed that GammaGateR produces highly similar results to a silver standard established through manual annotation. Furthermore, we demonstrated its effectiveness in identifying biological signals, achieved by mapping known spatial interactions between CD68 and MUC5AC cells in the colon and by accurately predicting survival in ovarian cancer patients using the phenotype probabilities as input for machine learning methods. GammaGateR is a highly efficient tool that can improve the replicability of marker gating results, while reducing the time of manual segmentation. Availability and ImplementationThe R package is available at https://github.com/JiangmeiRubyXiong/GammaGateR. ContactPlease address correspondence to jiangmei.xiong@vanderbilt.edu and simon.vandekar@vumc.org. Key PointsO_LIGammaGateR is the first semi-automated marker gating tool for mIF image, and it will help to diminish the inconsistency with manual marker gating. C_LIO_LIWith novel cfGMM, GammaGateR can fit flexibly across slides with different distributions and incorporate biology priors. C_LIO_LIGammaGateR is proven to reveal credible prognostic information, and it can quantify known findings in tumor cell populations. C_LI

bioinformatics↗

bioRxiv2023

Golgi organization is a determinant of stem cell function in the small intestine

Cell-to-cell signalling between niche and stem cells regulates tissue regeneration. While the identity of many mediating factors is known, it is largely unknown whether stem cells optimize their receptiveness to niche signals according to the niche organization. Here, we show that Lgr5+ small intestinal stem cells (ISCs) regulate the morphology and orientation of their secretory apparatus to match the niche architecture, and to increase transport efficiency of niche signal receptors. Unlike the progenitor cells lacking lateral niche contacts, ISCs orient Golgi apparatus laterally towards Paneth cells of the epithelial niche, and divide Golgi into multiple stacks reflecting the number of Paneth cell contacts. Stem cells with a higher number of lateral Golgi transported Epidermal growth factor receptor (Egfr) with a higher efficiency than cells with one Golgi. The lateral Golgi orientation and enhanced Egfr transport required A-kinase anchor protein 9 (Akap9), and was necessary for normal regenerative capacity in vitro. Moreover, reduced Akap9 in aged ISCs renders ISCs insensitive to niche-dependent modulation of Golgi stack number and transport efficiency. Our results reveal stem cell-specific Golgi complex configuration that facilitates efficient niche signal reception and tissue regeneration, which is compromised in the aged epithelium.

cell biology↗

bioRxiv2023

Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Leveraging spatial molecular information to construct a phylogeographic map of tumor evolution can reveal individualized growth trajectories with diagnostic and therapeutic potential. Integrative analysis of spatial multi-omic data from 31 colorectal specimens revealed simultaneous microenvironmental and clonal alterations as a function of progression. Copy number variation served to re-stratify microsatellite stable and unstable tumors into chromosomally unstable (CIN+) and hypermutated (HM) classes. Phylogeographical maps classified tumors by their evolutionary dynamics, and clonal regions were placed along a global pseudotemporal progression trajectory. Cell-state discovery from a single-cell cohort revealed recurring epithelial gene signatures and infiltrating immune states in spatial transcriptomics. Charting these states along progression pseudotime, we observed a transition to immune exclusion in CIN+ tumors as characterized by a novel gene expression signature comprised of DDR1, TGFBI, PAK4, and DPEP1. We demonstrated how these genes and their protein products are key regulators of extracellular matrix components, are associated with lower cytotoxic immune infiltration, and show prognostic value in external cohorts. Through high-dimensional data integration, this atlas provides insights into co-evolution of tumors and their microenvironments, serving as a resource for stratification and targeted treatment of CRC.

systems biology↗

bioRxiv2023

Consensus tissue domain detection in spatial -omics data using MILWRM

Spatially resolved molecular assays provide high dimensional genetic, transcriptomic, proteomic, and epigenetic information in situ and at various resolutions. Pairing these data across modalities with histological features enables powerful studies of tissue pathology in the context of an intact microenvironment and tissue structure. Increasing dimensions across molecular analytes and samples require new data science approaches to functionally annotate spatially resolved molecular data. A specific challenge is data-driven cross-sample domain detection that allows for analysis within and between consensus tissue compartments across high volumes of multiplex datasets stemming from tissue atlasing efforts. Here, we present MILWRM - multiplex image labeling with regional morphology - a Python package for rapid, multi-scale tissue domain detection and annotation. We demonstrate MILWRMs utility in identifying histologically distinct compartments in human colonic polyps and mouse brain slices through spatially-informed clustering in two different spatial data modalities. Additionally, we used tissue domains detected in human colonic polyps to elucidate molecular distinction between polyp subtypes. We also explored the ability of MILWRM to identify anatomical regions of mouse brain and their respective distinct molecular profiles.

systems biology↗

bioRxiv2022

Inhibition of the BMP pathway suppresses tumor growth via downregulation of EGFR in MEK/ERK-dependent colorectal cancer

The bone morphogenetic protein (BMP) pathway promotes differentiation and induces apoptosis in normal colorectal epithelial cells. However, the effect of the BMP pathway in colorectal cancer (CRC) is controversial; it can either be tumor promoting or tumor suppressing, depending on the study. In this study, we found that CRC cells reside in a BMP-rich environment based on RNA-sequencing database analysis. Suppression of BMP using a specific BMP inhibitor, LDN193189, suppresses the growth of organoids in some CRC cases. CRC organoids treated with LDN193189 exhibited a decrease in epidermal growth factor receptor, which was, at least in part, mediated by protein degradation induced by leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1). Among CRC organoid panels from 18 different patients, suppression of organoid growth by BMP inhibition correlated with the induction of LRIG1 gene expression. Notably, knockdown of LRIG1 in organoids diminished the growth-suppressive effect of LDN193189. Furthermore, simultaneous treatment with LDN192189 and trametinib, an FDA-approved MEK inhibitor, resulted in a combination effect in both in vivo and in vitro xenograft tumor treatment in CRC organoids, which are susceptible to growth suppression by LDN193189. Taken together, the simultaneous inhibition of BMP and MEK can be a novel treatment option in CRC cases, and evaluating in vitro growth suppression and LRIG1 induction by BMP inhibition using patient-derived organoids could offer functional biomarkers for predicting potential responders.

cancer biology↗

bioRxiv2022

Lrig3 restricts the size of the colon stem cell compartment

The cellular census of the colonic crypt is tightly regulated, yet the molecular mechanisms that regulate this census are not fully understood. Lrig3, a transmembrane protein, is expressed in colonic crypt epithelial cells, including the stem, progenitor, and differentiated cell types. Mice missing Lrig3 have a disruption in their cellular census: using a novel Lrig3-/- mouse we demonstrate that Lrig3-/- mice have more cells per crypt, a greater mucosal area, and longer colons compared to wildtype mice, suggesting the expression of Lrig3 is required for both the total number of epithelial cells in the mouse colon, as well as colon length. In addition, we show Lrig3-/- mice have significantly more stem, progenitor, and deep crypt secretory cells, yet harbor a normal complement of enteroendocrine, Tuft, and absorptive cells. Lrig3-/- mice also have a concomitant decrease in phosphorylated Extracellular signal-related kinases, indicating the loss of Lrig3 leads to an expansion of the colonic stem cell compartment, in an Erk-dependent manner. Our study describes the expression of Lrig3 within the colon, defines perturbations in mice lacking Lrig3, and supports a role for Lrig3 in the establishment of both colonic crypt structure and cellular census, defined as the epithelial cell type and number in colon crypts. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/483523v3_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@244c29org.highwire.dtl.DTLVardef@1045457org.highwire.dtl.DTLVardef@13d4134org.highwire.dtl.DTLVardef@83db4e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Quantifying and correcting slide-to-slide variation in multiplexed immunofluorescence images

MotivationThe multiplexed imaging domain is a nascent single-cell analysis field with a complex data structure susceptible to technical variability that disrupts inference. These in situ methods are valuable in understanding cell-cell interactions, but few standardized processing steps or normalization techniques of multiplexed imaging data are available. ResultsWe implement and compare data transformations and normalization algorithms in multiplexed imaging data. Our methods adapt the ComBat and functional data registration methods to remove slide effects in this domain, and we present an evaluation framework to compare the proposed approaches. We present clear slide-to-slide variation in the raw, unadjusted data, and show that many of the proposed normalization methods reduce this variation while preserving and improving the biological signal. Further, we find that dividing this data by its slide mean, and the functional data registration methods, perform the best under our proposed evaluation framework. In summary, this approach provides a foundation for better data quality and evaluation criteria in the multiplexed domain. Availability and ImplementationSource code is provided at https://github.com/statimagcoll/MultiplexedNormalization. Contactcoleman.r.harris@vanderbilt.edu Supplementary informationSupplementary information is available online.

bioinformatics↗

bioRxiv2021

MCMICRO: A scalable, modular image-processing pipeline for multiplexed tissue imaging

Highly multiplexed tissue imaging makes molecular analysis of single cells possible in a preserved spatial context. However, reproducible analysis of the underlying data poses a substantial computational challenge. Here we describe a modular and open-source computational pipeline (MCMICRO) for performing the sequential steps needed to transform large, multi-channel whole slide images into single-cell data. We demonstrate use of MCMICRO on images of different tissues and tumors acquired using multiple imaging platforms, thereby providing a solid foundation for the continued development of tissue imaging software.

bioinformatics↗

bioRxiv2021

Human colorectal pre-cancer atlas identifies distinct molecular programs underlying two major subclasses of pre-malignant tumors

Most colorectal cancers (CRCs) develop from either adenomas (ADs) or sessile serrated lesions (SSLs). The origins and molecular landscapes of these histologically distinct pre-cancerous polyps remain incompletely understood. Here, we present an atlas at single-cell resolution of sporadic conventional tubular/tubulovillous ADs, SSLs, hyperplastic polyps (HPs), microsatellite stable (MSS) and unstable (MSI-H) CRC, and normal colonic mucosa. Using single-cell transcriptomics and multiplex imaging, we studied 69 datasets from 33 participants. We also examined separate sets of 66 and 274 polyps for RNA and targeted gene sequencing, respectively. We performed multiplex imaging on a tissue microarray of 14 ADs and 15 CRCs, and we integrated pre-cancer polyp data with published single-cell and The Cancer Genome Atlas (TCGA) bulk CRC data to establish potential polyp-cancer relationships. Striking differences were observed between ADs and SSLs that extended to MSS and MSI-H CRCs, respectively, reflecting their distinct origins and trajectories. ADs arose from WNT pathway dysregulation in stem cells, which aberrantly expanded and expressed a Hippo and ASCL2 regenerative program. In marked contrast, SSLs were depleted of stem cell-like populations and instead exhibited a program of gastric metaplasia in the setting of elevated cytotoxic inflammation. Using subtype-specific gene regulatory networks and shared genetic variant analysis, we implicated serrated polyps, including some HPs conventionally considered benign, as arising from a metaplastic program in committed absorptive cells. ADs and SSLs displayed distinct patterns of immune cell infiltration that may influence their natural history. Our multi-omic atlas provides novel insights into the malignant potential of colorectal polyps and serves as a framework for precision surveillance and prevention of sporadic CRC.

cancer biology↗