Most colorectal cancers (CRCs) develop from either adenomas (ADs) or sessile serrated lesions (SS... more 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, respectivel...
The emerging phenomenon of cellular heterogeneity in tissue requires single-cell resolution studi... more The emerging phenomenon of cellular heterogeneity in tissue requires single-cell resolution studies. A specific challenge for suspension-based single-cell analysis is the preservation of intact cell states when single cells are isolated from tissue contexts, in order to enable downstream analyses to extract accurate, native information. We have developed DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue) coupled to mass cytometry (CyTOF: Cytometry by Time-of-Flight), an experimental approach for profiling intact signaling states of single cells from epithelial tissue specimens. We have previously applied DISSECT-CyTOF to fresh mouse intestinal samples and to Formalin-Fixed, Paraffin-Embedded (FFPE) human colorectal cancer specimens. Here, we present detailed protocols for each of these procedures, as well as a new method for applying DISSECT to cryopreserved tissue slices. We present example data for using DISSECT on a cryopreserved specimen of the human colon to profile its immune and epithelial composition. These techniques can be used for high-resolution studies for monitoring disease-related alternations in different cellular compartments using specimens stored in cryopreserved or FFPE tissue banks.
Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigatio... more Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigation of cellular diversity. As a number of computational tools have been developed to identify and visualize cell populations within a single scRNA-seq dataset, there is a need for methods to quantitatively and statistically define proportional shifts in cell population structures across datasets, such as expansion or shrinkage or emergence or disappearance of cell populations. Here we present sc-UniFrac, a framework to statistically quantify compositional diversity in cell populations between single-cell transcriptome landscapes. sc-UniFrac enables sensitive and robust quantification in simulated and experimental datasets in terms of both population identity and quantity. We have demonstrated the utility of sc-UniFrac in multiple applications, including assessment of biological and technical replicates, classification of tissue phenotypes and regional specification, identification and defi...
Cellular and molecular gastroenterology and hepatology, 2018
Function at the organ level manifests itself from a heterogeneous collection of cell types. Cellu... more Function at the organ level manifests itself from a heterogeneous collection of cell types. Cellular heterogeneity emerges from developmental processes by which multipotent progenitor cells make fate decisions and transition to specific cell types through intermediate cell states. Although genetic experimental strategies such as lineage tracing have provided insights into cell lineages, recent developments in single-cell technologies have greatly increased our ability to interrogate distinct cell types, as well as transitional cell states in tissue systems. From single-cell data that describe these intermediate cell states, computational tools have been developed to reconstruct cell-state transition trajectories that model cell developmental processes. These algorithms, although powerful, are still in their infancy, and attention must be paid to their strengths and weaknesses when they are used. Here, we review some of these tools, also referred to as , and their associated assumpti...
Intestinal tuft cells are a morphologically unique cell type, best characterized by striking micr... more Intestinal tuft cells are a morphologically unique cell type, best characterized by striking microvilli that form an apical tuft. These cells represent approximately 0.5% of gut epithelial cells depending on location. While they are known to express chemosensory receptors, their function has remained unclear. Recently, numerous groups have revealed startling insights into intestinal tuft cell biology. Here, we review the latest developments in understanding this peculiar cell type's structure and function. Recent advances in volumetric microscopy have begun to elucidate tuft cell ultrastructure with respect to its cellular neighbors. Moreover, single-cell approaches have revealed greater diversity in the tuft cell population than previously appreciated and uncovered novel markers to characterize this heterogeneity. Finally, advanced model systems have revealed tuft cells' roles in mucosal healing and orchestrating type 2 immunity against eukaryotic infection. While much rema...
Modern single-cell technologies allow multiplexed sampling of cellular states within a tissue. Ho... more Modern single-cell technologies allow multiplexed sampling of cellular states within a tissue. However, computational tools that can infer developmental cell-state transitions reproducibly from such single-cell data are lacking. Here, we introduce p-Creode, an unsupervised algorithm that produces multi-branching graphs from single-cell data, compares graphs with differing topologies, and infers a statistically robust hierarchy of cell-state transitions that define developmental trajectories. We have applied p-Creode to mass cytometry, multiplex immunofluorescence, and single-cell RNA-seq data. As a test case, we validate cell-state-transition trajectories predicted by p-Creode for intestinal tuft cells, a rare, chemosensory cell type. We clarify that tuft cells are specified outside of the Atoh1-dependent secretory lineage in the small intestine. However, p-Creode also predicts, and we confirm, that tuft cells arise from an alternative, Atoh1-driven developmental program in the colo...
Flow of fluids through the gut, such as milk from a neonatal diet, generates a shear stress on th... more Flow of fluids through the gut, such as milk from a neonatal diet, generates a shear stress on the unilaminar epithelium lining the lumen. We report that exposure to physiological levels of fluid shear stress leads to the formation of large vacuoles, containing extracellular content, within polarizing intestinal epithelial cell monolayers. These observations lead to two questions: how can cells lacking primary cilia transduce shear stress, and what molecular pathways support the formation of vacuoles that can exceed 80% of the cell volume. We find that shear forces are sensed by these cells through actin-rich microvilli that eventually generate the apical brush border, providing one of the first evidence that these structures possess mechanosensing ability. Importantly, we identified the molecular pathway that regulates large vacuole formation downstream of mechanostimulation to involve central components of the autophagy pathway, including ATG5 and LC3, but not Beclin. Together...
Intestinal tuft cells are a rare, poorly understood cell type recently shown to be a critical med... more Intestinal tuft cells are a rare, poorly understood cell type recently shown to be a critical mediator of type 2 immune response to helminth infection. Here, we present advances in segmentation algorithms and analytical tools for multiplex immunofluorescence (MxIF), a platform that enables iterative staining of over 60 antibodies on a single tissue section. These refinements have enabled a comprehensive analysis of tuft cell number, distribution, and protein expression profiles as a function of anatomical location and physiological perturbations. Based solely on DCLK1 immunoreactivity, tuft cell numbers were similar throughout the mouse small intestine and colon. However, multiple subsets of tuft cells were uncovered when protein coexpression signatures were examined, including two new intestinal tuft cell markers, Hopx and EGFR phosphotyrosine 1068. Furthermore, we identified dynamic changes in tuft cell number, composition, and protein expression associated with fasting and refeed...
Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support t... more Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing antimicrobial peptides, including lysozyme and defensins. PC dysfunction is associated with disorders such as Crohn's disease and necrotizing enterocolitis, but the specific pathways regulating PC development and function are not fully understood. Here we tested the role of the neuregulin receptor ErbB3 in control of PC differentiation and the ISC niche. Intestinal epithelial ErbB3 knockout caused precocious appearance of PCs as early as postnatal day 7, and substantially increased the number of mature PCs in adult mouse ileum. ErbB3 loss had no effect on other secretory lineages, but increased expression of the ISC marker Lgr5. ErbB3-null intestines had elevated levels of the Atoh1 transcription factor, which is required for secretory fate determination, while Atoh1(+) cells had reduce...
A systems-level analysis of signaling in the mouse intestine identifies critical mediators of inf... more A systems-level analysis of signaling in the mouse intestine identifies critical mediators of inflammation and regionally specific responses.
Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of sig... more Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single-cell resolution. However, probing signaling in epithelial tissues using cytometry-based single-cell analysis has been confounded by the necessity of single-cell dissociation, where disrupting cell-to-cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue-DISSECT) that preserves native signaling for Cytometry Time-of-Flight (CyTOF) and fluorescent flow cytometry applications. A 21-plex CyTOF analysis encompassing core signaling and cell-identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor-alpha (TNF-α) stimulation. Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF-α-induced apoptosis ...
Most colorectal cancers (CRCs) develop from either adenomas (ADs) or sessile serrated lesions (SS... more 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, respectivel...
The emerging phenomenon of cellular heterogeneity in tissue requires single-cell resolution studi... more The emerging phenomenon of cellular heterogeneity in tissue requires single-cell resolution studies. A specific challenge for suspension-based single-cell analysis is the preservation of intact cell states when single cells are isolated from tissue contexts, in order to enable downstream analyses to extract accurate, native information. We have developed DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue) coupled to mass cytometry (CyTOF: Cytometry by Time-of-Flight), an experimental approach for profiling intact signaling states of single cells from epithelial tissue specimens. We have previously applied DISSECT-CyTOF to fresh mouse intestinal samples and to Formalin-Fixed, Paraffin-Embedded (FFPE) human colorectal cancer specimens. Here, we present detailed protocols for each of these procedures, as well as a new method for applying DISSECT to cryopreserved tissue slices. We present example data for using DISSECT on a cryopreserved specimen of the human colon to profile its immune and epithelial composition. These techniques can be used for high-resolution studies for monitoring disease-related alternations in different cellular compartments using specimens stored in cryopreserved or FFPE tissue banks.
Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigatio... more Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigation of cellular diversity. As a number of computational tools have been developed to identify and visualize cell populations within a single scRNA-seq dataset, there is a need for methods to quantitatively and statistically define proportional shifts in cell population structures across datasets, such as expansion or shrinkage or emergence or disappearance of cell populations. Here we present sc-UniFrac, a framework to statistically quantify compositional diversity in cell populations between single-cell transcriptome landscapes. sc-UniFrac enables sensitive and robust quantification in simulated and experimental datasets in terms of both population identity and quantity. We have demonstrated the utility of sc-UniFrac in multiple applications, including assessment of biological and technical replicates, classification of tissue phenotypes and regional specification, identification and defi...
Cellular and molecular gastroenterology and hepatology, 2018
Function at the organ level manifests itself from a heterogeneous collection of cell types. Cellu... more Function at the organ level manifests itself from a heterogeneous collection of cell types. Cellular heterogeneity emerges from developmental processes by which multipotent progenitor cells make fate decisions and transition to specific cell types through intermediate cell states. Although genetic experimental strategies such as lineage tracing have provided insights into cell lineages, recent developments in single-cell technologies have greatly increased our ability to interrogate distinct cell types, as well as transitional cell states in tissue systems. From single-cell data that describe these intermediate cell states, computational tools have been developed to reconstruct cell-state transition trajectories that model cell developmental processes. These algorithms, although powerful, are still in their infancy, and attention must be paid to their strengths and weaknesses when they are used. Here, we review some of these tools, also referred to as , and their associated assumpti...
Intestinal tuft cells are a morphologically unique cell type, best characterized by striking micr... more Intestinal tuft cells are a morphologically unique cell type, best characterized by striking microvilli that form an apical tuft. These cells represent approximately 0.5% of gut epithelial cells depending on location. While they are known to express chemosensory receptors, their function has remained unclear. Recently, numerous groups have revealed startling insights into intestinal tuft cell biology. Here, we review the latest developments in understanding this peculiar cell type's structure and function. Recent advances in volumetric microscopy have begun to elucidate tuft cell ultrastructure with respect to its cellular neighbors. Moreover, single-cell approaches have revealed greater diversity in the tuft cell population than previously appreciated and uncovered novel markers to characterize this heterogeneity. Finally, advanced model systems have revealed tuft cells' roles in mucosal healing and orchestrating type 2 immunity against eukaryotic infection. While much rema...
Modern single-cell technologies allow multiplexed sampling of cellular states within a tissue. Ho... more Modern single-cell technologies allow multiplexed sampling of cellular states within a tissue. However, computational tools that can infer developmental cell-state transitions reproducibly from such single-cell data are lacking. Here, we introduce p-Creode, an unsupervised algorithm that produces multi-branching graphs from single-cell data, compares graphs with differing topologies, and infers a statistically robust hierarchy of cell-state transitions that define developmental trajectories. We have applied p-Creode to mass cytometry, multiplex immunofluorescence, and single-cell RNA-seq data. As a test case, we validate cell-state-transition trajectories predicted by p-Creode for intestinal tuft cells, a rare, chemosensory cell type. We clarify that tuft cells are specified outside of the Atoh1-dependent secretory lineage in the small intestine. However, p-Creode also predicts, and we confirm, that tuft cells arise from an alternative, Atoh1-driven developmental program in the colo...
Flow of fluids through the gut, such as milk from a neonatal diet, generates a shear stress on th... more Flow of fluids through the gut, such as milk from a neonatal diet, generates a shear stress on the unilaminar epithelium lining the lumen. We report that exposure to physiological levels of fluid shear stress leads to the formation of large vacuoles, containing extracellular content, within polarizing intestinal epithelial cell monolayers. These observations lead to two questions: how can cells lacking primary cilia transduce shear stress, and what molecular pathways support the formation of vacuoles that can exceed 80% of the cell volume. We find that shear forces are sensed by these cells through actin-rich microvilli that eventually generate the apical brush border, providing one of the first evidence that these structures possess mechanosensing ability. Importantly, we identified the molecular pathway that regulates large vacuole formation downstream of mechanostimulation to involve central components of the autophagy pathway, including ATG5 and LC3, but not Beclin. Together...
Intestinal tuft cells are a rare, poorly understood cell type recently shown to be a critical med... more Intestinal tuft cells are a rare, poorly understood cell type recently shown to be a critical mediator of type 2 immune response to helminth infection. Here, we present advances in segmentation algorithms and analytical tools for multiplex immunofluorescence (MxIF), a platform that enables iterative staining of over 60 antibodies on a single tissue section. These refinements have enabled a comprehensive analysis of tuft cell number, distribution, and protein expression profiles as a function of anatomical location and physiological perturbations. Based solely on DCLK1 immunoreactivity, tuft cell numbers were similar throughout the mouse small intestine and colon. However, multiple subsets of tuft cells were uncovered when protein coexpression signatures were examined, including two new intestinal tuft cell markers, Hopx and EGFR phosphotyrosine 1068. Furthermore, we identified dynamic changes in tuft cell number, composition, and protein expression associated with fasting and refeed...
Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support t... more Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing antimicrobial peptides, including lysozyme and defensins. PC dysfunction is associated with disorders such as Crohn's disease and necrotizing enterocolitis, but the specific pathways regulating PC development and function are not fully understood. Here we tested the role of the neuregulin receptor ErbB3 in control of PC differentiation and the ISC niche. Intestinal epithelial ErbB3 knockout caused precocious appearance of PCs as early as postnatal day 7, and substantially increased the number of mature PCs in adult mouse ileum. ErbB3 loss had no effect on other secretory lineages, but increased expression of the ISC marker Lgr5. ErbB3-null intestines had elevated levels of the Atoh1 transcription factor, which is required for secretory fate determination, while Atoh1(+) cells had reduce...
A systems-level analysis of signaling in the mouse intestine identifies critical mediators of inf... more A systems-level analysis of signaling in the mouse intestine identifies critical mediators of inflammation and regionally specific responses.
Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of sig... more Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single-cell resolution. However, probing signaling in epithelial tissues using cytometry-based single-cell analysis has been confounded by the necessity of single-cell dissociation, where disrupting cell-to-cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue-DISSECT) that preserves native signaling for Cytometry Time-of-Flight (CyTOF) and fluorescent flow cytometry applications. A 21-plex CyTOF analysis encompassing core signaling and cell-identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor-alpha (TNF-α) stimulation. Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF-α-induced apoptosis ...
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Papers by Ken Lau