Biology subjects

Southard-Smith, A. N.

Publications and source records attributed to Southard-Smith, A. N..

7 recordsLinked to original sources

bioRxiv2023

Differential chromatin accessibility and transcriptional dynamics define breast cancer subtypes and their lineages

Breast cancer is a heterogeneous disease, and treatment is guided by biomarker profiles representing distinct molecular subtypes. Breast cancer arises from the breast ductal epithelium, and experimental data suggests breast cancer subtypes have different cells of origin within that lineage. The precise cells of origin for each subtype and the transcriptional networks that characterize these tumor-normal lineages are not established. In this work, we applied bulk, single-cell (sc), and single-nucleus (sn) multi-omic techniques as well as spatial transcriptomics and multiplex imaging on 61 samples from 37 breast cancer patients to show characteristic links in gene expression and chromatin accessibility between breast cancer subtypes and their putative cells of origin. We applied the PAM50 subtyping algorithm in tandem with bulk RNA-seq and snRNA-seq to reliably subtype even low-purity tumor samples and confirm promoter accessibility using snATAC. Trajectory analysis of chromatin accessibility and differentially accessible motifs clearly connected progenitor populations with breast cancer subtypes supporting the cell of origin for basal-like and luminal A and B tumors. Regulatory network analysis of transcription factors underscored the importance of BHLHE40 in luminal breast cancer and luminal mature cells, and KLF5 in basal-like tumors and luminal progenitor cells. Furthermore, we identify key genes defining the basal-like (PRKCA, SOX6, RGS6, KCNQ3) and luminal A/B (FAM155A, LRP1B) lineages, with expression in both precursor and cancer cells and further upregulation in tumors. Exhausted CTLA4-expressing CD8+ T cells were enriched in basal-like breast cancer, suggesting altered means of immune dysfunction among breast cancer subtypes. We used spatial transcriptomics and multiplex imaging to provide spatial detail for key markers of benign and malignant cell types and immune cell colocation. These findings demonstrate analysis of paired transcription and chromatin accessibility at the single cell level is a powerful tool for investigating breast cancer lineage development and highlight transcriptional networks that define basal and luminal breast cancer lineages.

cancer biology↗

bioRxiv2022

A contamination focused approach for optimizing thesingle-cell RNA-seq experiment

Achieving high data quality in single-cell RNA-seq (scRNA-seq) experiments has always been a significant challenge stemming from minute signal that can be detected in individual cells. Droplet-based scRNA-seq additionally suffers from ambient contamination, comprising nucleic acid materials released by dead cells into the loading buffer and co-encapsulated with real cells, which further washes out real biological signals. Here, we developed quantitative, ambient contamination-based metrics and an associated software package that can both evaluate current datasets and guide new experimental optimizations. We performed a series of experimental optimizations using the inDrops platform to address the mechanical and microfluidic cell encapsulation aspect of an scRNA-seq experiment, with a focus on minimizing ambient contamination. We report improvements that can be achieved via cell fixation, microfluidic loading, microfluidic dilution, and nuclei versus cell preparation; many of these parameters are inaccessible on commercial platforms. We provide insights into previously obscured factors that can affect scRNA-seq data quality and suggest mitigation strategies that can guide future experiments.

genomics↗

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↗

bioRxiv2020

Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBACKGROUND & AIMSC_ST_ABSThe enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (EN). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions. METHODSHuman myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA-Seq. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA-Seq (snRNA-Seq), totaling over four billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse snRNA-Seq data. Comparative informatics between human and mouse datasets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction (HCR). RESULTSSeveral EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of one or more marker genes. CONCLUSIONSOur combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human GI studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/187211v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1b41277org.highwire.dtl.DTLVardef@12856feorg.highwire.dtl.DTLVardef@1674dadorg.highwire.dtl.DTLVardef@11ca1c8_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

neuroscience↗

bioRxiv2019

Dual indexed design of in-Drop single-cell RNA-seq libraries improves sequencing quality and throughput

The increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated. Here, we engineered a new dual-indexed library structure, called TruDrop, on top of the inDrop scRNA-seq platform to solve these compatibility challenges, such that TruDrop libraries and standard Illumina libraries can be sequenced alongside each other on the NovaSeq. We overcame the index-hopping issue, demonstrated significant improvements in base-calling accuracy, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior library structures. Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.

molecular biology↗

bioRxiv2019

An Inter-Species Translation Model Implicates Integrin Signaling in Infliximab-Resistant Colonic Crohn’s Disease

Anti-TNF therapy resistance is a major clinical challenge in Crohns Disease (CD), partly due to insufficient understanding of disease-site, protein-level mechanisms of CD and anti-TNF treatment resistance. Although some proteomics data from CD mouse models exists, data type and phenotype discrepancies contribute to confounding attempts to translate between preclinical animal models of disease and human clinical cohorts. To meet this important challenge, we develop and demonstrate here an approach called Translatable Components Regression (TransComp-R) to overcome inter-species and trans-omic discrepancies between CD mouse models and human subjects. TransComp-R combines CD mouse model proteomic data with patient pre-treatment transcriptomic data to identify molecular features discernable in the mouse data predictive of patient response to anti-TNF therapy. Interrogating the TransComp-R models predominantly revealed upregulated integrin pathway signaling via collagen-binding integrin ITGA1 in anti-TNF resistant colonic CD (cCD) patients. Toward validation, we performed single-cell RNA sequencing on biopsies from a cCD patient and analyzed publicly available immune cell proteomics data to characterize the immune and intestinal cell types contributing to anti-TNF resistance. We found that ITGA1 is indeed expressed in colonic T-cell populations and that interactions between collagen-binding integrins on T-cells and colonic cell types expressing secreted collagens are associated with anti-TNF therapy resistance. Biologically, TransComp-R linked previously disparate observations about collagen and ITGA1 signaling to a potential therapeutic avenue for overcoming anti-TNF therapy resistance in cCD. Methodologically, TransComp-R provides a flexible, generalizable framework for addressing inter-species, inter-omic, and inter-phenotypic discrepancies between animal models and patients to deliver translationally relevant biological insights.\n\nOne Sentence SummaryBrubaker et al. implicate dysregulated collagen-binding integrin signaling in resistance to anti-TNF therapy in Crohns Disease by developing a mouse-proteomic to human-transcriptomic translation model and confirm the associated inter-cellular signaling network using single-cell RNA sequencing.

systems biology↗

bioRxiv2019

Commensal-derived succinate enhances tuft cell specification and suppresses ileal inflammation

Longitudinal analysis of Crohn's disease (CD) incidence has identified an inverse correlation with helminth infestation and recent studies have revealed that intestinal tuft cell hyperplasia is critical for helminth response. Tuft cell frequency was decreased in the inflamed ilea of CD patients and a mouse model of TNF-induced Crohn's-like ileitis (TNF{Delta}ARE). Single-cell RNA sequencing paired with unbiased differential trajectory analysis of the tuft cell lineage in a genetic model of tuft cell hyperplasia (AtohKO) demonstrated that the tuft cell lineage had increased tricarboxylic acid (TCA) cycle gene signatures. Commensal microbiome-derived succinate was detected in the ileal lumen of these animals while microbiome depletion suppressed tuft cell hyperplasia. Therapeutic succinate treatment in TNF{Delta}ARE animals reduced pathology in correlation with induced tuft cell specification. We provide evidence implicating the modulatory role of intestinal tuft cells in chronic intestinal inflammation, which could facilitate leveraging this rare and elusive cell type for CD treatment.

cell biology↗