Biology subjects

Sears, C. L.

Publications and source records attributed to Sears, C. L..

10 recordsLinked to original sources

bioRxiv2025

Synergistic role of riboflavin-auxotrophic Enterococcus for MR1 expression and intra-tumoral mucosal associated invariant T (MAIT) cell activation

The role of mucosal invariant T cells (MAITs), in the lung tumor microenvironment re-mains poorly understood, especially in the setting of immune checkpoint inhibitors. We identified intratumoral MAIT cells from paired single cell RNA and TCR sequencing datasets of tumor infil-trating CD3 T cells isolated from non-small cell lung cancer tumors in patients receiving neoadju-vant PD-1 blockade therapy. MAIT cells were subclustered to identify conventional MAIT-associ-ated TCR clonotypes predicted to recognize intratumoral bacteria, which we then tested for func-tional recognition using a MAIT TCR capture functional assay. Strikingly, although not directly recognized by MAIT cells, previously identified probiotic Enterococcus spp and detected in the intratumoral microbiome of lung cancer patients, selectively synergized with exogenous riboflavin biosynthesis-derived metabolites to induce expression of MR1 by antigen presenting cells, includ-ing dendritic cells, B cells and mononuclear phagocytes. Boosting of MR1 cell surface expression resulted from perturbation of endo-lysosomal vacuolar pathway by Enterococcus and recycling of early endosomal MR1 to the cytoplasmic membrane. Riboflavin auxotrophic Enterococcus spp may therefore exercise their beneficial immunomodulatory functions upon immune checkpoint blockade treatment, at least in part, by promoting intratumoral MR1 expression and innate like T cell activation. Our results indicate that composition of the intratumoral microbiome during im-mune checkpoint inhibitor treatment has the potential to impact the function of human intratumoral MAIT cells.

immunology↗

bioRxiv2025

Antibiotic-induced microbiota depletion impairs the pro-regenerative response to a biological scaffold in mice

Therapeutic biological scaffolds promote tissue repair primarily through the induction of type 2 immunity. However, systemic immunological factors-including aging, sex, and previous infections-can modulate this response. The gut microbiota is a well-established modulator of immune function across organ systems, yet its influence on type 2-mediated repair remains underexplored. Here, we establish a bidirectional relationship between the gut microbiota and biological scaffold-mediated tissue repair. Utilizing a conventionalized germ-free mouse, we demonstrate that scaffold implantation induces compositional and functional changes in the gut microbiome, particularly affecting amino acid biosynthesis. Additionally, in a model of antibiotic-induced microbiota depletion (AIMD), we show that dysbiosis disrupts key immune regulators of type 2 immunity, including reductions in eosinophils, pro-regenerative macrophages, and IL-4-producing CD4+ T cells. At 6 weeks post-scaffold implantation, we observed a significant decrease in myocytes with centrally located nuclei alongside an upregulation in pro-fibrotic gene expression with antibiotic treatment. These findings provide insights into the influence of the gut microbiota on type 2-mediated tissue repair. Significance StatementAntibiotics are routinely administered perioperatively to prevent infection during surgeries and biomaterial implantation. Here, we demonstrate that antibiotic-induced microbiota depletion disrupts the type 2 immune response critical for biomaterial-mediated tissue repair. Our findings highlight the gut microbiota as a determinant of constructive healing and a potential contributor to inter-individual variability in responses to biologic scaffolds.

immunology↗

bioRxiv2025

An Anaerobic Pathogen Rewires Host Metabolism to Fuel Oxidative Growth in the Inflamed Gut

To colonize their host and cause disease, enteric pathogens must deploy their virulence factors to establish distinct nutrient niches. How obligate anaerobic pathogens construct nutrient niches in the densely populated large intestine remains poorly understood. Enterotoxigenic Bacteroides fragilis (ETBF) is considered an obligate anaerobic bacterium and has been implicated in inflammation-associated diseases, including colitis and colorectal cancer. Here we show that ETBF uses its virulence factor, Bacteroides fragilis toxin, to reprogram colonic epithelial cell metabolism to colonize the inflamed gut. Bacteroides fragilis toxin activates colonic epithelial signaling and hijacks the host bile acid recycling pathway, inducing a metabolic shift in the epithelium from oxidative phosphorylation to glycolysis. This shift increases local concentrations of lactate and oxygen, nutrients that support an oxidative metabolism in ETBF. These findings reveal an unexpected strategy by which a pathogenic organism, previously considered to be an obligate anaerobic bacterium, generates and exploits an oxidative niche in the inflamed gut.

microbiology↗

bioRxiv2025

Cooperation Between Genotoxic Bacteria Accelerates Tumorigenesis in a Mouse Model of Colon Carcinogenesis

To identify causal links between gut microbes and tumorigenesis, we colonized groups of germ-free colon tumor-susceptible mice (ApcMin/+;Il10-/-) with 15 cultured human fecal microbiotas from healthy individuals, as well as patients with inflammatory bowel disease and colorectal cancer. The number of colonic tumors in ApcMin/+;Il10-/- mice varied by donor microbiota but not by the health status of the donor. In vitro screens of host cell proliferation, genotoxicity, and inflammation in bacteria-mammalian cell cocultures revealed that genotoxicity best predicted tumorigenic microbes in vivo with genotoxic microbes present in all tested individuals. The genotoxic subset of strains from each donor induced more tumors than the complete community - even when the complete community was not tumorigenic. Combining genotoxic microbes from multiple sources increased tumor number and decreased the time to tumor onset. Together these results suggest that most individuals harbor genotoxic bacterial strains and the balance of genotoxic to protective strains determines the timing and severity of tumorigenesis in vivo.

microbiology↗

bioRxiv2025

Fusobacterium nucleatum is enriched in invasive biofilms in colorectal cancer

Fusobacterium nucleatum is an oral bacterium known to colonize colorectal tumors, where it is thought to play an important role in cancer progression. Recent advances in sequencing and phenotyping of F. nucleatum have revealed important differences at the subspecies level, but whether these differences impact the overall tumor ecology, and tumorigenesis itself, remain poorly understood. In this study, we sought to characterize Fusobacteria in the tumor microbiome of a cohort of individuals with CRC through a combination of molecular, spatial, and microbiologic analyses. We assessed for relative abundance of F. nucleatum in tumors compared to paired normal tissue, and correlated abundance with clinical and pathological features. We demonstrate striking enrichment of F. nucleatum and the recently discovered subspecies animalis clade 2 (Fna C2) specifically in colon tumors that have biofilms, highlighting the importance of complex community partnerships in the pathogenesis of this important organism.

microbiology↗

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↗

bioRxiv2021

Prevalence, persistence, and genetics of antibody responses to protein toxins and virulence factors

Microbial exposures are crucial environmental factors that impact healthspan by sculpting the immune system and microbiota. Antibody profiling via programmable Phage ImmunoPrecipitation Sequencing (PhIP-Seq) provides a high-throughput, costeffective approach for multiplexed detection of exposure and response to thousands of microbial protein products. Here we designed and constructed a library of 95,601 56 amino acid peptide tiles spanning a subset of environmental proteins more likely to be associated with immune responses: those with "toxin" or "virulence factor" keyword annotations. PhIP-Seq was used to profile the circulating antibodies of ~1,000 individuals against this "ToxScan" library of 14,430 toxins and virulence factors from 1,312 genera of organisms. In addition to a detailed analysis of six commonly encountered human commensals and pathogens, we study the age-dependent stability of the ToxScan profile and use a genome-wide association study (GWAS) to find that the MHC-II locus modulates the selection of bacterial epitopes. We detect previously described anti-flagellin antibody responses in a Crohns disease cohort and identify a novel association between anti-flagellin antibodies and juvenile dermatomyositis (JDM). PhIP-Seq with the ToxScan library provides a new window into exposure and immune responses to environmental protein toxins and virulence factors, which can be used to study human health and disease at cohort scale.

immunology↗

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

Clinically adaptable polymer enables simultaneous spatial analysis of colonic tissues and biofilms

Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multi-modal analyses of both microbes and host cells in a spatially-resolved fashion would enable studies into these complex interactions in native tissue environments, potentially in clinical specimens. While techniques to preserve each of the microbial and host cell compartments have been used to examine tissues and microbes separately, we endeavored to develop approaches to simultaneously analyze both compartments. Herein, we established an original method for mucus preservation using Poloxamer 407 (also known as Pluronic F-127), a thermoreversible polymer with mucus-adhesive characteristics. We demonstrate that this approach can preserve spatially-defined compartments of the mucus bi-layer in the colon and the bacterial communities within, compared with their marked absence when tissues were processed with traditional formalin-fixed paraffin-embedded (FFPE) pipelines. Additionally, antigens for antibody staining of host cells were preserved and signal intensity for 16S rRNA fluorescence in situ hybridization (FISH) was enhanced in Poloxamer-fixed samples. This in turn enabled us to integrate multi-modal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and crosslink at room temperature for use in clinical workflows. These results suggest that the fixative formulation of Poloxamer 407 can be integrated into biospecimen collection pipelines for simultaneous analysis of microbes and host cells.

cell biology↗

bioRxiv2018

Epigenetic changes induced by Bacteroides fragilis toxin

Enterotoxigenic Bacteroides fragilis (ETBF) is a gram negative, obligate anaerobe member of the gut microbial community in up to 40% of healthy individuals. This bacterium is found more frequently in people with colorectal cancer (CRC) and causes tumor formation in the distal colon of mice heterozygous for the adenomatous polyposis coli gene (Apc+/-); tumor formation is dependent on ETBF-secreted Bacteroides fragilis toxin (BFT). Though some of the immediate downstream effects of BFT on colon epithelial cells (CECs) are known, we still do not understand how this potent exotoxin causes changes in CECs that lead to tumor formation and growth. Because of the extensive data connecting alterations in the epigenome with tumor formation, initial experiments attempting to connect BFT-induced tumor formation with methylation in CECs have been performed, but the effect of BFT on other epigenetic processes, such as chromatin structure, remains unexplored. Here, the changes in chromatin accessibility (ATAC-seq) and gene expression (RNA-seq) induced by treatment of HT29/C1 cells with BFT for 24 and 48 hours is examined. Our data show that several genes are differentially expressed after BFT treatment and these changes correlate with changes in chromatin accessibility. Also, sites of increased chromatin accessibility are associated with a lower frequency of common single nucleotide variants (SNVs) in CRC and with a higher frequency of common differentially methylated regions (DMRs) in CRC. These data provide insight into the mechanisms by which BFT induces tumor formation. Further understanding of how BFT impacts nuclear structure and function in vivo is needed.\n\nImportanceColorectal cancer (CRC) is a major public health concern; there were approximately 135,430 new cases in 2017, and CRC is the second leading cause of cancer-related deaths for both men and women in the US (1). Many factors have been linked to CRC development, the most recent of which is the gut microbiome. Pre-clinical models support that enterotoxigenic Bacteroides fragilis (ETBF), among other bacteria, induce colon carcinogenesis. However, it remains unclear if the virulence determinants of any pro-carcinogenic colon bacterium induce DNA mutations or changes that initiate clonal CEC expansion. Using a reductionist model, we demonstrate that BFT rapidly alters chromatin structure and function consistent with capacity to contribute to CRC pathogenesis.

genomics↗