Biology subjects

Rogers, K. L.

Publications and source records attributed to Rogers, K. L..

12 recordsLinked to original sources

bioRxiv2026

CXCL9-Dependent Immune Networks of Spontaneous Tertiary Lymphoid Structures in a Murine Subcutaneous Tumor Model

Recent discoveries have identified human tumor tertiary lymphoid structures (TLS) as organized clusters of innate and adaptive immune cells and stromal cells. Cancer-associated TLS can promote tumor clearance and enhance responses to immune checkpoint blockade (ICB) immunotherapy. However, the absence of TLS in conventional mouse tumor models, particularly subcutaneous transplant models, limits experimental manipulation and study of TLS development. By tuning tumor antigen expression, we have generated a subcutaneous tumor model that naturally forms TLS within the immune-hot, vasculature-rich peritumoral adipose tissue. These TLS were identified as clusters of B and T cells in close proximity to high endothelial venules. TLS organization was underpinned by spatially compartmentalized chemokine expression, with CCL21 produced in the T cell zone of perivascular TLS and CXCL13 present in the B cell region. Single cell transcriptomic sequencing showed that the immune composition in the TLS forming peritumoral tissue resembled human cancer TLS signatures. Upon anti-PD-L1 ICB treatment, TLS underwent expansion, which was associated with increased interferon responses in macrophages and coincided with tumor clearance. We further identified the interferon-inducible chemokine CXCL9 as a key regulator of the TLS microenvironment. CXCL9-expressing macrophages populated TLS, interacting with B cells following ICB treatment. CXCL9 deficiency reduced B cell recruitment to peritumoral tissue and TLS formation. Further T and B cell receptor sequencing indicated a CXCL9-dependent promotion of antigen-specific responses within the tumor and TLS niche. Combined, these findings establish a tractable subcutaneous model of naturally arising tumor TLS and define the cellular and molecular networks that regulate their formation and function. This provides a platform to dissect how TLS develop and test strategies that promote TLS formation to enhance cancer therapy.

immunology↗

bioRxiv2026

SpatialBench: Comparative cross-platform benchmarking of high-resolution spatial transcriptomics using matched mouse lymphoid tissue

Spatial transcriptomics (ST) has rapidly expanded with the introduction of multiple high-resolution platforms, yet cross-platform benchmarking remains limited and largely focused on technical performance. Here we present SpatialBench, a matched multi-platform resource comprising Visium HD, Xenium and MERSCOPE data together with single-cell and single-nucleus references from a malaria-challenged wild-type and B cell-specific Tbx21 knockout mouse spleen model. In this system, loss of T-bet in B cells disrupts germinal center (GC) polarization and antibody maturation, providing a biologically grounded benchmark for technology comparison. We leveraged this system to systematically evaluate ST platform performance using technical and biological readouts. Across platforms, immune organization and Tbx21-associated programs were consistently recovered, indicating robustness of major biological signals. Platforms instead differed in the level of biological resolution accessible. Visium HD enabled transcriptome-scale GC characterization and, together with Xenium, resolved dark and light zone organization, whereas GC zonation was not resolved in MERSCOPE, consistent with differences in transcript detection sensitivity. SpatialBench provides a biologically defined reference dataset for evaluation of ST technologies, method development, computational benchmarking, and studies of GC spatial organization in lymphoid tissue.

bioinformatics↗

bioRxiv2026

Formation of the moving junction is the nexus for host cytoskeletal remodelling during Plasmodium falciparum invasion of human erythrocytes

Plasmodium falciparum invasion of human erythrocytes is a complex and tightly coordinated process, involving host cell attachment, moving junction formation and engagement of the parasites actomyosin motor. The temporal precision of these events is mediated by distinct ligand-receptor interactions and the sequential release of the merozoites apical organelles. What remains unclear is how these molecular and biophysical interactions enable Plasmodium to bypass the stable erythrocyte membrane-cytoskeletal complex. Here, several P. falciparum lines expressing different fluorescently tagged apical organelle proteins, were imaged with lattice light sheet microscopy (LLSM) to determine the timing of cytoskeletal disassembly and apical organelle release. Blocking the AMA1-RON2 interaction has no effect on the PfRh5-basigin Ca2+ flux but prevents host cytoskeleton disassembly. In contrast, the inhibition of parasite actin polymerisation had no effect on cytoskeletal clearance but caused a sustained Ca2+ response. We further demonstrate that establishment of the moving junction is temporally linked to clearance of the host cytoskeleton. Collectively, our findings support the existence of an association between the RON complex and components of the host cytoskeleton, which mediates the localised disruption of the erythrocyte-membrane cytoskeletal complex during invasion.

microbiology↗

bioRxiv2026

MMV687794 blocks Plasmodium falciparum invasion of red blood cells by targeting a Surface-associated Lipid-Interacting Rhoptry Protein, PfSLIRP

Invasion of red blood cells (RBCs) by the human malaria parasite, Plasmodium falciparum, drives disease. During invasion, the parasite pushes its way into the RBC while wrapping the RBC membrane around itself to establish the parasitophorous vacuole, a stable niche within the RBC where the parasite grows. To better understand invasion, we investigated the mechanism of action of an invasion-inhibitory compound, MMV687794 (MMV794). Lattice light-sheet microscopy revealed that MMV794 blocks parasite entry by preventing parasitophorous vacuole formation. In vitro drug resistance selection of parasites with MMV794 found mutations to the /{beta} hydrolase, PF3D7_0403800, and engineering one of these mutations (C36W) into parasites by CRISPR/Cas9 recapitulated the resistance phenotype. Expansion microscopy demonstrated that this protein is expressed in schizonts, localising to the surface of rhoptries, which are specialised apical secretory organelles that function during invasion. Lipidomics and proteomics analyses of C36W parasites uncovered widespread changes to lipid composition/homeostasis and altered abundance of proteins involved in invasion, indicating a role for PF3D7_0403800 in invasion-associated lipid metabolism. Finally, we used solvent-induced proteome profiling and reactivity assays to confirm drug-target engagement. Together, our findings identify a novel Surface-associated Lipid-Interacting Rhoptry Protein (PfSLIRP) that coordinates lipid metabolism at the rhoptries to enable RBC invasion.

microbiology↗

bioRxiv2025

Genetic barcoding uncovers the clonal makeup of solid and liquid biopsies

Intra-tumoral heterogeneity (ITH) is fueling tumor progression in breast cancer, as specific clones present within a tumor may have a selective advantage to colonize distant organs and escape therapy. Accurate sampling of ITH is therefore a pressing challenge in clinical oncology, to adequately predict recurrence and inform rational and personalized therapies. Here we used cellular barcoding to track the spatio-temporal composition of human breast cancer clones in six preclinical models - across two cell lines and four patient derived xenografts (PDXs). This allowed direct side-by-side quantitative comparison not only of intra-tumor clonal composition, but also of how that composition was reflected in needle biopsies and cell-free DNA (cfDNA). These analyses highlighted several clinically relevant findings. First, the use of orthogonal genetic and optical barcoding revealed that clonal diversity in the center of non-necrotic primary tumors was significantly higher compared to their periphery. Second, cfDNA barcode analysis suggested that DNA shedding in the vasculature varied largely, not only depending on necrosis and tumor burden, but also between models. Third, combining information captured in both solid and liquid biopsies can provide a more robust assessment of tumor clonal composition. Taken together, these results showcases the utility of these barcoded models to optimize the use of solid and liquid biopsies as surrogate markers of ITH.

cancer biology↗

bioRxiv2024

A necroptotic-to-apoptotic signaling axis underlies inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic condition caused by altered cytokine signaling, maladaptive immunity, dysbiosis, and intestinal barrier dysfunction. Patients with IBD receive therapy to correct these imbalances and achieve remission. However, most patients relapse, suggesting that pathological mechanisms persist during remission. Here, we show that excess epithelial cell death is an underlying feature of IBD that arises in patients in remission and on advanced therapy. Mechanistically, nascent inflammation reprograms epithelial cells into a macrophage-like state that promotes RIPK1-independent necroptotic signaling, then triggers iNOS-mediated mitochondrial apoptosis of absorptive epithelial cells and PUMA-mediated intestinal stem cell death. These findings reveal aberrant epithelial cell death signaling as a hallmark of IBD that occurs early in mucosal lesion development and persists despite current therapeutic approaches. One-Sentence SummaryEpithelial cell death is dysregulated in patients with inflammatory bowel disease.

cell biology↗

bioRxiv2024

Myeloid-instructed CD14+CD4+ T cells within the tumor microenvironment are associated with TNFα signaling and prolonged survival in non-small cell lung cancer

The tumor microenvironment (TME) harbors a diverse array of innate and adaptive immune cells that can either support anti-tumor immunity or facilitate tumor progression. Among these, myeloid cells are key drivers of immunosuppression, yet therapeutic strategies targeting their function have achieved limited clinical success. A deeper understanding of the mechanisms by which immunosuppressive myeloid cells promote tumor progression is needed to guide the development of more effective treatments. Using spatial multi-omics analyses, we identified a population of myeloid-instructed CD14+ T cells present in both the tumor core and adjacent non-malignant lung tissue of patients with non-small cell lung cancer (NSCLC). CD4+ T cells acquired CD14 from myeloid cells by trogocytosis, resulting in an atypical T cell phenotype. High infiltration of CD14+CD4+ T cells in the tumor core was associated with poor patient survival. Spatial transcriptomics profiling revealed that tumors enriched in CD14+CD4+ T cells exhibited increased TNF signaling. Functional assays demonstrated that TNF enhanced trogocytosis, promoting the transfer of myeloid membrane components to CD4{square} T cells and driving the accumulation of CD14{square}CD4{square} T cells. These findings uncover a novel TNF-mediated mechanism of immunosuppression in the TME, whereby TNF promotes aberrant myeloid-T cell interactions that may contribute to tumor progression. This work highlights a previously unrecognized axis of innate-adaptive immune crosstalk in NSCLC and suggests that targeting TNF could disrupt this pathway, restore effective T cell function, and improve therapeutic outcomes.

cancer biology↗

bioRxiv2024

Spotlight on 10x Visium: a multi-sample protocol comparison of spatial technologies

BackgroundSpatial transcriptomics allows gene expression to be measured within complex tissue contexts. Among the array of spatial capture technologies available is 10x Genomics Visium platform, a popular method which enables transcriptomewide profiling of tissue sections. Visium offers a range of sample handling and library construction methods which introduces a need for benchmarking to compare data quality and assess how well the technology can recover expected tissue features and biological signatures. ResultsHere we present SpatialBench, a unique reference dataset generated from spleen tissue of mice responding to malaria infection spanning several tissue preparation protocols (both fresh frozen and FFPE samples, with and without CytAssist tissue placement). We noted better quality control metrics in reference samples prepared using probe-based capture methods, particularly those processed with CytAssist, validating the improvement in data quality produced with the platform. Our analysis of replicate samples extends to explore spatially variable gene detection, the outcomes of clustering and cell deconvolution using matched single-cell RNA-sequencing data and publicly available reference data to identify cell types and tissue regions expected in the spleen. Multi-sample differential expression analysis recovered known gene signatures related to biological sex or gene knockout. ConclusionsWe framed a comprehensive multi-sample analysis workflow that allowed us to generate consistent results both within and between different subsets of replicate samples, enabling broader comparisons and interpretations to be made at the group-level. Our SpatialBench dataset, analysis, and workflow can serve as a practical guide for Visium users and may prove valuable in other benchmarking studies.

genomics↗

bioRxiv2020

A toolbox for imaging RIPK1, RIPK3 and MLKL in mouse and human cells

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3 and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol-is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3 and MLKL signals.

cell biology↗

bioRxiv2020

Chromosomes distribute randomly to, but not within, human neutrophil nuclear lobes

The proximity pattern and radial distribution of chromosome territories within spherical nuclei are well understood to be random and non-random, respectively. Whether this distribution pattern is conserved in the partitioned or lobed nuclei of polymorphonuclear cells is unclear. Here we use chromosome paint technology and a novel high-throughput imaging analysis pipeline to examine the chromosome territories of all 46 chromosomes in hundreds of single human neutrophils - an abundant and famously polymorphonuclear immune cell. By comparing the distribution of chromosomes to randomly shuffled controls, and validating with orthogonal chromosome conformation capture technology, we show for the first time that all human chromosomes randomly distribute to neutrophil nuclear lobes, while maintaining a non-random radial distribution within these lobes. Furthermore, by leveraging the power of this vast dataset, we are able to reveal characteristics of chromosome territories not detected previously. For example, we demonstrate that chromosome length correlates with three-dimensional volume not only in neutrophils but other human immune cells. This work demonstrates that chromosomes are largely passive passengers during the neutrophil lobing process, but are able to maintain their macro-level organisation within lobes. Furthermore, the random distribution of chromosomes to the naturally partitioned nuclear lobes suggests that specific transchromosomal interactions are unimportant in mature neutrophils.

immunology↗

bioRxiv2020

Macrophages provide a transient muscle stem cell niche via NAMPT secretion

Skeletal muscle is paradigmatic of a regenerative tissue that repairs itself via the activation of a resident stem cell1. Termed the satellite cell, these normally quiescent cells are induced to proliferate by ill-defined wound-derived signals2. Identifying the source and nature of these pro-regenerative cues has been hampered by an inability to visualise the complex cellular interactions that occur within the wound environment. We therefore developed a zebrafish muscle injury model to systematically capture satellite cell interactions within the injury site, in real time, throughout the repair process. This analysis identified that a specific subset of macrophages dwells within the injury, establishing a transient but obligate stem cell niche required for stem cell proliferation. Single cell profiling identified specific signals secreted from dwelling macrophages that include the cytokine, Nicotinamide phosphoribosyltransferase (NAMPT/Visfatin/PBEF). Here we show that NAMPT secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (CCR5) expressed on muscle stem cells. This analysis reveals that along with their well-described ability to modulate the pro-inflammatory and anti-inflammatory phases of wound repair, specific macrophage populations also provide a transient stem cell-activating niche, directly supplying pro-proliferative cues that govern the timing and rate of muscle stem cell-mediated repair processes.

cell biology↗

bioRxiv2019

Quantitative high-resolution imaging of live microbial cells at high hydrostatic pressure

The majority of the Earths microbial biomass exists in the Deep Biosphere, in the deep ocean and within the Earths crust. While other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high-pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold pressure on the Earths surface. The mechanisms of pressure adaptation are also important to in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here we describe implementation of a high-pressure sample chamber with a 2-photon scanning microscope system allowing for the first time, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for Fluorescence Lifetime Imaging Microscopy with Phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon, Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning Number and Brightness (sN&B) and Raster scanning Imaging Correlation Spectroscopy (RICS)) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in E. coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. SIGNIFICANCEThe majority of the Earths microbial biomass exists in high-pressure environments where pressures can reach over 100 MPa. The molecular mechanisms that allow microbes to flourish under such extreme conditions remain to be discovered. The high pressure, high resolution imaging system presented here revealed pressure dependent changes in metabolism and protein interactions in live microbial cells, demonstrating great promise for understanding deep life.

microbiology↗