Biology subjects

Kroehling, L.

Publications and source records attributed to Kroehling, L..

12 recordsLinked to original sources

bioRxiv2026

Age-based shifts in multicellular community organization and abundance in HPV-Negative Head and Neck Cancer

Aging represents a fundamental driver of cancer risk and progression, yet the mechanisms by which aging reshapes the tumor microenvironment remain incompletely understood. To interrogate how aging alters cellular and microenvironmental organization in cancer, we developed an integrated single-cell atlas of HPV-negative head and neck squamous cell carcinoma (HNSCC) encompassing 73 patients aged 18-91 years. Using a multi-layered computational framework combining NMF-based module discovery and partial correlation network analysis, we reveal that age-associated differences in tumor composition are driven primarily by selective shifts in cell type abundance and organization rather than widespread age-dependent transcriptional rewiring. We identify an age-enriched cellular community centered on basal-like squamous epithelial states linked to epithelial-mesenchymal transition, matrix-associated myofibroblast-like CAFs, and FOLR2+ macrophages, which secrete immunosuppressive and ECM-remodeling factors. Cross-dataset in-silico validation via TCGA transcriptomes, spatial transcriptomics, and mouse tumor models confirms the correlation of these cell types across patients, specific ligand-receptor usage, and increased proportions of epithelial and stromal cells in aged mouse models. Among epithelial cell-intrinsic age-associated transcriptional changes, midkine (MDK) emerges as a rare but robust tumor-derived signal linked to stromal remodeling, immunosuppression, and invasive epithelial programs. These findings reveal conserved mechanisms of age-dependent cellular reorganization in the tumor microenvironment and identify MDK and associated stromal-remodeling pathways as potential intervention points for age-stratified cancer therapy.

cancer biology↗

bioRxiv2025

Dietary Serine Restriction Impacts H3K27 and H3K4 Methyl Epigenomes to Impede Head and Neck Cancer Cell Plasticity and Tumor Growth

Oral squamous cell carcinoma (OSCC) is an aggressive head and neck malignancy characterized by high morbidity, therapeutic resistance and intratumoral heterogeneity driven by plastic cell states. Given that metabolic inputs can shape cell identities via epigenetic mechanisms, we investigated how metabolism of a non-essential amino acid, serine, affects histone modifications with key roles in cell plasticity: H3K27me3, which represses differentiation genes, and H3K4me3 which activates stemness and epithelial-to-mesenchymal transition (EMT) genes. Using a panel of human OSCC patient-derived cell lines and an orthotopic murine isograft model, we show that OSCC cells depend on exogenous serine for proliferation. Dietary serine deprivation induced de novo serine synthesis with a concomitant increase in -ketoglutarate (KG), a cofactor for KDM6B and KDM5A/B demethylases of H3K27me3 and H3K4me3, respectively. RNA-seq-derived serine deprivation gene signatures revealed activation of keratinization program and suppression of EMT and proliferation genes and tracked with good OSCC patient outcomes in TCGA. Furthermore, CUT & RUN profiling showed site-specific losses of H3K27me3 at differentiation genes and reduction of H3K4me3 at stemness, EMT and cell cycle genes. However, inhibition of KG with 2-hydroxyglutarate was not sufficient to rescue cell proliferation. Instead, genome-wide analysis revealed widespread H3K27me3-H3K4me3 bivalency, with extensive transcriptional repression of proliferation and oncogenic programs. Functionally, serine deprivation impaired orthotopic tumor growth and improved the immune landscape in syngeneic mice. Our studies identify a metabolic serine-KG-KDM-H3K27me3/H3K4me3 bivalency axis that globally reprograms OSCC chromatin as a potential therapeutic strategy to impede tumor plasticity and evolution to advanced disease. ImplicationsThis study identifies the tumor-suppressive role of dietary serine deprivation through epigenetic reprogramming of chromatin in OSCC.

cancer biology↗

bioRxiv2025

LSD1 inhibition corrects dysregulated MHC-I and dendritic cells activation through IFNγ-CXCL9-CXCR3 axis to promote antitumor immunity in HNSCC

Poor infiltration of CD8+ T cells and dysregulated MHC-I confer resistance to anticancer clinical therapies. This study aimed to elucidate the mechanisms of lysine-specific demethylase 1 (LSD1, encoded by KDM1A gene) in antitumor immunity in Head and Neck Squamous cell carcinoma (HNSCC). LSD1 inhibition in syngeneic and chronic tobacco carcinogen-induced HNSCC mice recruited activated dendritic cells (DCs), CD4+ and CD8+ T cells, enriched interferon-gamma (IFN{gamma}) in T cells, CXCL9 in DCs, and CXCR3 in T cells, as evaluated using flow cytometry and single-cell RNA-seq analysis. Humanized HNSCC mice and TCGA data validated the inverse correlation of KDM1A with DC markers, CD8+ T cells, and their activating chemokines. Kdm1a knockout in mouse HNSCC and LSD1 inhibitor treatment to co-culture of human HNSCC cells with human peripheral blood mononuclear cells (PBMCs) resulted in MHC-I upregulation in cancer cells for efficient antigen presentation in tumors. Overall, LSD1 inhibition in tumor cells upregulates MHC class I and induces DCs to produce CXCL9, which in turn activates CD8+ T cells through the CXCL9-CXCR3 axis to produce IFN{gamma}. Finally, we identified a novel mechanism by which LSD1 inhibition promotes the activation of H3K4me2 and its direct interaction with MHC-I to induce antitumor immunity. This may have implications in poorly immunogenic and immunotherapy-resistant cancers. Statement of SignificanceLSD1-mediated unique mechanisms have impact on epigenetic therapy, MHC-I resistant HNSCC therapies, and poor CD8+ and dendritic cell infilterated tumors.

cancer biology↗

bioRxiv2025

A highly resolved integrated transcriptomic atlas of human breast cancers

In this study, we developed an integrated single cell transcriptomic (scRNAseq) atlas of human breast cancer (BC), the largest resource of its kind, totaling > 600,000 cells across 138 patients. Rigorous integration and annotation of publicly available scRNAseq data enabled a highly resolved characterization of epithelial, immune, and stromal heterogeneity within the tumor microenvironment (TME). Within the immune compartment we were able to characterize heterogeneity of CD4, CD8 T cells and macrophage subpopulations. Within the stromal compartment, subpopulations of endothelial cells (ECs) and cancer associated fibroblasts (CAFs) were resolved. Within the cancer epithelial compartment, we characterized the functional heterogeneity of cells across the axes of stemness, epithelial-mesenchymal plasticity, and canonical cancer pathways. Across all subpopulations observed in the TME, we performed a multi-resolution survival analysis to identify epithelial cell states, and immune and stromal cell types, which conferred a survival advantage in both The Cancer Genome Atlas (TCGA), METABRIC, and SCANB. We also identified robust associations between TME composition and clinical phenotypes such as tumor subtype and grade that were not discernible when the analysis was limited to individual datasets, highlighting the need for atlas-based analyses. This atlas represents a valuable resource for further high-resolution analyses of TME heterogeneity within BC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/643025v3_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@4c51d3org.highwire.dtl.DTLVardef@8ffc01org.highwire.dtl.DTLVardef@e9a6f7org.highwire.dtl.DTLVardef@1d0c058_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

A highly resolved integrated single-cell atlas of HPV-negative head and neck cancer

Head and Neck Squamous Cell Carcinomas (HNSCC) are the seventh most prevalent form of cancer and are associated with human papilloma virus infection (HPV-positive) or with tobacco and alcohol use (HPV-negative). HPV-negative HNSCCs have a high recurrence rate, and individual patients responses to treatment vary greatly due to the high level of cellular heterogeneity of the tumor and its microenvironment. Here, we describe a HNSCC single cell atlas, which we created by integrating six publicly available datasets encompassing over 230,000 cells across 54 patients. We contextualized the relationships between existing signatures and cell populations, identified new subpopulations, and show the power of this large-scale resource to robustly identify associations between transcriptional signatures and clinical phenotypes that would not be possible to discover using fewer patients. We reveal a previously undefined myeloid population, sex-associated changes in cell type proportions, and novel interactions between CXCL8-positive fibroblasts and vascular endothelial cells. Beyond our findings, the atlas will serve as a public resource for the high-resolution characterization of tumor heterogeneity of HPV-negative HNSCC.

cancer biology↗

bioRxiv2021

Antigen presentation by type 3 innate lymphoid cells instructs the differentiation of gut microbiota-specific regulatory T cells

The mutualistic relationship of gut-resident microbiota and cells of the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a poised, but largely non-aggressive, immune cell compartment1, 2. Consequences of disturbing this balance, by environmental or genetic factors, include proximal inflammatory conditions, like Crohns disease, and systemic illnesses, both metabolic and autoimmune. One of the means by which this equilibrium is achieved is through induction of both effector and suppressor or regulatory arms of the adaptive immune system. In mice, Helicobacter species induce regulatory (iTreg) and follicular helper (Tfh) T cells in the colon-draining mesenteric lymph nodes under homeostatic conditions, but can instead induce inflammatory Th17 cells when iTreg cells are compromised3, 4. How Helicobacter hepaticus and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here, we investigated which cells and molecular components are required to convey the microbial instruction for the iTreg differentiation program. We found that antigen presentation by cells expressing ROR{gamma}t, rather than by classical dendritic cells, was both required and sufficient for iTreg induction. These ROR{gamma}t+ cells, likely to be type 3 innate lymphoid cells (ILC3) and/or a recently-described population of Aire+ cells termed Janus cells5, require the MHC class II antigen presentation machinery, the chemokine receptor CCR7, and v integrin, which activates TGF-{beta}, for iTreg cell differentiation. In the absence of any of these, instead of iTreg cells there was expansion of microbiota-specific pathogenic Th17 cells, which were induced by other antigen presenting cells (APCs) that did not require CCR7. Thus, intestinal commensal microbes and their products target multiple APCs with pre-determined features suited to directing appropriate T cell differentiation programs, rather than a common APC that they endow with appropriate functions. Our results illustrate the ability of microbiota to exploit specialized functions of distinct innate immune system cells, targeting them to achieve the desired composition of equipoised T cells, thus maintaining tolerance.

immunology↗

bioRxiv2021

c-MAF dependent perivascular macrophages regulate diet induced metabolic syndrome

Macrophages are an essential part of tissue development and physiology. Perivascular macrophages have been described in tissues and appear to play a role in development and disease processes, although it remains unclear what are the key features of these cells. Here, we identify a subpopulation of perivascular macrophages in several organs, characterized by their dependence on the transcription factor c-MAF, displaying non-conventional macrophage markers including LYVE1, Folate receptor 2 and CD38. Conditional deletion of c-MAF in macrophage lineages caused ablation of perivascular macrophages in the brain and altered muscularis macrophages program in the intestine. In the white adipose tissue (WAT), c-MAF deficient perivascular macrophages displayed an altered gene expression profile, which was linked to an increased vascular branching into the tissue. Upon feeding on high fat diet (HFD), mice with c-MAF deficient macrophages showed improved metabolic parameters compared to wild-type mice, including less weight gain, greater glucose tolerance and reduced inflammatory cell profile in WAT. These results define c-MAF as a central regulator of perivascular macrophages cell identity and transcriptional program in vivo and reveal a novel role for this tissue resident macrophage population in the regulation of metabolic syndrome.

immunology↗

bioRxiv2020

RORα enforces stability of the T-helper-17 cell effector program

T helper 17 (Th17) cells regulate mucosal barrier defenses, but also promote multiple autoinflammatory diseases. Although many molecular determinants of Th17 cell differentiation have been described, the transcriptional programs that sustain Th17 cells in vivo remain obscure. The transcription factor ROR{gamma}t is critical for Th17 cell differentiation, but a distinct role of the closely-related ROR, which is co-expressed in Th17 cells, is not known. Here we demonstrate that, although dispensable for Th17 cell differentiation, ROR governs optimal Th17 responses in peripheral tissues. Thus, the absence of ROR in T cells led to significant reductions in both ROR{gamma}t expression and effector function amongst Th17 cells, due to need for cooperative ROR and ROR{gamma}t binding to a newly-identified Rorc enhancer element that is essential for Th17 lineage maintenance in vivo. Altogether, these data point to a non-redundant role of ROR in Th17 lineage maintenance via reinforcement of the ROR{gamma}t transcriptional program.

immunology↗

bioRxiv2019

Selective metabolic redundancy of Gpi1 allows for specific inhibition of inflammatory Th17 cells

SummaryTargeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.

immunology↗

bioRxiv2019

VIP-producing enteric neurons interact with innate lymphoid cells to regulate feeding-dependent intestinal epithelial barrier functions

The intestinal mucosa serves as both a conduit for uptake of food-derived nutrients and microbiome-derived metabolites and as a barrier that prevents tissue invasion by microbes and tempers inflammatory responses to the myriad contents of the lumen. How the intestine coordinates physiological and immune responses to food consumption to optimize nutrient uptake while maintaining barrier functions remains unclear. Here, we describe how a gut neuronal signal triggered by food intake is integrated with intestinal antimicrobial and metabolic responses controlled by type 3 innate lymphoid cells (ILC3)1-3. Food consumption rapidly activates a population of enteric neurons that express vasoactive intestinal peptide (VIP)4. Projections of VIP-producing neurons (VIPergic neurons) in the lamina propria are in close proximity to clusters of ILC3 that selectively express VIP receptor type 2 (VIPR2 or VPAC2). ILC3 production of IL-22, which is up-regulated by commensal microbes such as segmented filamentous bacteria (SFB)5-7, is inhibited upon engagement of VIPR2. As a consequence, there is a reduction in epithelial cell-derived antimicrobial peptide, but enhanced expression of lipid-binding proteins and transporters8. During food consumption, activation of VIPergic neurons thus enhances growth of epithelial-associated SFB and increases lipid absorption. Our results reveal a feeding- and circadian-regulated dynamic intestinal neuro-immune circuit that promotes a trade-off between IL-22-mediated innate immune protection and efficiency of nutrient absorption. Modulation of this pathway may hence be effective for enhancing resistance to enteropathogen2,3,9 and for treatment of metabolic diseases.

immunology↗

bioRxiv2019

Serum Amyloid A Proteins Induce Pathogenic TH17 Cells and Promote Inflammatory Disease

Lymphoid cells that produce IL-17 cytokines protect barrier tissues from pathogenic microbes, but are also prominent effectors of inflammation and autoimmune disease. T-helper (TH17) cells, defined by ROR{gamma}t-dependent production of IL-17A and IL-17F, exert homeostatic functions in the gut upon microbiota-directed differentiation from naive CD4+ T cells. In the non-pathogenic setting, their cytokine production is regulated by serum amyloid A proteins (SAA1 and SAA2) secreted by adjacent intestinal epithelial cells. However, TH17 cell behaviors vary markedly according to their environment. Here we show that SAAs additionally direct a pathogenic pro-inflammatory TH17 cell differentiation program, acting directly on T cells in collaboration with STAT3-activating cytokines. Using loss- and gain-of-function mouse models, we show that SAA1, SAA2, and SAA3 have distinct systemic and local functions in promoting TH17-mediated inflammatory diseases. These studies suggest that T cell signaling pathways modulated by the SAAs may be attractive targets for anti-inflammatory therapies.

immunology↗

bioRxiv2018

Skin inflammation driven by differentiation of quiescent tissue-resident ILCs into a spectrum of pathogenic effectors

Psoriasis pathology is driven by the type 3 cytokines IL-17 and Il-22, but little is understood about the dynamics that initiate alterations in tissue homeostasis. Here, we use mouse models, single-cell RNA-seq (scRNA-seq), computational inference and cell lineage mapping to show that psoriasis induction reconfigures the functionality of skin-resident ILCs to initiate disease. Tissue-resident ILCs amplified an initial IL-23 trigger and were sufficient, without circulatory ILCs, to drive pathology, indicating that ILC tissue remodeling initiates psoriasis. Skin ILCs expressed type 2 cytokines IL-5 and IL-13 in steady state, but were epigenetically poised to become ILC3-like cells. ScRNA-seq profiles of ILCs from psoriatic and naive skin of wild type (WT) and Rag1-/- mice form a dense continuum, consistent with this model of fluid ILC states. We inferred biological \"topics\" underlying these states and their relative importance in each cell with a generative model of latent Dirichlet allocation, showing that ILCs from untreated skin span a spectrum of states, including a naive/quiescent-like state and one expressing the Cd74 and Il13 but little Il5. Upon disease induction, this spectrum shifts, giving rise to a greater proportion of classical Il5- and Il13- expressing \"ILC2s\" and a new, mixed ILC2/ILC3-like subset, expressing Il13, Il17, and Il22. Using these key topics, we related the cells through transitions, revealing a quiescence-ILC2-ILC3s state trajectory. We demonstrated this plasticity in vivo, combining an IL-5 fate mouse with IL-17A and IL-22 reporters, validating the transition of IL-5-producing ILC2s to IL-22- and IL-17A-producing cells during disease initiation. Thus, steady-state skin ILCs are actively repressed and cued for a plastic, type 2 response, which, upon induction, morphs into a type 3 response that drives psoriasis. This suggests a general model where specific immune activities are primed in healthy tissue, dynamically adapt to provocations, and left unchecked, drive pathological remodeling.

immunology↗