Biology subjects

Anderson, A.

Publications and source records attributed to Anderson, A..

At least 19 recordsLinked to original sources

bioRxiv2026

CAR T cell targeting of inflammatory myeloid progenitors in the bone marrow remodels border-associated macrophages and reverses cognitive aging

Aging is associated with progressive neuroinflammation and cognitive decline, yet the cellular sources that sustain this process and whether they can be targeted peripherally remain unclear. Here, we identify inflammatory border-associated macrophages (BAMs) as key drivers of neuroinflammation in aging and show that their therapeutic and prophylactic elimination through intrathecal CAR T cell therapy restores cognitive performance in mouse models of aging and Alzheimer's disease. Furthermore, targeting of aged inflammatory bone marrow myeloid progenitors through either intravenous CAR T cells, which do not infiltrate the brain, or through transplantation of CAR T-treated progenitors is sufficient to reduce neuroinflammation and cognitive impairment. These findings reveal that bone marrow myeloid progenitors harbor a heritable inflammatory transcriptional state that is transmitted to their BAM progeny, driving neuroinflammation and cognitive deterioration, and conserved in human aging. Critically, this proinflammatory state is marked by the upregulation of surface proteins, enabling precise peripheral CAR T cell targeting of these progenitors for long-lasting therapeutic effects in cognitive aging.

cell biology↗

bioRxiv2026

Breaking the link between demyelination and axon loss: SARM1 inhibition as a neuroprotective strategy for multiple sclerosis

Axonal degeneration is a principal driver of irreversible neurological disability in multiple sclerosis (MS), yet current treatments fail to target this neurodegenerative phase directly. Sterile alpha and TIR domain-containing protein 1 (SARM1) has emerged as an executioner of programmed axon destruction and is overexpressed in grey and white matter MS tissue, making its inhibition a promising therapeutic strategy. However, the effects of SARM1 inhibition on localized neurodegeneration versus systemic and central inflammation remain poorly understood in complex autoimmune environments like MS. Here, we investigated SARM1 pathology using global SARM1-/- knockout (SARM1-/-) mice, AAV-mediated CRISPR knockdown and overexpression of SARM1 in retinal ganglion cells (RGCs), and the small-molecule SARM1 inhibitor 5-iodoisoquinoline (5IIQ), across optic nerve crush (ONC) and experimental autoimmune encephalomyelitis (EAE) models. While global SARM1-/- did not alter the overall clinical course of EAE, it revealed a complex phenotype characterized by an altered peripheral inflammatory cytokine profile and persistent CNS immune infiltration, alongside preserved axonal and myelin integrity. RGC-restricted SARM1 knockdown partially preserved RGCs and axons during EAE, whereas SARM1 overexpression worsened both retinal function and axonal injury. Pharmacological inhibition with 5IIQ preserved axonal integrity and restored visual function in both the ONC and EAE models, as confirmed by electrophysiology, and reduced serum neurofilament light chain (NfL) in EAE, without affecting demyelination. Together, these findings demonstrate that SARM1 inhibition uncouples axonal self-destruction from demyelination and gross neuroinflammation while providing robust structural and functional neuroprotection. This study establishes SARM1 as a viable target for neuroprotective co-therapies designed to complement existing immunomodulatory and remyelinating regimens in MS and related neurodegenerative disorders.

neuroscience↗

bioRxiv2026

Ex vivo human tumor slices more accurately predict patient responses to an oncolytic virus than in vivo mouse models

Immunotherapies, including oncolytic viruses (OV), are promising therapies that can enhance anti-tumor immune responses. However, preclinical success of immunotherapies in mouse models has not always translated to clinical benefit in cancer patients. This study compared preclinical efficacy and mechanism of action for ASP9801, a vaccinia virus expressing IL-7 and IL-12, using mouse models of colorectal cancer (CRC) in vivo and in human organotypic tumor slice models ex vivo. The murine surrogate for ASP9801 significantly reduced tumor volumes in treated and abscopal tumors in two different CRC models in vivo (MC38 and RO100). Treatment efficacy was accentuated when combined with anti-PD1 treatment, and single-cell RNA sequencing analysis revealed depletion of tumor cells and increased T cell infiltration and activation in both treated and abscopal tumors. However, human tissue analysis ex vivo (E-slices) using PDX models and patient samples showed that ASP9801 is not effective in CRC, consistent with clinical trial results. On the other hand, ASP9801 was highly effective in GBM, indicating indication-specific efficacy of ASP9801, and how E-slice assays can be used to identify treatment-sensitive indications. This study demonstrates the superiority of E-slices over mouse models for predicting clinical response and its utility in planning clinical trials.

cancer biology↗

bioRxiv2026

Cardiomyocyte-specific loss of Smyd5 leads to a robust activation of inflammatory signaling and heart failure in mice.

Background: Cardiomyocytes respond to stress by undergoing hypertrophic growth driven by dynamic changes in gene expression. Epigenetic mechanisms, including histone methylation, play critical roles in regulating these transcriptional programs, yet the enzymes controlling these modifications during cardiac disease remain largely unknown. The SMYD family of histone methyltransferases regulates gene expression in multiple biological contexts, but the function of SMYD5 in the mammalian heart has never been investigated. Methods: SMYD5 expression was assessed in human heart failure samples and in a mouse model of cardiac hypertrophy. To define its functional role in vivo, we generated inducible cardiomyocyte-specific Smyd5 knockout mice and characterized their cardiac phenotype using molecular, histological, and functional analyses. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) was performed to examine histone H4 lysine 20 trimethylation (H4K20me3) at the Il-6 promoter. Results: SMYD5 expression was altered in diseased human and mouse hearts. Under basal conditions, cardiomyocyte-specific deletion of Smyd5 resulted in baseline structural cardiac remodeling and transcriptional signatures characteristic of pathological stress. Smyd5-deficient hearts exhibited marked inflammatory activation resembling a cytokine storm with immune cell infiltration and heart failure. Notably, Smyd5 knockout mice displayed a 100-fold increase in Il-6 expression, accompanied by a global reduction in H4K20me3. ChIP-qPCR analysis of the Il-6 promoter, together with loss- and gain-of-function analysis of SMYD5, supports a direct epigenetic role of SMYD5 in regulating Il-6 expression through H4K20me3 in cardiomyocytes. Conclusions: SMYD5 is a previously unrecognized epigenetic regulator of cardiac homeostasis that restrains inflammatory signaling in cardiomyocytes under normal conditions. Loss of Smyd5 disrupts H4K20me3, leading to derepression of Il-6 in cardiomyocytes and a robust inflammatory response characterized by immune cell recruitment and fibrosis, accompanied by rapid progression of cardiac remodeling and heart failure. These findings identify SMYD5 as a critical regulator of intrinsic cardiomyocyte inflammatory signaling and reveal a novel chromatin-based mechanism contributing to inflammatory cardiomyopathies.

molecular biology↗

bioRxiv2026

Paralemmin 2 is a shared, alternatively-spliced regulator of axon initial segments and paranodal junctions in oligodendrocytes

Nervous system function depends on highly specialized axonal membrane domains. For example, axon initial segments (AIS), nodes of Ranvier, and paranodal junctions are essential for action potential initia-tion and saltatory conduction. These domains use shared molecular machinery converging on the master scaffolding protein AnkyrinG (AnkG). Using endogenous AnkG-TurboID proximity proteomics, we identify Paralemmin 2 (Palm2), a previously uncharacterized CNS protein, as a shared component of the AIS and nodes of Ranvier in neurons, and paranodes in oligodendrocytes. Palm2 localizes transiently to the AIS and nodes during development, but is found at paranodes throughout life, reflecting cell type-specific splicing of a lipid-modified domain both necessary and sufficient for AnkG interaction. Mice lacking oli-godendroglial Palm2 assemble normal paranodes during development, but aged mice lose paranode in-tegrity due to loss of paranodal AnkG and Neurofascin-155. Our findings identify Palm2 as a lipid raft-associated regulator of axonal and oligodendroglial membrane domains and reveal cell type-specific pro-tein variants as a recurring principle for specializing axonal and glial membrane architectures.

neuroscience↗

bioRxiv2026

Metabolic signatures of cone-dominant and cone-degenerating retinas

Cone photoreceptors are essential for daylight vision, and their degeneration has more profound visual consequences than rod loss in retinal degenerative diseases. Metabolic dysfunction is closely associated with cone degeneration, however the relatively small population of cones in mice and humans have limited our understanding of cone-specific metabolism. Here, we leveraged cone-dominant and cone-degeneration mouse models including Nrl-/-, Cnga3-/-, and high-dose Triiodothyronine (T3) treatment to investigate cone-specific metabolism and their metabolic impacts on the retinal pigment epithelium (RPE). Across models, cone-dominant retinas consistently showed lower pyruvate abundance alongside increases in glutathione, purines, pentose phosphate pathway intermediates, and one-carbon metabolites. Increases in several key amino acids were also associated with higher cone abundance, such as proline, arginine, alanine, valine, leucine, and hypotaurine. Strikingly, aminoadipate, an intermediate in lysine catabolism, was the most robustly changed metabolite in the retina, showing highly consistent increases across models. Relative cone increases were also associated with metabolic changes in the RPE/choroid. Like the retina, RPE/choroids showed consistent increases in aminoadipate, proline, and hypotaurine, as well as xanthosine and betaine, alongside decreases in uracil. Moreover, proteomic analysis of Nrl-/- mice showed decreases in many key metabolite transporters in the RPE/choroid, including carriers for glucose, lactate, aspartate, glutamate, serine, lysine, taurine, and proline. Collectively, these findings further our understanding of cone-specific metabolism and highlight potential cone-specific metabolic vulnerabilities in retinal degeneration.

biochemistry↗

bioRxiv2026

Decoding human sperm signalling: Phosphoproteomic discovery of kinases governing fertilization competency

Capacitation, the process whereby sperm gain the functional competence to fertilize an egg in the absence of de novo transcription and translation, is orchestrated by a hierarchy of kinases driving the phosphorylation of sperm proteins. While increased phosphorylation, in particular tyrosine phosphorylation, is a revered hallmark of fertilization competency in our species, only a limited repository of phosphorylated substrates and kinases have ever been reported from human sperm. To broaden therapeutic targets for sperm targeted contraceptives and infertility therapies, we adapted a contemporary phosphoproteomic technique termed EasyPhos to generate bespoke methodology for the investigation of human sperm signalling. This approach yielded high depth phosphoproteomes of non-capacitated and capacitated human spermatozoa with in silico investigation of the phosphosites revealing 52 kinases with previously uncharacterized roles in sperm capacitation. Investigating the function of the putative sperm capacitation kinases identified yielded several kinases with novel roles in the regulation of sperm function. Of particular interest, polo like kinase 1 (PLK1) inhibition significantly reduced progressive sperm motility, attenuated capacitation-associated tyrosine phosphorylation and reduced the sperm acrosome reaction, an essential step to achieve fertilization. These findings reveal extensive phosphoproteome remodelling during human sperm capacitation, expanding the landscape of molecular targets for fertility control.

Cell Biology↗

bioRxiv2026

Pathway-selective mitophagy regulates retinal physiology and neurogenic transitions in Muller glia

Mitochondrial quality control (MQC) is essential for retinal homeostasis, yet how distinct mitophagy pathways are coordinated within specialized retinal cell types remains poorly understood. Here, we show that Muller glia engage distinct mitophagy programmes that are differentially activated across physiological, metabolic stress, and differentiation contexts. Using pathway-resolved analyses supported by mouse and human single-cell transcriptomic datasets, we demonstrate that PINK1-dependent and receptor-mediated mitophagy pathways coexist within Muller glia and exhibit distinct functional and spatial regulation. To enable precise, time-resolved interrogation of these processes, we developed MQ-MG2, a spontaneously immortalised Muller glial model stably expressing the Mito-QC reporter while preserving endogenous mitophagy adaptors and metabolic features of primary Muller cells. Using this system, we identify context-dependent activation of mitophagy pathways with spatial relevance in vivo and reveal transient coordination of PINK1-dependent and receptor-associated mitophagy during Muller glial neurogenic differentiation. Suppression of fission-dependent mitophagy impaired the acquisition of complex neurite features in MQ-MG2, with a comparable phenotype observed following targeted PINK1 deletion in human neurogenic cells. Together, these findings position Muller glia as active integrators of mitochondrial quality control, capable of engaging distinct mitophagy programmes according to cellular context.

cell biology↗

bioRxiv2026

Spatially defined axonal guidance in neural organoids with micropatterned microfluidic channels

Three-dimensional stem cell-derived neural organoids provide a promising platform for investigating early brain development and interregional circuit formation. Although co-culture of region-specific organoids into assembloids has enabled the study of cortical and subcortical interactions, these models lack directional specificity and spatial control, limiting their ability to recapitulate canonical circuit architecture. Here, we present a microfluidic platform for constructing directional and tunable interregional circuits while preserving anatomical distinction. This system, which we term "directoids" incorporates micropatterned polydimethylsiloxane (PDMS) microstructures to control uni- and bidirectional axonal growth between cortical and thalamic organoids. We observed a 70.4% success rate of axons traversing the full channel length in the permissive direction and reaching the opposing organoid, whereas no neurites successfully crossed the probative direction. These results demonstrate robust directionally bias in axon outgrowth and establish a scalable, reproducible strategy for controlling asymmetric connectivity between anatomically distinct neural organoids. Using high-density CMOS microelectrode arrays, we further validated directional tuning of extracellular action potential propagation within directoid microchannels, a feature not observed in straight-channel connectoid controls. Directoids also exhibited significant asymmetry in firing rates between channel entry and exit sites, consistent with engineered bias in signal flow. This provides an experimental paradigm for dissecting how anatomical connectivity and functional activity converge to shape neuronal networks. Together, these findings establish a microfluidic platform for investigating the mechanisms underlying hierarchical circuit formation, regional specification, and functional integration in developing human neural organoid models at cellular resolution not possible in vivo.

bioengineering↗

bioRxiv2026

Translational toolkit for reproducible, cross-study profiling of human ageing hallmarks in human blood and tissue

BackgroundAgeing is a complex, multi-dimensional process, underpinned by interacting biological hallmarks that collectively contribute to functional decline and increased susceptibility to disease. While considerable progress has been made in delineating individual ageing pathways, translation into human studies has been hindered by methodological heterogeneity and a lack of standardised, multi-system approaches. Here, we describe a validated, high-resolution toolkit for the simultaneous quantification of multiple ageing hallmarks in clinically accessible human samples, encompassing cellular senescence, immune ageing, inflammation, mitochondrial function, mTOR signalling, autophagy, genomic instability, and stem cell exhaustion. MethodsBlood (25ml) was obtained from young and aged donors (26-81y). Deep immunophenotyping was performed using a novel 30-colour spectral flow cytometry panel. T-cell mTOR activation and autophagic flux were assessed by flow cytometry. Metabolic flux was measured by Seahorse. From whole blood (4 ml), muscle, and adipose tissue (AT) (obtained during elective hip arthroplasty) RNA, DNA, AT stem cells, and myoblasts were isolated. DNA copy number and senescent cell burden were assessed by q-PCR and SA-{beta}-gal staining, respectively. FindingsUtilising this toolkit, we identified pronounced age-related immune remodelling, increased senescent T-cell burden, diminished mitochondrial capacity and altered mTOR-autophagy signalling between healthy young and aged donors. Furthermore, metabolism was significantly affected by anti-coagulant and freezing sample before analysis. InterpretationThis integrated platform provides a foundation for reproducible, cross-study analyses and facilitates translational investigation of interventions targeting health-span extension. FundingWellcome Leap Dynamic Resilience program (co-funded by Temasek Trust).

immunology↗

bioRxiv2026

Ventricular Forebrain Organoids Reproduce Macroscale Geometry of the Developing Telencephalon

During development, the size of the neuroepithelial cell pool plays a key role in establishing brain size, determining the numbers of derived progenitors and subsequent neuronal cell types. While early histogenesis is well modelled in brain organoids, the organ-scale geometry of the telencephalon is not accurately recapitulated. Herein, we present a new approach for generating ventral and dorsal forebrain organoids which develop a large ventricular neuroepithelium, characteristic of the closed telencephalic vesicle. Using a growth medium that supports aerobic glycolysis and is typically used for endothelial cells, we modulate neuroepithelial expansion to induce a more anatomically accurate neuroepithelial layer which, upon maturation, thickens physiologically to generate the typical neurogenic layered architecture. In addition, we present a new method for embedding organoids in miniature collagen spheres which mimics native extracellular matrix, stabilizes the ventricular geometry for dynamic culture conditions, and provides a means for incorporating vascular cells for neurovascular development. Finally, we demonstrate that human organoids grown under these conditions exhibit dramatically enlarged ventricles and delayed maturation compared to mouse. Together, this approach provides a model of the forebrain neuroepithelium with morphogenetic macroscale geometry and tissue architecture, suitable for investigating neurodevelopment and disease.

bioengineering↗

bioRxiv2026

Guarding versus self-guarding in innate immunity

Hosts have evolved a variety of innate immune responses to pathogens. In many cases, hosts directly detect pathogen-associated molecular patterns (PAMPs) or pathogen effectors to trigger an immune response. However, hosts may also detect pathogens indirectly through guarding, whereby immune receptors ( guards) monitor the effects of pathogens (e.g., modification of target cells) rather than the pathogens themselves. Guarding poses a different evolutionary challenge for pathogens than direct recognition of PAMPs, as replication may necessitate the modification or disruption of guarded host proteins ( guardees). Recently, self-guarding has been discovered, in which the host target functions as both guard and guardee. Self-guarding appears to present an intractable problem for pathogens: modification of the host target may benefit replication, but also triggers an immune response. If self-guarding creates an apparently inescapable detection mechanism, why has self-guarding only recently been discovered? Here, we use mathematical models of within-host pathogen and immune dynamics to compare guarding and self-guarding architectures. We show that self-guarding leads to a more rapid immune response and faster pathogen suppression, but is also more prone to false-positive immune responses, likely imposing greater costs through autoimmunity. We therefore hypothesise that the greater potential for false-positive immune responses may limit the conditions under which self-guarding evolves.

evolutionary biology↗

bioRxiv2025

Genomic and morphometric evidence for Austronesian-mediated pig translocation in the Pacific

Human mediated translocation of non-native pig species (genus Sus) to the islands of Wallacea and Oceania has significantly altered local ecosystems. To investigate the timing and trajectory of these introductions, we conducted both genomic analyses of 576 pig nuclear genomes and a geometric morphometric analysis of 714 modern and ancient dental remains. Our analyses demonstrate that feral and domestic pigs in Wallacea and Oceania possess diverse ancestries resulting from the introduction of multiple, sequential pig populations followed by gene flow. Despite the variability in their genomic ancestry these pigs all possess a distinct tooth morphology, and a genetic link to the Chinese domestic pig populations that accompanied the dispersal of Austronesian language speakers [~]4,000-3,000 years ago via Taiwan and the Philippines.

evolutionary biology↗

bioRxiv2025

Reprogramming of Cellular Plasticity via ETS and MYC Core-regulatory Circuits During Response to MAPK Inhibition in BRAF-mutant Colorectal Cancer

BackgroundAberrant enhancer dynamics play a critical role in the initiation and progression of colorectal cancer (CRC). BRAFV600E-mutated metastatic CRC may be a unique subtype, exhibiting a strong epigenetic phenotype. Interestingly, bromodomain 2, a reader protein for H3K27ac-marked enhancers, was found to be synthetically lethal in CRC with BRAF + EGFR inhibition. DesignWe evaluated the effectiveness of targeting aberrant enhancers with bromodomain and extraterminal (BET) + MAPK pathway inhibitors in patient-derived xenograft models of metastatic CRC, followed by comprehensive profiling of transcriptomic and chromatin dynamics upon BET inhibitor combination treatment. ResultsThe combination of BET and standard MAPK inhibitors has demonstrated improved efficacy against BRAFV600E CRC and selective improvements against RAS-mutant CRC in vivo. We showed that BET + MAPK inhibition induced a profound downregulation of the MAPK signaling pathway compared to MAPK inhibition alone. The loss of activation signal on enhancers, as determined by H3K27ac, led to dysregulation of core-regulatory circuitries of CRC, especially loss of the auto-regulatory mechanism of the MAPK downstream E26 transformation-specific transcription factor family. Single nucleus multiome (RNA + ATAC) sequencing further distinguished differential transcriptomic and chromatin dynamics at cell type levels. Profound downregulation of well-differentiated cell types confirmed deep inhibition of MAPK signaling and downstream transcription factors. On the other hand, dedifferentiated cell populations were abundant after MAPK or combination inhibition, suggesting therapy-induced cell state switching and adaptation. ConclusionWe are evaluating BET + BRAF + EGFR inhibition in patients with treatment-refractory BRAFV600E metastatic CRC. ClinicalTrial.gov identifier: NCT06102902. What is already known on this topicEnhancer aberrations emerge as critical epigenetic features in the progression of colorectal cancer (CRC). However, the dynamics of active enhancer and the therapeutic potential of enhancer blockade, particularly in CRC tumors with BRAFV600E mutation, is not well understood. What this study addsThis study demonstrates improved efficacy of BET inhibitor combination therapies in diverse patient-derived models and reveals epigenetic reprogramming driven cellular plasticity in BRAFV600E-mutated CRC. How this study might affect research, practice or policyOur findings support treatment with BET + BRAF + EGFR inhibitors for patients with BRAFV600E-mutant mCRC [NCT06102902]. This study also highlights the potential of combining epigenetic agents to standard targeted therapy, offering a novel treatment option for this subset of patients.

cancer biology↗

bioRxiv2025

Whole-Proteome Screening and Multi-Modal Profiling of Antigen-Specific CD4+ T Cells at Single-Cell Resolution

Systematic whole-proteome screening and comprehensive profiling of antigen-specific CD4+ T cells are crucial for advancing our understanding of CD4+ T cell immunity, yet such efforts remain technically challenging. Here, we present a high-throughput platform that employs large-scale class II single-chain trimer libraries to detect antigen-specific CD4+ T cells, while simultaneously profiling their antigen specificity, TCR/{beta} sequences, MHC restriction, whole transcriptomes, and patient/timepoint origins at single-cell resolution. We benchmarked SCTs against conventional pMHCs and validated the SCT library-based approach in direct ex vivo identification of antigen-specific CD4+ T cells in healthy donors. We then applied the platform to screen the entire SARS-CoV-2 receptor-binding domain in a longitudinal patient cohort, identifying 2,188 antigen-specific CD4+ T cells and revealing key features that define antigen immunogenicity. Extending to cancer, we performed whole-proteome screening of HPV-16 E6/E7 for TCR repertoire profiling in a precancerous cohort, uncovering functional heterogeneity of HPV-specific TCRs. By integrating high-throughput antigen screening with high-dimensional, multi-modal cellular characterization, our approach offers an unprecedented window into CD4+ T cell immunity across diverse disease contexts and empowers the development of new therapies.

bioengineering↗

bioRxiv2025

Computational Platform for streamlining the success of sequential antibiotic therapy

The scarcity of antibiotics and the need for swift decision-making present significant challenges for healthcare practitioners. When confronted with such circumstances, practitioners must prioritize their approach based on several key factors. By leveraging the recent biological discovery of collateral sensitivity, we have devised an open-source computational platform. This platform utilizes the drug resistance profile of an evolved strain and initial population conditions to potentially predict the failure of cycling therapy in eradicating or restraining a multi-drug resistant bacterial population. We demonstrate how this framework can anticipate potential failures for a range of antibiotics in chronic pseudomonas aeruginosa infections. This innovative methodology lays the foundation for evolutionary therapies that can facilitate the selection of appropriate treatments, thereby reducing antibiotic resistance.

evolutionary biology↗

bioRxiv2025

Clinical isolates of Enterobacter species can persist in human macrophages without bacterial replication and minimal cellular cytotoxicity

BackgroundThe Enterobacter cloacae complex (Ecc) encompasses opportunistic Gram-negative bacteria demonstrating considerable phenotypic and genotypic diversity. Bloodstream, respiratory and urinary tract infections by Ecc bacteria are associated with morbidity and mortality worldwide. These infections are often difficult to treat since Ecc bacteria are resistant to penicillins, quinolones, aminoglycosides, and third-generation cephalosporins. Resistance also extends to carbapenems, leaving only polymyxins, such as colistin, as a last resort antibiotic for treatment. However, colistin resistance in Ecc isolates is also unexpectedly frequent. Despite extensive information on antibiotic resistance by Enterobacter species, much less is known about their infection biology. There are few reports on the survival and persistence of selected Enterobacter species in macrophages and epithelial cells, but how Enterobacter isolates interact with innate immune host cells upon engulfment remains unexplored. In this study, we have investigated the intracellular trafficking of a subset of antimicrobial resistant Ecc clinical isolates, including colistin-resistant strains, within human macrophages, and determined the macrophage response to the intracellular infection. MethodsPhagocytosis of 11 clinical Ecc isolates, including E. cloacae, E. bugandensis, E. kobei, E. xiangfangensis, E. roggenkampii, E. hoffmannii, and E. ludwigii was investigated in THP-1 and human monocyte derived macrophages (HMDMs). Confocal fluorescence microscopy was used to ascertain intracellular trafficking via co-localisation of cell markers with fluorescent bacteria. Intracellular bacterial replication was assessed by bacterial enumeration in cell lysates after killing extracellular bacteria and by a fluorescence dilution approach to follow the synthesis of the bacterial cell wall over time. Macrophage cell cytotoxicity was investigated by quantifying the release of lactate dehydrogenase during infection with all isolates. Two prototypic isolates, the E. cloacae ATCC13047 type strain and the E. bugandensis 104107, were used to explore in more detail the response of macrophages to the intracellular infection by determining cleavage of the proinflammatory markers caspase-1, gasdermin D and pro-interleukin-1{beta}. FindingsWe found that Ecc isolates do not replicate in human macrophages but survive within a modified late phagolysosome compartment. Survival occurred in all species investigated and did not correlate with colistin resistance, lipopolysaccharide modifications, or bacterial pathogenicity in the Galleria mellonella infection model. All isolates induced macrophage cell cytotoxicity at significantly lower levels than controls treated with lipopolysaccharide and nigericin treatment (to induce a proinflammatory response). Low cytotoxicity also correlated with absence of cleavage of proinflammatory markers in infected macrophages. InterpretationEcc species can survive without replication inside human macrophages with minimal effects on cell viability and inflammation. These observations could have implications in the clinical outcome of patients that cannot readily clear infecting Ecc bacteria. This can potentially lead to prolonged intracellular survival and infection relapse. FundingBiotechnology and Biological Sciences Research Council grants BB/T005807/1 and BB/S006281/1. Research In contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed with the terms "Enterobacter" AND "macrophage", "Enterobacter" AND "monocytes", and "Enterobacter" AND "intracellular" for original articles published in English up to July 9, 2024. The search excluded terms "sakazakii" and "aerogenes" since E. aerogenes and E. sakazakii have been moved to the genus Klebsiella and Cronobacter, respectively. Of the 55, 15, and 181 studies we identified, respectively, only one reported testing Enterobacter cloacae phagocytosis. Another study reported intracellular bacterial communities in uroepithelial cells, which serve as a reservoir during urinary tract infection. One other study reported the isolation of E. hormaechei from human atherosclerotic tissue and described studies in THP-1 monocytic macrophages. A few earlier studies also reported Enterobacter cytotoxins affecting immune cells, and an E. cloacae polysaccharide capable of inducing apoptosis in epithelial cells. These studies did not investigate mechanisms and there have been no more recent follow-ups; importantly, it remains unclear if the strains employed in these studies were properly identified as Enterobacter species. Therefore, despite isolated observations of survival of Enterobacter isolates surviving in immune and epithelial cells, there is an overall knowledge gap in our understanding of these pathogens concerning intracellular survival compartments, kinetics of survival, and induction of macrophage cytotoxicity and inflammatory responses. Added value of this studyOur study is the first to investigate in detail how clinical isolates of various Enterobacter species can survive intracellularly in human macrophages. All isolates display multidrug antimicrobial resistance, including some with colistin resistance, and can survive intracellularly in human macrophages. Our data demonstrate that intracellular Enterobacter resides in vacuoles for up to 44 hours without replication. Colocalization experiments with various fluid phase and membrane cellular markers revealed that bacteria-containing vacuoles are modified late phagolysosomes, which do not accumulate the autophagosome marker LC3B. Intracellular bacterial survival did not associate to any Enterobacter species tested, the presence of colistin resistance, lipopolysaccharide modifications, or virulence in the Galleria mellonella infection model. Moreover, intracellular infection caused minimal cytotoxicity in macrophages without evidence of macrophage proinflammatory cell death by pyroptosis. Implications of all the available evidenceOur findings underscore the capacity of Enterobacter species, traditionally viewed as extracellular bacteria, to hideout in macrophages without inducing a significant inflammatory response. These properties may further complicate the treatment of antibiotic-resistant Enterobacter infections in susceptible populations such as the elderly and neonates. These findings open a door to the development of host-directed therapeutics to enhance bacterial clearance by macrophage-mediated killing.

microbiology↗

bioRxiv2025

Adaptive Plasticity Tumor Cells Modulate MAPK-Targeting Therapy Response in Colorectal Cancer

MAPK pathway inhibitors (MAPKi) are increasingly used in the treatment of advanced colorectal cancer, but often produce short-lived responses in patients. Although acquired resistance by de novo mutations in tumors have been found to reduce response in some patients, additional mechanisms underlying the limited response durability of MAPK targeting therapy remain unknown. Here, we denote new contributory tumor biology and provide insight on the impact of tumor plasticity on therapy response. Analysis of MAPKi treated patients revealed activation of stemness programs and increased ASCL2 expression, which are associated with poor outcomes. Greater ASCL2 with MAPKi treatment was also seen in patient-derived CRC models, independent of driver mutations. We find ASCL2 denotes a distinct cell population, arising from phenotypic plasticity, with a proliferative, stem-like phenotype, and decreased sensitivity to MAPKi therapy, which were named adaptive plasticity tumor (APT) cells. MAPK pathway suppression induces the APT phenotype in cells, resulting in APT cell enrichment in tumors and limiting therapy response in preclinical and clinical data. APT cell depletion improved MAPKi treatment efficacy and extended MAPKi response durability in mice. These findings uncover a cellular program that mitigates the impact of MAPKi therapies and highlights the importance of addressing tumor plasticity to improve clinical outcomes.

cancer biology↗