Biology subjects

Marques, M.

Publications and source records attributed to Marques, M..

14 recordsLinked to original sources

bioRxiv2026

CCR4+low-density neutrophils define a novel immunosuppressive subset associated with breast cancer progression and early mortality

Neutrophils are increasingly recognized as key players in cancer progression, yet their functional heterogeneity limits their therapeutic exploitation. In breast cancer (BC), elevated circulating and tumor-associated neutrophils have been associated with poor prognosis, and low-density neutrophils (LDN), particularly, have been linked to immunosuppression. However, the specific neutrophil subsets driving these effects remain undefined. Here, we identify a previously unrecognized CCR4-expressing neutrophil subset, strongly enriched among LDN, whose frequency rises with disease stage and correlates with faster progression and shorter survival. Serial blood sampling further showed that CCR4+LDN levels fluctuate in line with clinical course, with increases indicating near-term risk in metastatic patients. Our findings also reveal that this subset has an immunosuppressive signature more pronounced than that of the remaining LDN, migrates preferentially toward tumor-derived chemokines, and dampens T cell-driven tumor killing even when PD-1 is blocked. Additionally, in a pilot cohort of Triple-Negative BC patients on anti-PD-1 therapy, early changes in this population were associated with treatment outcome. Together, these results reveal a distinct neutrophil population that actively contributes to immune escape and tumor progression, establishing CCR4+LDN as a minimally invasive real-time biomarker of disease course and treatment response, and as a promising target for neutrophil-directed therapies in BC.

immunology↗

bioRxiv2026

Warming exacerbates fungal pathogenicity to drive physiological collapse and microbiome reorganization in octocorals

Corals are increasingly threatened by climate change, yet the interplay between ocean warming and fungal infection remains experimentally underexplored. Here, we demonstrate that thermal stress exacerbates the pathogenicity of the fungus Aspergillus sydowii, driving physiological collapse and functional reorganization in the octocoral Sclerophytum sp. We deployed a 34-day mesocosm heat-stress experiment and quantified performance, mortality, and taxonomic and functional microbiome shifts. Combined heat and fungal exposure proved highly lethal, causing 45% mortality compared to 7% under heat exposure alone. Re-isolation from diseased nubbins implicates A. sydowii as the causal agent. Metagenomic profiling indicated that physiological collapse was underpinned by a functional transition from mutualism to antagonism in the microbiome. Specifically, combined heat and fungal stress triggered a depletion of ankyrin- and WD40-repeat proteins - hallmarks of symbiotic stability - concurrent with a surge in genes involved in fungal cell wall degradation and secondary metabolite biosynthesis. While surviving holobionts recovered full photosynthetic efficiency after combined stress, their microbiomes did not revert to baseline. Instead, they assembled into a taxonomically distinct configuration characterized by enrichments of sulfate-reducers (Thermodesulfobacteriota) and thermotolerant phototrophs (Thermosynechococcales). This suggests that despite the rapid photosystem recovery, the holobiont retained a complex legacy of thermal and biotic stress across both its internal chemical microenvironment and its microbiome. These findings highlight the decoupled recovery processes of different holobiont components, demonstrating that even though some corals may survive severe climate-driven disease, they emerge as ecologically reorganized entities.

ecology↗

bioRxiv2026

GATA4 loss promotes mutant Kras-driven pancreatic ductal adenocarcinoma in the absence of canonical precursor lesions

ABSTRACTGATA6 and GATA4 play key roles in pancreatic development and are essential to maintain the classical transcriptional program in pancreatic ductal adenocarcinoma (PDAC). Using genetic mouse models we show that, in contrast to GATA6, GATA4 is dispensable for the maintenance of acinar homeostasis in the adult pancreas. Deletion of Gata4 in mice expressing mutant Kras in the embryonic pancreas (KG4C) leads to PDAC development in the absence of tissue remodeling, pancreatic intraepithelial neoplasia (PanIN), or other canonical precursor lesions present in Gata4-proficient (KC) mice. Similar observations were made when Gata4 was selectively inactivated in adult, Kras-mutant, acinar cells. We identify Pale Acinar Lesions (PALes) as a previously unrecognized pancreatic lesion, distinct from acino-ductal metaplasia (ADM) and PanINs, present in KC and KG4C mice but not in wild type mice. PALes display weak expression of acinar and ductal markers and lack mucins; they have lower proliferation rates than PanINs. RNA-seq and ChIP-seq reveal that GATA4 and GATA6 partially share genomic binding sites and transcriptomic effects, but they exert opposing influences on mutant Kras-induced, haematopoietic cell-dependent, transcriptional inflammatory programs. Adenoviral-mediated pancreatic expression of IL17 restored the formation of ductal lesions in KG4C mice but failed to rescue PanIN development. Our data indicate that GATA4 functions through the coordinated action of multiple inflammatory factors that are required for ADM/PanIN formation but are dispensable for PDAC development. Collectively, these findings challenge current paradigms of PDAC initiation and progression.

cancer biology↗

bioRxiv2026

Nerve growth factor receptor identifies a basal subpopulation linked to poor prognosis and reduced immunotherapy responses in bladder cancer

PurposeBladder cancer (BLCA) is a heterogeneous tumor type. Only one third of muscle-invasive (MIBC) patients respond to immune checkpoint inhibitors (ICIs). Reliable resistance markers are needed to guide clinical decisions. We investigated the nerve growth factor receptor (NGFR) in BLCA and analyzed its correlation with disease progression and response to immunotherapy. Experimental DesignWe analyzed NGFR expression in BLCA cell lines, organoids, mouse models and patient samples. The cohorts used were The Cancer Genome Atlas (TCGA), enriched in muscle-invasive bladder cancer (MIBC) (n=407); IMvigor210, representing MIBC patients treated with ICIs (n=348); and UROMOL2, as a non-muscle-invasive bladder cancer (NMIBC)-specific cohort (n=535). IMvigor010 was also included (n=728). Patients were stratified by NGFR expression quartiles. We analyzed survival and tumor subtypes and performed stromal deconvolution and functional profiling. We assessed stemness- and invasion-related features in SCaBER cells. ResultsNGFR marks a basal tumor cell subcluster and is independently associated with poor prognosis in TCGA and IMvigor210. NGFR-high tumors show stromal content enriched in cancer-associated fibroblasts, lower neoantigen burden, higher CD8+ T effector signature together with an immune-excluded phenotype, and a CAF-specific TGF{beta} signature. In the immunotherapy-treated cohort, high NGFR expression was also associated with poorer outcome. Functionally, NGFR appears to promote a stem-like/pro-invasive program in BLCA cells. ConclusionsNGFR identifies a basal-like BLCA subpopulation linked to poor survival, while its association with immunotherapy response requires further validation. In addition, our in vitro analyses support a role of NGFR in stem-like and invasive traits, highlighting its relevance as a biomarker in BLCA.

cancer biology↗

bioRxiv2026

Fatty acids from adipocyte lipolysis stimulate insulin secretion

White adipose tissue and pancreatic islets play central roles in the regulation of metabolic homeostasis. Although ectopic lipid accumulation is established as a driver of impaired insulin secretion, the acute contribution of adipocyte lipolysis to islet function remains poorly documented. Here, we investigated a mouse model with inducible adipocyte-specific deletion of both adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), which leads to defective adipocyte lipolysis. Despite preserved ex vivo islet function, these mice displayed a marked reduction in insulin secretion in response to stimulation of adipocyte {beta}3-adrenoceptors, as well as following glucose and arginine challenges. Mechanistically, we identified non-esterified fatty acids as critical mediators of lipolysis-driven insulin secretion, engaging pancreatic signaling of the free fatty acid receptors FFAR4 (a.k.a. GPR120) and FFAR1 (a.k.a. GPR40). The regulation of insulin secretion by adipocyte lipolysis was preserved in high-fat diet-induced obesity. These findings identify an underappreciated adipose-islet crosstalk that couples adipocyte lipolysis to insulin secretion and links lipid and glucose metabolism.

physiology↗

bioRxiv2026

Capillary-based Subcellular Sampling Uncovers the Stress Granule Proteome in Single Cells

Many diseases arise from dysfunction within specific organelles or biomolecular condensates, highlighting the value of analysing proteins at subcellular resolution to uncover new biological mechanisms. We report a novel capillary-based subcellular sampling workflow coupled with liquid chromatography-mass spectrometry (LC-MS) for proteomic analysis of defined subcellular regions of individual cells. We applied this methodology to stress granules (SGs), membrane-less biomolecular condensates that form in response to cellular stress (including viral infection), and are implicated in infection, neuropathology and cancer. Comprehensive characterisation of SG protein composition remains limited by technical challenges associated with bulk purification, including loss of spatial context, dynamic behaviour and contamination from cytosolic material. Using our novel method, we identified a high-confidence set of 405 SG-associated proteins, including 46 established SG residents alongside numerous previously unreported candidates. Functional enrichment analysis revealed pathways consistent with known SG biology, while comparison with an independent cytosolic proteome dataset demonstrated minimal overlap, supporting the specificity of the sampling strategy. Selected novel SG protein candidates (AHNAK2, DDX39B, NUDT1 and FKBP2) were validated using immunofluorescence microscopy. These findings establish capillary-based subcellular sampling as a viable approach for proteomic analysis of SGs with preserved spatial context and provide a framework for analysing other subcellular compartments. Table of contentsWe report an LC-MS-based capillary sampling workflow for proteomic analysis of subcellular structures within single cells. This methodology identified 405 high-confidence stress granule-associated proteins, including 46 previously established and numerous novel candidates. The approach demonstrated high specificity and preserved spatial context, expanding the capabilities of subcellular proteomics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/724230v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1fa0bb0org.highwire.dtl.DTLVardef@1158524org.highwire.dtl.DTLVardef@1d82812org.highwire.dtl.DTLVardef@2ee4d9_HPS_FORMAT_FIGEXP M_FIG C_FIG Figure made in Biorender.com.

cell biology↗

bioRxiv2025

The assembly of stress granule-like foci during foot-and-mouth disease virus infection is uncoupled from activation of cellular intrinsic antiviral signalling

Foot-and-mouth disease virus (FMDV) is highly contagious among cloven-hoofed animals and poses a major threat to the livestock industry worldwide. A fundamental gap in knowledge for high consequence viruses such as FMDV is understanding how the virus evolved to evade cellular antiviral responses. FMDV belongs to the Picornaviridae, a family of positive-sense single-stranded RNA viruses. The detection of viral double-stranded viral RNA intermediates during infection can trigger both the assembly of cytoplasmic stress granules (SGs) and the activation of the RIG-I-like receptors (RLR)-mediated innate immune response (IIR). FMDV has been proposed to antagonize these mechanisms, suggesting that both can limit viral replication. In this study, we investigate the dynamic and importance of SG assembly for IIR activation upon dsRNA stimulation or FMDV replication in porcine epithelial kidney cells. First, we show that the formation of SG following a challenge with poly(I:C), a viral dsRNA mimic, does not modulate the activation of IIR. Our data further reveal transient assembly of SG during FMDV infection followed by virus-induced cleavage of G3BP1, a core SG protein. While SG assembly does not impact viral replication or antiviral response activation, we demonstrate that preventing their disassembly negatively impacts FMDV replication. Overall, our data suggests that while SGs assembly is uncoupled from IIR activation, manipulating their disassembly limits replication and might serve as a potential therapeutic target to prevent FMDV infection. ImportanceBiomolecular condensates, including stress granules, are key regulatory compartments that control fundamental cellular processes. By condensing RNAs and proteins, these structures enable cells to rapidly adapt to stress, such as viral infection. Different biomolecular condensates with specific dynamics or compositions have been described during infection, and many viruses are known to disrupt or highjack their components. Moreover, the cell-autonomous innate immune response is proposed to be regulated by biomolecular condensates. However, the molecular mechanisms underpinning these functions remain unclear and controversial. Here we investigated the interplay between stress granules and the innate response upon the stimulation with a viral dsRNA mimic or foot-and-mouth disease virus infection. We demonstrate that infection triggers the formation of stress granules independently from activation of innate signalling pathways. We also show that stress granules persistence attenuates viral replication posing them as part of the cells response that viruses must overcome or subvert to replicate.

microbiology↗

bioRxiv2025

Genetic interference of distinctive Mycobacterium tuberculosis peptidoglycan modifications enhances β-lactam susceptibility and reveals expression-sensitive host immune dynamics

The high mortality associated with tuberculosis (TB), alongside the lack of efficient therapeutics against emerging multidrug-resistant Mycobacterium tuberculosis (Mtb) strains, emphasizes the need to identify novel antitubercular targets. Mycobacterial peptidoglycan, displaying characteristic modifications comprising the amidation of D-iso-glutamate (D-iGlu) and the N-glycolylation of muramic acid, is a promising therapeutic target. The genes encoding the enzymes mediating these PG modifications (murT/gatD and namH) were silenced in Mtb using CRISPR interference (CRISPRi) to investigate their impact on {beta}-lactam susceptibility and host immune responses. First, qRT-PCR confirmed successful target mRNA knockdown, with variable repression efficiency based on the selected sgRNA, PAM strength, and target site. Phenotypic characterization through spotting dilution and growth curve assays corroborated the essentiality of D-iGlu amidation for mycobacterial survival, in contrast to the N-glycolylation of muramic acid. Moreover, susceptibility assays showed that both PG modifications contribute to {beta}-lactam resistance, with sgRNA2-mediated murT knockdown substantially increasing {beta}-lactam and isoniazid susceptibility. Furthermore, checkerboard assays showed reductions in the minimum fractional inhibitory concentration index (FICImin) value for AMX/MEM+CLA and EMB combinations following the depletion of both PG modifications, with significant differences observed upon namH knockdown. Additionally, D-iGlu amidation was uncovered as a determinant of Mtb survival within THP-1-derived macrophages at 6 days post-infection. Infection of THP-1-derived macrophages with MurT/GatD-depleted Mtb upregulated IL-1{beta} and downregulated IL-10, whereas NamH depletion caused upregulation of both IL-1{beta} and IL-10. Altogether, our findings unveiled the potential of targeting these PG modifications for the development of innovative therapeutic regimens against TB.

microbiology↗

bioRxiv2025

STAG2 cohesin cooperates with DREAM to maintain quiescence and suppress tumourigenesis in the urothelium

The maintenance of quiescence is essential for tissue homeostasis. STAG2 is one of the few genes mutated in the normal urothelium of organ donors, with mutant cells undergoing positive selection 1. STAG2 is also a major tumour suppressor gene 2-4 and its inactivation is an early event in bladder carcinogenesis 1,3. However, how STAG2, a cohesin component, regulates urothelial homeostasis remains largely unknown. Here, we demonstrate that Stag2 inactivation in normal murine urothelial cells interferes with differentiation programs, triggers transient cell cycle entry, and primes cells for clonal expansion under stress. Moreover, STAG2 loss enhances tumor formation in urothelial cells expressing mutant FGFR3 - the key oncogene in bladder cancer 5. We reveal that STAG2 cooperates with the DREAM transcriptional complex, a master regulator of quiescence 6,7, by binding to shared genomic sites, including cell cycle control genes. STAG2 loss alters DREAM target expression, complex composition, and chromatin distribution, and leads to rewiring of chromatin interactions involving DREAM binding motifs in genes critical for cell cycle entry. Our findings provide compelling evidence that STAG2 loss disrupts in 3D genome organization through a novel mechanism involving the DREAM complex, thereby impairing homeostatic quiescence and increasing oncogenic sensitivity.

cancer biology↗

bioRxiv2024

Sickness behaviour within cleaning interactions

When facing diseases, strategies such as social distancing have proven effective in preventing disease spread. Cleaning interactions are social interactions where cleaner organisms remove ectoparasites from clients. These interactions can be potential vectors of infectious diseases in reef fishes. Here, we aimed to determine if cleaner wrasses (Labroides dimidiatus) employ social distancing and other behavioural tactics to prevent disease spread within the context of cleaning interactions. To this end, we used bacterial lipopolysaccharides (LPS) immunostimulation to induce "sickness" behaviour in cleaners. After LPS injection, we performed four behavioural trials: social preference (from cleaner and client perspectives), social interaction, and bystander cooperativeness tests. In the social preference test, LPS-injected cleaners showed lower activity rates but no change in their social preference. However, some clients preferred "sick" cleaners. Contrarily, during the social interaction test, cleaners reduced the number of interactions, despite clients willingness to interact. For the bystander test, LPS-injected cleaners were unable to adjust their behaviour to be more cooperative. Reduced activity levels revealed lethargy, which may lead to passive self-isolation, allowing for some social distancing of healthy individuals. This suggests that immune-stimulated cleaners undergo changes in their behaviour which can be viewed as a passive social distancing preventing disease spread.

animal behavior and cognition↗

bioRxiv2023

Influenza A virus activates the unfolded protein response and induces the accumulation of insoluble protein aggregates that are essential for efficient viral propagation

Influenza A virus (IAV) is one of the main causes of annual respiratory epidemics in humans. IAV employs multiple strategies to evade host immunity and hijack cellular mechanisms to support proper virion formation and propagation. Some of these strategies encompass the manipulation of pathways involved in protein homeostasis, leading to changes in the host proteome and protein distribution within the cell. In this study, we performed a detailed analysis of the interplay between IAV and the host cells proteostasis mechanisms throughout the entire infectious cycle. We reveal that IAV infection induces the activation of the inositol requiring enzyme 1 (IRE1) branch of the unfolded protein response (UPR), at an infection stage that coincides with high rates of viral protein translation. This activation is particularly important for infection, as attenuation of virus production was observed upon IRE1 inhibition. Concomitantly to UPR activation, we observed the accumulation of virus-induced insoluble protein aggregates, which contain both viral and host proteins and are associated with a dysregulation of the host cell RNA metabolism. We demonstrate that this accumulation is important for IAV propagation, as its prevention using a quinoline-steroid hybrid compound significantly reduces the number of produced infectious virus particles. Our data suggests that the formation of these insoluble protein aggregates favors the final steps of the infection cycle, more specifically the virion assembly. Our findings reveal additional mechanisms by which IAV disrupts the host cell proteostasis to favor infection and uncover new cellular targets that can be explored for the development of host-directed antiviral strategies.

microbiology↗

bioRxiv2022

Human cytomegalovirus vMIA inhibits MAVS oligomerization at peroxisomes in an MFF-dependent manner

Upon intracellular recognition of viral RNA, RIG-I-like proteins interact with MAVS at peroxisomes and mitochondria, inducing its oligomerization and the downstream production of direct antiviral effectors. The human cytomegalovirus (HCMV) is able to specifically evade this antiviral response, via its antiapoptotic protein vMIA. Besides suppressing the programmed cell death of infected cells, vMIA inhibits the antiviral signalling at mitochondria by inducing the organelles fragmentation, consequently hindering the interaction between MAVS and the endoplasmic reticulum protein STING. Here, we demonstrate that vMIA interferes with the peroxisomal antiviral signalling via a distinct mechanism that is independent of the organelles morphology and does not affect STING. vMIA interacts with MAVS at peroxisomes and inhibits its oligomerization, restraining downstream signalling, in an MFF-dependent manner. This study also demonstrates that vMIA is totally dependent on the organelles fission machinery to induce peroxisomal fragmentation, while this dependency is not observed at mitochondria. Furthermore, although we demonstrate that vMIA is also able to inhibit MAVS oligomerization at mitochondria, our results indicate that this process, such as the whole vMIA-mediated inhibition of the mitochondrial antiviral response, is independent of MFF. These observed differences in the mechanisms of action of vMIA towards both organelles, likely reflect their intrinsic differences and roles throughout the viral infection. This study uncovers specific molecular mechanisms that may be further explored as targets for antiviral therapy and highlights the relevance of peroxisomes as platforms for antiviral signalling against HCMV.

cell biology↗

bioRxiv2019

Immunohistochemistry-based taxonomical classification of bladder cancer predicts response to neoadjuvant chemotherapy

Platinum-based neoadjuvant chemotherapy (NAC) increases the survival of patients with organ-confined urothelial bladder cancer (UBC). Because not all patients benefit from treatment, NAC has not been widely applied in the clinical setting. There is strong evidence, based on retrospective studies, that patients with Basal/Squamous (BASQ)-like tumours present with more advanced disease and have worse prognosis; global transcriptomics can identify tumour subtypes associated with response to NAC. We aimed to investigate whether tumour immunohistochemical (IHC) subtyping predicts NAC response. Patients with muscle-invasive UBC having received platinum-based NAC were identified in two hospitals in Spain. Tissue microarrays were constructed; RNA and DNA were extracted from full sections. Nanostring analysis and immunohistochemistry to identify BASQ-like tumours and mutational analysis of UBC oncogenes. We used hierarchical clustering to classify 126 tumours and adjusted logistic regression to assess association with treatment response. Outcomes were progression-free survival and disease-specific survival; univariable and multivariate Cox regression models were applied. We found very high concordance between mRNA and protein for the 4 markers analyzed. We identified three main subgroups: BASQ-like (FOXA1/GATA3 low; KRT5/6/14 high), Luminal-like (FOXA1/GATA3 high; KRT5/6/14 low), and mixed-cluster (FOXA1/GATA3 high; KRT5/6 high; KRT14 low). Patients with BASQ-like tumours were more likely to achieve a pathological response to NAC, displaying a disease-specific survival similar to that of the remaining patients. In conclusion, patients with BASQ-like tumours - identified through simple and robust immunohistochemistry - have a higher likelihood of undergoing a pathological complete response to NAC. Prospective validation in independent series is required.\n\nNovelty and impactNeoadjuvant chemotherapy is an important component of the management of patietns with muscle-invasive bladder cancer but improved stratification is necessary. This retrospective study shows that patients with BASQ-like tumors can be identified using immunohistochemistry on paraffin-embedded tissue and are 4-fold more likely to achieve a pathological complete response to platinum-based NAC. The disease-specific survival of patients with BASQ-like tumours treated with NAC was not different from that of other tumour subtypes.

pathology↗

bioRxiv2019

Overexpression of osmosensitive Ca2+-activated channel TMEM63B promotes migration in HEK293T cells

The recent discovery of the osmosensitive calcium (Ca2+) channel OSCA has revealed the potential mechanism by which plant cells sense diverse stimuli. Osmosensory transporters and mechanosensitive channels can detect and respond to osmotic shifts that play an important role in active cell homeostasis. TMEM63 family of proteins are described as the closest homologues of OSCAs. Here, we characterize TMEM63B, a mammalian homologue of OSCAs, recently classified as mechanosensitive. In HEK293T cells TMEM63B localizes to the plasma membrane and is associated to F-actin. This Ca2+-activated channel specifically induces Ca2+ influx across the membrane in response to extracellular Ca2+ concentration and hyperosmolarity. In addition, overexpression of TMEM63B in HEK293T cells significantly enhanced cell migration and wound healing. The link between Ca2+ osmosensitivity and cell migration might help to establish TMEM63Bs pathogenesis, for example in cancer in which it is frequently overexpressed.

biochemistry↗