Biology subjects

Azuma, M.

Publications and source records attributed to Azuma, M..

11 recordsLinked to original sources

bioRxiv2026

NECTIN4 defines a subtype-dependent tumor state coupling enfortumab vedotin targetability to antitumor immunity in bladder cancer

NECTIN4 is the target of enfortumab vedotin (EV), but its biological role in bladder cancer (BC) and its relationship to EV plus PD-1 blockade remain unclear. Integrating clinical multi-omics, spatial and single-cell immune profiling, and experimental models, we identify NECTIN4 as a subtype-dependent determinant of tumor state and antitumor immunity. In basal BC cells, NECTIN4 suppressed inflammatory and mesenchymal programs. Consistently, NECTIN4-high basal/squamous muscle-invasive BCs showed favorable survival, higher checkpoint inhibitor response rates, and a coordinated CD8+ T cell-inflamed microenvironment with no comparable associations in luminal papillary tumors. In orthotopic models, murine Nectin4 promoted CD8+ T cell-dependent tumor control, whereas human NECTIN4 conferred EV sensitivity. EV also attenuated NECTIN4-dependent TIGIT binding. Conversely, NECTIN4-low models were preferentially sensitive to HDAC inhibition. NECTIN4 therefore couples antibody-drug conjugate targetability to a subtype-specific tumor-immune state permissive for checkpoint blockade, whereas the NECTIN4-low state defines a distinct inflammatory-mesenchymal state with an alternative therapeutic vulnerability.

cancer biology↗

bioRxiv2026

Deep learning-mediated detection of accelerated water drinking after aquaresis in V1b vasopressin receptor knockout mice

How water intake is initiated and maintained following V2 vasopressin receptor antagonism remains poorly understood. To elucidate the role of the V1b receptor in managing dehydration stress induced by V2 antagonism, we used deep learning-based computer vision to analyze drinking behavior in V1b knockout (V1bKO) and wild-type (WT) mice. While total water access and intake volume were comparable between genotypes, V1bKO mice exhibited distinct temporal dynamics. Modeling cumulative intake with the Hill equation revealed that the time required to reach 50\% of maximal water access was significantly shorter in V1bKO mice than in WT mice. This accelerated drinking effectively mitigated increases in serum osmolality and body weight loss. A reduced Hill's coefficient in V1bKO mice indicates a reduction of the rapid, cooperative-like water accumulation seen in WT mice. Furthermore, elevated basal hemoglobin levels in V1bKO mice were independent of dehydration, as confirmed via bone marrow transplant. Analysis of movement trajectories revealed that V1bKO mice exhibit a lower proportion of vertical movement (required for nozzle access) despite similar total distances traveled. Collectively, our results demonstrate that the V1b receptor critically regulates water-seeking behavior and osmotic homeostasis.

pharmacology and toxicology↗

bioRxiv2026

A combination of EWSR1-FLI1 with loss of one EWSR1 allele promotes damage in the mitotic spindle and sensitization to microtubule-destabilizing agent

Microtubule-destabilizing agents (MDAs) and microtubule-stabilizing agents (MSAs) are commonly used chemotherapeutic agents due to its activity to induce cell death by compromising the dynamics of spindles during mitosis. Ewing sarcoma, the second most common pediatric bone cancer, is known to selectively respond to MDAs as a first-line treatment, but not to MSAs. Ewing sarcoma cells carry an aberrant EWSR1-FLI1 fusion gene and only one wildtype EWSR1 allele. To investigate the origin of this MDA sensitivity, we used an (AID-EWSR1/wt: EWSR1-FLI1-mCherry/wt) cell line that enables conditional induction of EWSR1-FLI1 expression (Tet-On system) and EWSR1 knockdown derived from one EWSR1 allele (auxin-degron system). A combination of EWSR1-FLI1 expression and EWSR1 knockdown induces apoptosis upon nocodazole treatment, a type of MDA. Our study revealed that the mitotic spindles of Ewing sarcoma cells (A673, RD-ES and SK-ES1) contain elevated levels of tubulin damage, visualized with GTP-tubulin, compared to mesenchymal stem cells (MSC). Consistently, the combination of EWSR1-FLI1 expression and EWSR1 knockdown in (AID-EWSR1/wt: EWSR1-FLI1-mCherry/wt) cell line also leads to an increased incidence of damage in mitotic spindles. Together, we propose that the sensitivity of Ewing sarcoma cells is derived from the increased levels of damage to mitotic spindles caused by EWSR1-FLI1 expression and EWSR1 knockdown.

Cancer Biology↗

bioRxiv2026

The combination of EWSR1-FLI1 and loss of one EWSR1 allele leads to the induction of trisomy 8

Ewing sarcoma is a pediatric cancer that develops in skeletal elements. The majority of Ewing sarcoma patients carry the aberrant EWSR1-FLI1 fusion gene. Despite trisomy 8 being an additional common aberration associated with a poor prognosis for patients, its induction mechanism remains unknown. When the EWSR1-FLI1 gene is formed, the cell loses one wildtype EWSR1 allele. To elucidate the induction mechanism of trisomy 8, we generated a cell line that allows for the conditional induction of EWSR1-FLI1 expression and EWSR1 knockdown (derived from a single EWSR1 allele. Specifically, the conditional cell line was generated by integrating the Tet-on EWSR1-FLI1 construct into the AAVS locus and adding a miniAID tag at the 5 end of the EWSR1 locus using auxin-degron system. A combination of the EWSR1-FLI1 expression and degradation of one allele-derived EWSR1 induced a high incidence of trisomy 8 within eight days, enhancing colony formation. Mechanistically, trisomy 8 is induced by the haploinsufficiency of EWSR1, and the remaining EWSR1 proteins are likely inhibited by interaction with EWSR1-FLI1. Our data showed that the knockout of EWSR1 alone was sufficient to increase the incidence of trisomy 8. Expression of wild-type EWSR1 in EWSR1 knockout cells rescued the high incidence of trisomy 8. In contrast, the EWSR1:R565A mutant, which lacks the ability to interact with Aurora B kinase, failed to rescue this phenotype. We propose that the combination of EWSR1-FLI1 expression and loss of EWSR1 contributes to the induction of trisomy 8 through the compromised EWSR1-Aurora B pathway. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/726567v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@999891org.highwire.dtl.DTLVardef@1ef6748org.highwire.dtl.DTLVardef@65e475org.highwire.dtl.DTLVardef@179da40_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2026

IntelliProfiler 2.0: An integrated R pipeline for long-term home-cage behavioral profiling in group-housed mice using eeeHive 2D

BackgroundLong-term home-cage monitoring is essential to quantify spontaneous locomotor and social behaviors in group-housed mice, but analysis of high-density RFID tracking data remains a barrier to reproducibility. New methodsWe developed IntelliProfiler 2.0, a fully R-based pipeline tailored to the eeeHive 2D floor-mounted RFID array. The workflow performs data import from text logs, preprocessing, coordinate reconstruction, missing-value handling, feature extraction, statistical testing, and visualization in a single environment. Behavioral metrics include travel distance, close contact ratio (CCR), and a newly implemented inter-individual distance metric. ResultsIn four-day recordings of group-housed C57BL/6J mice (8 males and 8 females), IntelliProfiler 2.0 captured circadian phase-dependent locomotion and proximity patterns and reproduced sex-dependent differences consistent with prior analyses while incorporating updated hardware specifications. Radar-chart summaries enabled intuitive comparison of multidimensional behavioral profiles and inter-individual variability across light/dark phases. Comparison with existing methodsCompared with IntelliProfiler 1.0 and multi-tool workflows, IntelliProfiler 2.0 consolidates analysis into a single, script-based R pipeline, reducing operational complexity and improving reproducibility. The updated implementation supports recent manufacturer-driven changes, including antenna renumbering and multi-USB data export. ConclusionsIntelliProfiler 2.0 provides a reproducible, extensible framework for high-throughput behavioral phenotyping of group-housed mice and is scalable across hardware configurations, including simplified single-board recordings. HighlightsO_LIEnd-to-end R pipeline for eeeHive 2D floor-based RFID tracking analysis C_LIO_LIStandardized setup with comprehensive manuals and protocols C_LIO_LIInter-individual distance metric to quantify group spatial structure C_LIO_LICircadian- and sex-dependent behavioral profiling in group-housed mice C_LIO_LIRadar-charts summarize multidimensional behavioral profiles and variability C_LI

animal behavior and cognition↗

bioRxiv2024

Expression of fibroblast activation protein-α in human deep venous thrombus

BackgroundFibroblast activation protein- (FAP), a type-II transmembrane serine protease, is expressed during wound healing, in cancer-associated fibroblasts, and in chronic fibrosing diseases. However, its expression in deep vein thrombus (DVT) remains unclear. Therefore, in this study, we investigated FAP expression and localization in DVT. MethodsFirst, we pathologically accessed aspirated thrombi of patients with DVT (n=14), classifying thrombotic areas as follows; fresh, cellular lysis, endothelialization, fibroblastic reaction. Endothelialization and fibroblastic reaction were defined as organizing reactions. We immunohistochemically examined FAP-expressed areas and expressed cells. Second, we analyzed FAP expression in cultured dermal fibroblasts. ResultsAll the aspirated thrombi showed a mixture of at least three of the four thrombotic areas. Specifically, 83 % of aspirated thrombi showed fresh and organizing reactions within each thrombus. Immunohistochemical expression of FAP was restricted in organizing area. Further, FAP expression in the thrombi was mainly found in vimentin-positive or -smooth muscle actin-positive fibroblasts in double immunofluorescence. Some CD163-positive macrophages also showed FAP expression. FAP mRNA and protein levels significantly increased in cultured fibroblasts with low- proliferative activity under 0.1% fetal bovine serum (FBS) than that in fibroblasts under 10% FBS. Fibroblasts cultured in 10% FBS showed a significant decrease in FAP mRNA levels following supplementation with hemin, but not with thrombin. ConclusionsThe heterogeneous composition of the venous thrombi suggests the existence of a multistep thrombus formation process in human DVT. Further, fibroblasts or myofibroblasts may express FAP in organizing process in DVT. FAP expression may increase in fibroblasts with low-proliferative activity.

pathology↗

bioRxiv2024

Computer vision analysis of mother-infant interaction identified efficient pup retrieval in V1b vasopressin receptor knockout mice

Close contact between lactating rodent mothers and their infants is essential for effective nursing. Whether the mothers effort to retrieve the infants to their nest requires the vasopressin-V1b vasopressin receptor axis has not been fully defined. To address this question, V1b receptor knockout (V1bKO) and control mice were analyzed in pup retrieval test. Because an exploring mother in a new test cage randomly accessed to multiple infants in changing backgrounds over time, a computer vision-based deep learning analysis was applied to continuously calculate the distances between the mother and the infants as a parameter of their relationship. In an open-field, a virgin female V1bKO mice entered fewer times into the center area and moved shorter distances than wild-type (WT). While this behavioral pattern persisted in V1bKO mother, the pup retrieval test demonstrated that total distances between a V1bKO mother and infants came closer in a shorter time than with a WT mother. Moreover, in the medial preoptic area, parts of the V1b receptor transcripts were detected in galanin- and c-fos-positive neurons following maternal stimulation by infants. This research highlights the effectiveness of deep learning analysis in evaluating the mother-infant relationship and the critical role of V1b receptor in pup retrieval during the early lactation phase. Highlights(1) Mother-infant relationship was calculated as distances by computer vision and object detection analysis. (2) V1b knockout female mice tended to avoid entering centerfield in a new cage compared to control female. (3) Lack of V1b vasopressin receptor in mice facilitated the pup retrieval in early infancy, even with shorter distances of total movements.

animal behavior and cognition↗

bioRxiv2023

Factor XI localization in human deep venous thrombus and function of activated factor XI on venous thrombus formation and hemostasis in rabbit

BackgroundNovel anticoagulants targeting coagulation factor XI (FXI)/activated FXI (FXIa) are under development. However, whether FXI is present in human deep vein thrombosis (DVT) and whether FXIa and activated factor X (FXa) play different roles in venous thrombus formation and hemostasis remain unclear. This study aimed to determine the presence of FXI in DVT and the effects of direct oral FXIa and FXa inhibitors on venous thrombus formation and hemostasis in rabbits and mural thrombus formation in flow chamber system. MethodsWe immunohistochemically assessed FXI localization in human aspirated DVT (n=15). Additionally, we compared thrombus formation induced by endothelial denudation and stenosis in jugular vein, and skin bleeding time and volume between rabbits treated with direct FXIa inhibitors (ONO-1600586) and FXa inhibitors (rivaroxaban). Ex vivo rabbit and human blood were perfused on a flow chamber under low shear rates (70/s). ResultFXI was localized in all DVT, predominantly in fibrin-rich areas. The FXI-immunopositive area in the non-organizing area was greater than that in the organizing area. Although FXIa and FXa inhibitors comparably inhibited venous thrombus formation, FXIa inhibitors did not affect bleeding time or volume in rabbits. FXIa or FXa inhibitors mildly or strongly inhibited fibrin formation at low shear rates respectively. Furthermore, the FXIa inhibitor suppressed human FXIa activity, thrombin generation, and fibrin formation during perfusion. ConclusionThe pathological findings of human DVT suggest FXIs role in human DVT. FXIa inhibitors may inhibit less fibrin formation than FXa inhibitors, and may explain the minor role of FXIa in hemostasis. EssentialO_LIPresence of factor XI (FXI) in venous thrombus and less bleeding in its inhibition are unclear. C_LIO_LIWe assessed FXI localization in deep vein thrombosis (DVT) and function of FXIa in rabbit. C_LIO_LIFXI localized in human DVT that provide a rationale for FXI inhibition in human DVT. C_LIO_LIFXIa inhibitor inhibited less fibrin formation than factor Xa inhibitor under low-shear rate. C_LI

pathology↗

bioRxiv2022

Cortical-wide impairment of glymphatic system after focal brain injury

In peripheral tissues, cells are maintained in the interstitial fluid that flows from capillaries to lymph system. However, the brain has no lymphatic capillaries, and the actual state of the interstitial fluid has long been unknown. Recently, a glymphatic system has been proposed in which part of the cerebrospinal fluid flowing on the surface of brain tissue enters the brain parenchyma via the peri-arterial space, becomes interstitial fluid, and then flows out again from the peri-venous space. Brain injury due to head contusion or stroke is thought to impair the intracerebral circulation and aggravate the extracellular environment, but the actual situation is unknown. Therefore, in this study, we examined the effects of focal brain tissue damage on intracerebral circulation using the light-injured mouse, an originally developed closed head injury model. In light-injured mice, the injury-making process does not affect intracerebral circulation because the cranium is maintained. However, this method has quantitative problems, so we developed a method to image cerebrospinal fluid and blood vessels from the surface of the cerebral cortex. After examining different injury sites and different time periods after injury, it was found that intracerebral circulation was reduced to the same extent on the ipsilateral and contralateral sides of the injury at one-week post-injury. This intracerebral circulatory deficit was still partially present at four-weeks post-injury. These results indicate that the intracerebral circulation is extensively impaired by local injury and neurodegenerative diseases.

neuroscience↗

bioRxiv2022

EWSR1 prevents the induction of aneuploidy by regulating the localization of Aurora B at inner centromere

EWSR1 (Ewing sarcoma breakpoint region 1) was originally identified as a part of an aberrant EWSR1/FLI1 fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the EWSR1/FLI1 fusion gene in the tumor genome, the cell loses one wild type EWSR1 allele. Our previous study demonstrated that the loss of ewsr1a (homologue of human EWSR1) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the tp53 mutant background. To dissect the molecular function of EWSR1, we successfully established a stable DLD-1 cell line that enables a conditional knockdown of EWSR1 using Auxin Inducible Degron (AID) system. When both EWSR1 genes of DLD-1 cell were tagged with mini-AID at its 5-end using CRISPR/Cas9 system, treatment of the (AID-EWSR1/AID-EWSR1) DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the EWSR1 knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. This defect was proceeded by a lower incidence of the localization of Aurora B at inner centromeres, and by a higher incidence of the protein at kinetochores compared to the control cells during pro/metaphase. Despite these defects, the EWSR1 knockdown cells did not undergo mitotic arrest, suggesting that the cell lacks the error correction mechanism. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of EWSR1-mCherry and EWSR1:R565A-mCherry (a mutant that has low affinity for Aurora B) in the (AID-EWSR1/AID-EWSR1) DLD-1 cells. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Together, we demonstrate that EWSR1 is essential to prevent aneuploidy through interaction with Aurora B, most likely by regulating the localization of Aurora B at centromere.

cell biology↗

bioRxiv2020

Genome sequence of Hydrangea macrophylla and its application in analysis of the double flower phenotype

Owing to its high ornamental value, the double flower phenotype of hydrangea (Hydrangea macrophylla) is one of its most important traits. In this study, genome sequence information was obtained to explore effective DNA markers and the causative genes for double flower production in hydrangea. Single molecule real-time sequencing data followed by a HiC analysis was employed. The resultant haplotype-phased sequences consisted of 3,779 sequences (2.256 Gb in length and N50 of 1.5 Mb), and 18 pseudomolecules comprising 1.08 Gb scaffold sequences along with a high-density SNP genetic linkage map. Using the genome sequence data obtained from two breeding populations, the SNPs linked to double flower loci (Djo and Dsu), were discovered for each breeding population. DNA markers J01 linked to Djo and S01 linked to Dsu were developed, and these could be used successfully to distinguish the recessive double flower allele for each locus respectively. The LEAFY gene was suggested as the causative gene for Dsu, since frameshift was specifically observed in double flower accession with dsu. The genome information obtained in this study will facilitate a wide range of genomic studies on hydrangea in the future.

genomics↗