Biology subjects

Kawaguchi, K.

Publications and source records attributed to Kawaguchi, K..

31 records · Page 2Linked to original sources

bioRxiv2023

Selective elimination of CD169+ macrophages in lymph nodes invaded by breast cancers

Breast cancer cells suppress the host immune system to efficiently invade the lymph nodes; however, the underlying mechanism remains incompletely understood. Here, we report that metastasized breast cancer cells selectively eliminate CD169+ lymph node sinus macrophages, an initiator of anti-cancer immunity, from the lymph nodes. The comparison between paired lymph nodes with and without metastasis from the same patients demonstrated that the number of CD169+ macrophages was reduced in metastatic lymph nodes, whereas the numbers of other major immune cell types were unaltered. We also detected the infiltration of CD169+ macrophages into metastasized cancer tissues depending on sections, suggesting that CD169+ macrophages were gradually eliminated after anti-cancer reactions. Furthermore, CD169+ macrophage elimination was prevalent in major breast cancer subtypes and correlated with breast cancer staging. Collectively, we propose that metastasized breast cancer cells dispel CD169+ macrophages from lymph nodes in a phased manner, disabling a critical step of anti-cancer immunity.

cancer biology↗

bioRxiv2023

mRNA vaccine induces protective immunity against the type III secretory virulence of Pseudomonas aeruginosa

Few studies have addressed mRNA vaccination against bacteria despite the desperate need to control antimicrobial resistance. Herein, we developed mRNA vaccines targeting the type III secretion system of Pseudomonas aeruginosa, which plays a critical role in the pathogenesis. Vaccination with a nucleoside-modified mRNA encapsulated in lipid nanoparticles improved mouse survival, reduced the bacterial load in the lung, and alleviated pathological changes in the lungs after bacterial challenge.

immunology↗

bioRxiv2023

Senescence triggers intracellular acidification and lysosomal alkalinization via ATP6AP2 attenuation in breast cancer cells

Several chemotherapy drugs induce the senescence of cancer cells; however, the mechanism underlying intracellular pH dysregulation in senescent cells remains unclear. Adenosine triphosphatase H+ transporting accessory protein 2 (ATP6AP2) plays a critical role in maintaining pH homeostasis in cellular compartments. We here report a new function of ATP6AP2 in senescent breast cancer cells induced by doxorubicin and abemaciclib treatment. ATP6AP2 expression was significantly downregulated in senescent cells, leading to aberrant pH levels that impaired lysosome function and caused immune response changes. The drugs caused cell cycle arrest and proliferation suppression through the upregulation of senescence-related genes. Additionally, senescent cells showed altered inflammatory and immune transcriptional profiles by reprogramming the senescence-associated secretory phenotype. These findings suggest that ATP6AP2-mediated pH regulation during therapy-induced senescence may be linked to immune changes in senescent cancer cells. These findings provide novel insights into understanding the cellular mechanisms underlying the response to anti-cancer drugs.

cancer biology↗

bioRxiv2023

Inducible expression of the restriction enzyme uncovered genome-wide distribution and dynamic behavior of histones H4K16ac and H2A.Z at DNA double-strand breaks in Arabidopsis

DNA double-strand breaks (DSBs) are among the most serious types of DNA damage and cause mutations and chromosomal rearrangements. In eukaryotes, DSBs are immediately repaired in coordination with chromatin remodeling for the deposition of DSB-related histone modifications and variants. To elucidate the details of DSB-dependent chromatin remodeling throughout the genome, artificial DSBs need to be reproducibly induced at various genomic loci. Recently, in mammals, a comprehensive method for elucidating chromatin remodeling at multiple DSB loci via chemically induced expression of a restriction enzyme was developed. However, this DSB induction system is not suitable for investigating chromatin remodeling during and after DSB repair, and such approach has not been performed in plants. Here, we established a transgenic Arabidopsis plant harboring a restriction enzyme gene Sbf I driven by a heat-inducible promoter. Using this transgenic plant, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of histones H4K16ac and H2A.Z and investigated dynamics of these histone marks around the endogenous 623 Sbf I recognition sites. We also precisely quantified DSB efficiency at all cleavage sites using the DNA resequencing data obtained by ChIP-seq procedures. From the results, Sbf I-induced DSBs were detected at 360 loci and induced the transient deposition of H4K16ac and H2A.Z around these regions. Interestingly, we also observed the co-localization of H4K16ac and H2A.Z at some DSB loci. Overall, DSB-dependent chromatin remodeling was found to be a highly conserved between plants and animals. These findings provide new insights into chromatin remodeling that occurs in response to DSBs in Arabidopsis.

molecular biology↗

bioRxiv2022

LapTrack: Linear assignment particle tracking with tunable metrics

MotivationParticle tracking is an important step of analysis in a variety of scientific fields, and is particularly indispensable for the construction of cellular lineages from live images. Although various supervised machine learning methods have been developed for cell tracking, the diversity of the data still necessitates heuristic methods that require parameter estimations from small amounts of data. For this, solving tracking as a linear assignment problem (LAP) has been widely applied and demonstrated to be efficient. However, there has been no implementation that allows custom connection costs, parallel parameter tuning with ground truth annotations, and the functionality to preserve ground truth connections, limiting the application to datasets with partial annotations. ResultsWe developed LapTrack, a LAP-based tracker which allows including arbitrary cost functions and inputs, parallel parameter tuning, and ground-truth track preservation. Analysis of real and artificial datasets demonstrates the advantage of custom metric functions for tracking score improvement. The tracker can be easily combined with other Python-based tools for particle detection, segmentation, and visualization. Availability and implementationLapTrack is available as a Python package on PyPi, and the notebook examples are shared at https://github.com/yfukai/laptrack. The data and code for this publication are hosted at https://github.com/NoneqPhysLivingMatterLab/laptrack-optimization. Contactysk@yfukai.net

bioinformatics↗

bioRxiv2022

Serum amyloid alpha 1-2 are not required for systemic inflammation in the 4T1 murine breast cancer model

Cancers induce the production of acute phase proteins such as serum amyloid alpha (SAA) in the liver and cause systemic inflammation. Despite the well-known coincidence of acute phase response and systemic inflammation, the direct roles of SAA proteins in systemic inflammation in the cancer context remains incompletely characterized, particularly in vivo. Here, we investigate the in vivo significance of SAA proteins in systemic inflammation in the 4T1 murine breast cancer model. 4T1 cancers elevate the expression of SAA1 and SAA2, the two major murine acute phase proteins in the liver. The elevation of Saa1-2 correlates with the up-regulation of immune cell-related genes including neutrophil markers. To examine this correlation in detail, we generate mice that lack Saa1-2 and investigate immune-cell phenotypes. RNA-seq experiments reveal that deletion of Saa1-2 does not strongly affect 4T1-induced activation of immune cell-related genes in the liver and bone marrow. Flow cytometry experiments demonstrate the dispensable roles of SAA1-2 in cancer-dependent neutrophil infiltration to the liver. This study clarifies the negligible contribution of SAA1-2 proteins in systemic inflammation in the 4T1 breast cancer model.

cancer biology↗

bioRxiv2022

Combined blockade of VEGF, Angiopoietin-2, and PD1 reprograms glioblastoma endothelial cells into quasi-antigen-presenting cells

Glioblastoma (GBM) remains a highly aggressive and uniformly fatal tumor, with a 5-year survival of <10%. Moreover, all randomized clinical trials with immune-checkpoint blockers have failed to date. Here we report that tumor endothelial cell (EC) dysfunction confers resistance to immunotherapy in preclinical GBM models. Anti-VEGF-therapy-induced vascular normalization is insufficient to fully restore the EC function and alleviate inflammatory edema induced by blocking programmed cell death protein 1 (PD1). Strikingly, concomitant blockade of angiopoietin 2 (Ang2), vascular endothelial growth factor (VEGF), and programmed cell death protein 1 (PD1) reprograms dysfunctional ECs to quasi-antigen presenting cells and upregulates receptors required for cytotoxic T lymphocyte entry into the tumor. Blocking VEGF, Ang2, and PD1 induces durable anti-tumor response while controlling edema. Upregulation of the transcription factor T-bet is necessary for generating resident memory T cells elicited by this combination therapy. Moreover, a human GBM organoid model formed from human cancer cells, ECs, CD14 and CD8 cells recapitulated these findings. Our study reveals the role of Ang2 in resistance to VEGF and/or PD1-blockade and provides a compelling rationale for clinical evaluation of this combination in GBM patients.

cancer biology↗

bioRxiv2021

Graph-based machine learning reveals rules of spatiotemporal cell interactions in tissues

Robustness in developing and homeostatic tissues is supported by various types of spatiotemporal cell-to-cell interactions. Although live imaging and cell tracking are powerful in providing direct evidence of cell coordination rules, extracting and comparing these rules across many tissues with potentially different length and timescales of coordination requires a versatile framework of analysis. Here we demonstrate that graph neural network (GNN) models are suited for this purpose, by showing how they can be applied to predict cell fate in tissues and utilized to infer the cell interactions governing the multicellular dynamics. Analyzing the live mammalian epidermis data, where spatiotemporal graphs constructed from cell tracks and cell contacts are given as inputs, GNN discovers distinct neighbor cell fate coordination rules that depend on the region of the body. This approach demonstrates how the GNN framework is powerful in inferring general cell interaction rules from live data without prior knowledge of the signaling involved.

cell biology↗

bioRxiv2021

A Drosophila Toolkit for Imaging of HA-tagged Proteins Unveiled a Block in Autophagy Flux in the Last Instar Larval Fat Body

For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with POI harboring different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: "HA Frankenbody". This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Autophagy was dispensable for the swelling of lysosomes, indicating that lysosomal activity is downregulated at this stage. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.

cell biology↗

bioRxiv2021

Gradual differentiation uncoupled from cell cycle exit generates heterogeneity in the epidermal stem cell layer.

High turnover tissues continually lose specialized cells that are replaced by stem cell activity. In the adult mammalian epidermis, it is unclear how molecularly heterogenous stem/progenitor cell populations fit into the complete trajectory of epidermal differentiation. We show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes. Differentiation-committed cells remain capable of dividing to produce daughter cells fated to further differentiate, demonstrating that differentiation is uncoupled from cell cycle exit. These cell divisions are not required as part of an obligate transit amplifying program but instead protect density in the stem cell layer. Thus, instead of distinct contributions from multiple progenitors, a continuous gradual differentiation process fuels homeostatic epidermal turnover. One sentence summaryHeterogeneity in the epidermal stem cell layer reflects a gradual differentiation program that is uncoupled from the loss of proliferative capacity.

cell biology↗

bioRxiv2019

Easily adaptable head-free training system of macaques for tasks requiring precise measurements of eye position

We describe a modified system for training macaque monkeys without invasive head immobilization on visuomotor tasks requiring the control of eye-movements. The system combines a conventional primate chair, a chair-mounted infrared camera for measuring eye-movements and a custom-made concave reward-delivery spout firmly attached to the chair. The animal was seated head-free inside the chair but the concavity of the spout stabilized its head during task performance. Training on visual fixation and discrimination tasks was successfully performed with this system. Eye-measurements, such as fixation-precision, pupil size as well as micro-saccades were comparable to those obtained using conventional invasive head-fixation methods. The system is inexpensive ([~]$40 USD material cost), easy to fabricate in a workshop (technical drawings are included), and readily adjustable between animals without the need to immobilize or sedate them for these adjustments.\n\nHighlightsO_LIWe developed an approach to train macaque monkeys head-free on visuomotor tasks requiring measurements of eye position\nC_LIO_LIThe setup is inexpensive, easy to build, and readily adjusted to the animal without the need for sedation\nC_LIO_LIThe system was tested for training on a visual fixation and a visual discrimination task\nC_LIO_LIEye measurements (fixation precision, pupil size, microsaccades) were comparable to those from head-fixed animals\nC_LI

neuroscience↗

bioRxiv2018

Using confidence inferred from pupil-size to dissect perceptual task-strategy: support for a bounded decision-formation process

During perceptual decisions subjects often rely more strongly on early rather than late sensory evidence even in tasks when both are equally informative about the correct decision. This early psychophysical weighting has been explained by an integration-to-bound decision process, in which the stimulus is ignored after the accumulated evidence reaches a certain bound, or confidence level. Here, we derive predictions about how the average temporal weighting of the evidence depends on a subjects decision-confidence in this model. To test these predictions empirically, we devised a method to infer decision-confidence from pupil size in monkeys performing a disparity discrimination task. Our animals data confirmed the integration-to-bound predictions, with different internal decision-bounds accounting for differences between animals. However, the data could not be explained by two alternative accounts for early psychophysical weighting: attractor dynamics either within the decision area or due to feedback to sensory areas, or a feedforward account due to neuronal response adaptation. This approach also opens the door to using confidence more broadly when studying the neural basis of decision-making.

neuroscience↗

bioRxiv2017

Epidermal stem cells self-renew upon neighboring differentiation

Many adult tissues are dynamically sustained by the rapid turnover of stem cells. Yet, how cell fates such as self-renewal and differentiation are orchestrated to achieve long-term homeostasis remains elusive. Studies utilizing clonal tracing experiments in multiple tissues have argued that while stem cell fate is balanced at the population level, individual cell fate - to divide or differentiate - is determined intrinsically by each cell seemingly at random ( 1 2 3 4 5). These studies leave open the question of how cell fates are regulated to achieve fate balance across the tissue. Stem cell fate choices could be made autonomously by each cell throughout the tissue or be the result of cell coordination ( 6 7). Here we developed a novel live tracking strategy that allowed recording of every division and differentiation event within a region of epidermis for a week. These measurements reveal that stem cell fates are not autonomous. Rather, direct neighbors undergo coupled opposite fate decisions. We further found a clear ordering of events, with self-renewal triggered by neighbor differentiation, but not vice-versa. Typically, around 1-2 days after cell delamination, a neighboring cell entered S/G2 phase and divided. Functional blocking of this local feedback showed that differentiation continues to occur in the absence of cell division, resulting in a rapid depletion of the epidermal stem cell pool. We thus demonstrate that the epidermis is maintained by nearest neighbor coordination of cell fates, rather than by asymmetric divisions or fine-tuned cell-autonomous stochastic fate choices. These findings establish differentiation-dependent division as a core feature of homeostatic control, and define the relevant time and length scales over which homeostasis is enforced in epithelial tissues.

cell biology↗