Biology subjects

Poon, C.

Publications and source records attributed to Poon, C..

12 recordsLinked to original sources

bioRxiv2023

Spatially resolved single-cell analysis uncovers protein kinase C-& expressing microglia with anti-tumor activity in glioblastoma

Glioblastoma (GBM) is a brain tumor that poses a formidable challenge to treatment options available. The tumor microenvironment (TME) in GBM is highly complex, marked by immunosuppression and cellular heterogeneity. Understanding the cellular interactions and their spatial organization within the TME is crucial for developing effective therapeutic strategies. In this study, we integrated single-cell RNA sequencing and spatial transcriptomics in a GBM mouse model to unravel the spatial landscape of the brain TME. We identified a previously unrecognized microglia subtype expressing protein kinase C{delta} (PKC{delta}) associated with potent anti-tumor functions. The presence of PKC{delta}-expressing microglia was confirmed in resected human GBM specimens. Elevating tumoral PKC{delta} expression using niacin or adeno-associated virus in mice enhanced the phagocytosis of GBM cells by microglia in culture and increased the lifespan of mice with intracranial GBM. These findings were corroborated in analyses of the TCGA GBM datasets where low PKC{delta} samples showed negative pathway enrichment for apoptosis, phagocytosis, and immune signaling pathways, as well as lower levels of immune cell infiltration overall. Our study underscores the importance of integrating spatial context to unravel the TME, resulting in the identification of previously unrecognized subsets of microglia with anti-tumor functions. These findings provide valuable insights for advancing innovative immunotherapeutic strategies in GBM.

immunology↗

bioRxiv2023

Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

Ubiquitin modifications alter protein function and stability, thereby regulating cell homeostasis and viability, particularly under stress. Ischemic stroke induces protein ubiquitination at the ischemic periphery, wherein cells remain viable, however the identity of ubiquitinated proteins is unknown. Here, we employed a proteomics approach to identify these proteins in mice undergoing ischemic stroke. The data are available in a searchable web interface (https://hochrainerlab.shinyapps.io/StrokeUbiOmics/). We detected increased ubiquitination of 198 proteins, many of which localize to the postsynaptic density (PSD) of glutamatergic neurons. Among these were proteins essential for maintaining PSD architecture, such as PSD95, as well as NMDA and AMPA receptor subunits. The largest enzymatic group at the PSD with elevated post-ischemic ubiquitination were kinases, such as CaMKII, PKC, Cdk5, and Pyk2, whose aberrant activities are well-known to contribute to post-ischemic neuronal death. Concurrent phospho-proteomics revealed altered PSD-associated phosphorylation patterns, indicative of modified kinase activities following stroke. PSD-located CaMKII, PKC, and Cdk5 activities were decreased while Pyk2 activity was increased after stroke. Removal of ubiquitin restored kinase activities to pre-stroke levels, identifying ubiquitination as the responsible molecular mechanism for post-ischemic kinase regulation. These findings unveil a previously unrecognized role of ubiquitination in the regulation of essential kinases involved in ischemic injury.

neuroscience↗

bioRxiv2022

Myeloid-specific KDM6B inhibition sensitizes Glioblastoma to PD1 blockade

Glioblastoma (GBM) tumors are enriched in immune-suppressive myeloid cells and are refractory to immune checkpoint therapy (ICT). Targeting epigenetic pathways to reprogram the functional phenotype of immune-suppressive myeloid cells to overcome resistance to ICT remains unexplored. Single-cell and spatial transcriptomic analyses of human GBM tumors demonstrated high expression of an epigenetic enzyme - histone 3 lysine 27 demethylase (KDM6B) in intra- tumoral immune-suppressive myeloid cell subsets. Importantly, myeloid-cell specific Kdm6b deletion enhanced pro-inflammatory pathways and improved survival in GBM tumor-bearing mice. Mechanistic studies elucidated that the absence of Kdm6b enhances antigen-presentation, interferon response and phagocytosis in myeloid cells by inhibiting mediators of immune suppression including Mafb, Socs3 and Sirpa. Further, pharmacological inhibition of KDM6B mirrored the functional phenotype of Kdm6b deleted myeloid cells and enhanced anti-PD1 efficacy. Thus, this study identified KDM6B as an epigenetic regulator of the functional phenotype of myeloid cell subsets and a potential therapeutic target to improve response to ICT.

immunology↗

bioRxiv2022

Oral Delivery of Kidney Targeting Nanotherapeutics for Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disorder. Although a variety of candidate drugs have been found to modulate cystogenesis in animal studies, results from clinical trials have often been unfavorable due to low renal bioavailability and drug-induced side effects. To mitigate this, nanoparticles can be designed to deliver drugs directly to the target organ to increase effective dose while limiting off-target side effects. Unfortunately, there are no kidney-targeted nanomedicines clinically available, and most of the existing FDA-approved nanoparticles require intravenous administration which is not suitable for ADPKD that require lifelong therapy. To address this, we developed an oral drug delivery system using chitosan nanoparticles (CS-NP) that were loaded with peptide amphiphile micelles carrying metformin (met), an ADPKD drug candidate (CS-KM-met). We previously showed that CS-NP can shield met in the gastrointestinal tract; thus, we hypothesized that CS-NP could also enhance bioavailability of kidney-targeting micelles (KMs) upon oral administration. Specifically, we measured the loading capacity of KM-met in CS-NP, evaluated the stability of CS-KM-met under acidic conditions that mimic the gastric environment, and measured in vitro therapeutic effects. Upon oral administration in C57BL/6J mice, CS-KM-met showed significantly greater bioavailability and accumulation in the kidneys as compared to KM-met without CS-NP or free met for up to 24 hours. As such, CS-KM-met showed enhanced therapeutic efficacy in vivo upon oral administration in PKD mice (Pkd1fl/fl; Pax8-rtTA; Tet-O-Cre) compared to KM-met only. Herein, we demonstrate the potential of an oral delivery nanoformulation for the treatment of chronic kidney diseases such as ADPKD for the first time.

bioengineering↗

bioRxiv2022

MiniVess: A dataset of rodent cerebrovasculature from in vivo multiphoton fluorescence microscopy imaging

We present MiniVess, the first annotated dataset of rodent cerebrovasculature, acquired using two-photon fluorescence microscopy. MiniVess consists of 70 3D image volumes with segmented ground truths. Segmentations were created using traditional image processing operations, a U-Net, and manual proofreading. Code for image preprocessing steps and the U-Net are provided. Supervised machine learning methods have been widely used for automated image processing of biomedical images. While much emphasis has been placed on the development of new network architectures and loss functions, there has been an increased emphasis on the need for publicly available annotated, or segmented, datasets. Annotated datasets are necessary during model training and validation. In particular, datasets that are collected from different labs are necessary to test the generalizability of models. We hope this dataset will be helpful in testing the reliability of machine learning tools for analyzing biomedical images.

neuroscience↗

bioRxiv2022

The Brain/MINDS Marmoset Connectivity Atlas: Exploring bidirectional tracing and tractography in the same stereotaxic space

We report on the implementation and features of the Brain/MINDS Marmoset Connectivity Atlas, BMCA, a new resource that provides access to anterograde neuronal tracer data in the prefrontal cortex of a marmoset brain. Neuronal tracers combined with fluorescence microscopy are a key technology for the systematic mapping of structural brain connectivity. We selected the prefrontal cortex for mapping due to its important role in higher brain functions. This work introduces the BMCA standard image preprocessing pipeline and tools for exploring and reviewing the data. We developed the BMCA-Explorer, which is an online image viewer designed for data exploration. Unlike other existing image explorers, it visualizes the data of different individuals in a common reference space at an unprecedented high resolution, facilitating comparative studies. To foster the integration with other marmoset brain image databases and cross-species comparisons, we added fiber tractography data from diffusion MRI, retrograde neural tracer data from the Marmoset Brain Connectivity Atlas project, and tools to map image data between marmoset and the human brain image space. This version of BMCA allows direct comparison between the results of 52 anterograde and 164 retrograde tracer injections in the cortex of the marmoset.

neuroscience↗

bioRxiv2021

A simple method of suspending implant wear particles for more physiological modelling of cell-particle interactions in vitro

Arthroplasty implants e.g. hip, knee, spinal disc sustain relatively high compressive loading and friction wear, which lead to the formation of wear particles or debris between articulating surfaces. Despite advances in orthopaedic materials and surface treatments, the production of wear debris from any part of a joint arthroplasty implant is currently unavoidable. Implant wear debris induces host immune responses and inflammation, which causes patient pain and ultimately implant failure through progressive inflammation-mediated osteolysis and implant loosening, where the severity and rate of periprosthetic osteolysis depends on the material and physicochemical characteristics of the wear particles. Evaluating the cytotoxicity of implant wear particles is important for regulatory approved clinical application of arthroplasty implants, as is the study of cell-particle response pathways. However, the particles of polymeric materials commonly used for arthroplasty implants tend to float when placed in culture media, which limits their contact with cell cultures. This study reports a simple means of suspending wear particles in liquid medium using sodium methylcellulose (NaCMC) to provide a more realistic proxy of the interaction between cells and tissues to wear particles in vivo, which are free-floating in synovial fluid within the joint cavity. Low concentrations of NaCMC dissolved in culture medium were found to be effective for suspending polymeric wear particles. Such suspensions may be used as more physiologically-relevant means for testing cellular responses to implant wear debris, as well as studying the combinative effects of shear and wear particle abrasion on cells in a dynamic culture environments such as perfused tissue-on-chip devices.

bioengineering↗

bioRxiv2020

Quantification of Hypoperfusion in Autism Assessed by Diffuse Correlation Spectroscopy and Frequency Domain Near Infrared Spectroscopy

There is a need for quantitative biomarkers for early diagnosis of autism. We showed previously that hemoglobin concentrations show contrast between autistic group vs control group in humans. Cerebral blood flow can provide additional sensitivity for improved characterization. Diffuse correlation spectroscopy (DCS) has been shown to be reliable method to obtain blood flow contrast in animals and humans. Thus, in this study we evaluated DCS in an established autism model. Our results indicate that autistic group had significantly lower blood flow compared to control group. These results confirm the previous results obtained from humans by positron emission tomography and magnetic resonance imaging.

bioengineering↗

bioRxiv2020

Nanoparticle-mediated microRNA-145 Delivery for Vascular Smooth Muscle Cell Phenotype Modulation and Atherosclerosis Treatment

Vascular smooth muscle cells (VSMCs) change from contractile to the synthetic phenotype during atherogenesis and 30-70% of cells that make up plaques have been elucidated to be of VSMC origin. MicroRNA-145 (miR-145) is responsible for regulating VSMC phenotypic switching, and low miR-145 levels in circulation have been linked with atherosclerosis. Hence, we developed nanoparticles for targeted delivery of miR-145 by synthesizing micelles co-assembled with miR-145 and the CCR2-binding peptides for plaque targeting. The miR cargo was protected in micelles from premature endosomal degradation and rescued contractile markers in synthetic VSMCs and SMCs isolated from patient arteries in vitro. In ApoE-/- mid-stage atherosclerotic mice, miR-145 micelles halted plaque growth and maintained contractile phenotypes similar to baseline levels. In early-stage atherosclerosis, a single dose of miR-145 micelles prevented lesion growth by 49%. We present the potential of miR-145 micelles as a therapeutic that can be applied longitudinally and intervene throughout atherosclerosis pathogenesis.

bioengineering↗

bioRxiv2020

Measuring the density and viscosity of culture media for optimized computational fluid dynamics analysis of in vitro devices

Culture medium is frequently modelled as water in computational fluid dynamics (CFD) analysis of in vitro culture systems involving flow, such as bioreactors and organ-on-chips. However, culture medium can be expected to have different properties to water due to its higher solute content. Furthermore, cellular activities such as metabolism and secretion of ECM proteins alter the composition of culture medium and therefore its properties during culture. As these properties directly determine the hydromechanical stimuli exerted on cells in vitro, these, along with any changes during culture must be known for CFD modelling accuracy and meaningful interpretation of cellular responses. In this study, the density and dynamic viscosity of DMEM and RPMI-1640 media supplemented with typical concentrations of foetal bovine serum (0, 5, 10 and 20% v/v) were measured to serve as a reference for computational design analysis. Any changes in the properties of medium during culture were also investigated with NCI-H460 and HN6 cell lines. The density and dynamic viscosity of the media increased proportional to the % volume of added foetal bovine serum (FBS). Importantly, the viscosity of 5% FBS-supplemented RPMI-1640 was found to increase significantly after 3 days of culture of NCI-H460 and HN6 cell lines, with distinct differences between magnitude of change for each cell line. Finally, these experimentally-derived values were applied in CFD analysis of a simple microfluidic device, which demonstrated clear differences in maximum wall shear stress and pressure between fluid models. Overall, these results highlight the importance of characterizing model-specific properties for CFD design analysis of cell culture systems.

bioengineering↗

bioRxiv2020

Biomimetic system design for engineering biofidelic 3-D respiratory tissues in vitro

Objective The structural and functional complexity of the respiratory system present significant challenges to capturing conditions vital for maintaining phenotypic cellular functions in vitro. Here we report a unique tissue engineering system that enables respiratory constructs to be cultured under physiological loading at an air-liquid interface (ALI).Methods The system consists of a porous poly-e-caprolactone scaffold mounted in a well insert, which articulates via magnetic coupling with a linear actuator device to strain attached scaffolds through a sterile barrier. For proof of concept, NCI-H460 human carcinoma cells were seeded on scaffold inserts which were subjected to 5-15% cyclic tensile strain at 0.2Hz within a six well plate. The dynamic constructs were cultured at an ALI in a standard incubator for up to 10 days along with unstimulated (static) ALI and static submerged control groups.Results High (near-100%) cell seeding efficiency was achieved within the scaffold-strain device. Both dynamic and static ALI groups yielded higher cell densities compared to the submerged control for all time points. Distinctly different patterns in cellular growth and behaviour between dynamic air-liquid interface and conventional static submerged culture groups were revealed by nuclei staining, where the actuated group displayed more uniform cellular distribution throughout the construct compared to both static controls.Conclusion Air-liquid interface culture and physiological strain are important for engineering respiratory tissue models.Significance The system described allows scalable and replicable culture of 3-D tissue engineered respiratory models under biologically-relevant conditions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗

bioRxiv2020

G9a methyltransferase governs cell identity in the lung and is required for KRAS G12D tumor development and propagation

Lung development, integrity and repair rely on precise Wnt signaling, which is corrupted in diverse diseases, including cancer. Here, we discover that G9a methyltransferase regulates Wnt signaling in the lung by controlling the transcriptional activity of chromatin-bound {beta}-catenin, through a non-histone substrate. Inhibition of G9a induces transcriptional, morphologic, and molecular changes consistent with alveolar type 2 (AT2) lineage commitment. Mechanistically, G9a activity functions to support regenerative properties of KrasG12D tumors and normal AT2 cells - the predominant cell of origin of this cancer. Consequently, G9a inhibition prevents KrasG12D lung adenocarcinoma tumor formation and propagation,and disrupts normal AT2 cell trans-differentiation. Consistent with these findings, low G9a expression in human lung adenocarcinoma correlates with enhanced AT2 gene expression and improved prognosis. These data reveal G9a as a critical regulator of Wnt signaling, implicating G9a as a potential target in lung cancer and other AT2-mediated lung pathologies.

cancer biology↗