Biology subjects

Chan, W. K.

Publications and source records attributed to Chan, W. K..

9 recordsLinked to original sources

bioRxiv2025

Cerebral Organoids with Integrated Endothelial Networks Emulate the Neurovascular Unit and Mitigate Core Necrosis

Cerebral organoids (COs) are multicellular, self-organized, in vitro, 3D brain-like tissues used for developmental biology, disease modelling and drug screening. However, their lack of vascularity renders them less physiologically accurate. Vascularization of COs remains challenging due to the different requirements between COs and vascular cells, limited vascular network penetration within the organoid, and the absence of luminal perfusion. Here, we devised an encapsulation approach in which human brain microvascular endothelial cells (HBMVECs) were delivered to developing COs from progressively degrading extracellular matrix (ECM)-based hydrogel droplets. By tuning this hydrogel concentration and media composition, we observed enhanced vascular-like network formation that expanded within the organoid tissue. Using pathway inhibitors, we showed that a subset of the endothelial cells (ECs) originated from the CO itself, promoting network integration. Endothelial networks displayed blood-brain barrier (BBB) features, including astrocytic end-footlike interactions, pericyte wrapping, and collagen-laminin basal lamina. Vascularized COs exhibited greater media internalization and up to three-fold lower apoptosis than non-vascularized COs. This comprehensive 3D neurovascular model is a promising platform for cerebrovascular research and drug testing applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/650161v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@175edb7org.highwire.dtl.DTLVardef@1499b91org.highwire.dtl.DTLVardef@184567borg.highwire.dtl.DTLVardef@149c8e4_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG HighlightsO_LIAddition and angiogenic stimulation of human brain microvascular endothelial cells (HBMVECs) co-cultured with cerebral organoids (COs) generate multicellular vascular-like networks C_LIO_LIVascularization induces changes in organoid morphology but not on tissue stiffness C_LIO_LIEndothelial networks morphological features are correlated with the concentration of the supporting matrix C_LIO_LIA number of the endothelial cells (ECs) in the networks originate from the organoid itself C_LIO_LIEndothelial networks integrate within the organoid tissue interacting with astrocytes and pericyte-like cells, and are surrounded by basement membrane-like depositions C_LIO_LIVascularized COs exhibit higher media diffusion, and a reduced necrotic core compared to non-vascularized COs C_LI

bioengineering↗

bioRxiv2024

Design of linked-domain protein inhibitors of UBE2D as tools to study cellular ubiquitination

Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC50 to cisplatin and whole-cell proteomics reveal an increased abundance of [~]20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2Ds central role in proteome management.

biochemistry↗

bioRxiv2024

Mitochondrial defects and metabolic vulnerabilities in Lynch syndrome-associated MSH2-deficient endometrial cancer

Lynch syndrome (LS) is defined by inherited mutations in DNA mismatch repair genes, including MSH2, and carries 60% lifetime risk of developing endometrial cancer (EC). Beyond hypermutability, specific mechanisms for LS-associated endometrial carcinogenesis are not well understood. Here, we assessed the effects of MSH2 loss on EC pathogenesis using a novel mouse model (PR-Cre Msh2flox/flox, abbreviated Msh2KO), primary cell lines established from this model, human tissues, and human EC cell lines with isogenic MSH2 knockdown. Beginning at eight months of age, 30% of Msh2KO mice exhibited endometrial atypical hyperplasia (AH), a precancerous lesion. At 12 to 16 months of age, 47% of Msh2KO mice exhibited either AH or ECs with histologic features similar to human LS-related ECs. Transcriptomic profiling of EC from Msh2KO mice revealed a transcriptomic signature for mitochondrial dysfunction. Studies in vitro and in vivo revealed mitochondrial dysfunction based upon two mechanisms: marked mitochondrial content reduction, along with pronounced disruptions to the integrity of retained mitochondria. Human LS-related ECs also exhibited mitochondrial content reduction compared with non-LS-related ECs. Functional studies revealed metabolic reprogramming of MSH2-deficient EC cells in vitro, including reduced oxidative phosphorylation and increased susceptibility to glycolysis suppression. We are the first to identify mitochondrial dysfunction and metabolic disruption as a consequence of MSH2 deficiency-related EC. Mitochondrial and metabolic aberrations should be evaluated as novel biomarkers for endometrial carcinogenesis or risk stratification and could serve as targets for cancer interception in women with LS. SignificanceThis is the first study to report mitochondrial dysfunction contributing to MSH2-deficient endometrial cancer development, identifying a noncanonical pathway for MSH2 deficient carcinogenesis, which also imparts vulnerability to metabolic targeting.

cancer biology↗

bioRxiv2023

Prdm1 Positively Regulates Liver Type 1 ILCs Cancer Immune Surveillance and Preserves Functional Heterogeneity

Group 1 innate lymphoid cells (ILCs) comprise conventional natural killer (cNK) cells and type 1 innate lymphoid cells (ILC1s). The main functions of liver cNK cells and ILC1s not only include directly killing target cells but also regulating local immune microenvironment of the liver through the secretion of cytokines. Uncovering the intricate mechanisms by which transcriptional factors regulate and influence the functions of liver cNK cells and ILC1s, particularly within the context of liver tumors, presents a significant opportunity to amplify the effectiveness of immunotherapies against liver malignancies. Using Ncr1-drived conditional knockout mouse model, our study reveals the regulatory role of Prdm1 in shaping the composition and maturation of cNK cells. Although Prdm1 did not affect the killing function of cNK cells in an in vivo cytotoxicity model, a significant increase in cancer metastasis was observed in Prdm1 knockout mice. Interferon-gamma (IFN-{gamma}), granzyme B, and perforin secretion decreased significantly in Prdm1 deficient cNK cells and liver ILC1s. scRNA sequencing data also provided evidences that Prdm1 maintains functional subsets of cNK cells and liver ILC1s and facilitates communications between cNK cells, liver ILC1s and macrophages. The present study unveiled a novel regulatory mechanism of Prdm1 in cNK cells and liver ILC1s, showing promising potential for developing innovative immune therapy strategies against liver cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/563222v4_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@194f9forg.highwire.dtl.DTLVardef@1ba5b0eorg.highwire.dtl.DTLVardef@1b02121org.highwire.dtl.DTLVardef@4f0385_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Loss of PAX6 alters the excitatory/inhibitory neuronal ratio in human cerebral organoids

The transcription factor PAX6 is a crucial regulator of multiple aspects of embryonic forebrain development. It has well-established roles in the regulation of excitatory and inhibitory neuron development in the embryonic cortex in mice but PAX6s roles during human forebrain development are less well understood. Using human cerebral organoids, we investigated PAX6s roles in human neurodevelopment. Homozygous PAX6 mutant (PAX6-/-) organoids were larger than controls and contained increased inhibitory cell types. Excitatory neurons were still generated in PAX6-/- organoids but they were less mature, and a subset showed dysregulated expression of inhibitory identity genes compared to PAX6+/+ controls. The inhibitory cells found in PAX6-/- organoids physically segregated from excitatory neurons and presented a distinct transcriptomic profile when compared to in vivo cortical inhibitory neurons. PAX6-/- organoids showed a dysregulated cellular response to PTN-PTPRZ1 signalling, which contributed to the observed increase in inhibitory neurons and the consequent altered excitatory to inhibitory neuronal ratio. Summary StatementHuman cerebral organoids lacking PAX6 expression show an altered excitatory to inhibitory neuronal ratio, concomitant with altered responses to intercellular PTN-PTPRZ1 signalling.

developmental biology↗

bioRxiv2022

PAX6 LIMITS THE COMPETENCE OF DEVELOPING CEREBRAL CORTICAL CELLS

The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6s main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side-effects of morphogens engaged contemporaneously in other essential functions.

neuroscience↗

bioRxiv2021

The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila homologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and spermatocytes, the only two Drosophila cell types bearing motile cilia/flagella. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly. SUMMARY STATEMENTDNAAF3 function as a dynein assembly factor for motile cilia is conserved in Drosophila.

cell biology↗

bioRxiv2020

Applications of genetic-epigenetic tissue mapping for plasma DNA in prenatal testing, transplantation and oncology

We developed Genetic-Epigenetic Tissue Mapping (GETMap) to determine the tissue composition of plasma DNA carrying genetic variants not present in the constitutional genome through comparing their methylation profiles with relevant tissues. We validated this approach by showing that, in pregnant women, circulating DNA carrying fetal-specific alleles was entirely placenta-derived. In lung-transplant recipients, we showed that, at 72 hours after transplantation, the lung contributed only a median of 17% to the plasma DNA carrying donor-specific alleles and hematopoietic cells contributed a median of 78%. In hepatocellular cancer patients, the liver was identified as the predominant source of plasma DNA carrying tumor-specific mutations. In a pregnant woman with lymphoma, plasma DNA molecules carrying cancer mutations and fetal-specific alleles were accurately shown to be derived from the lymphocytes and placenta, respectively. Analysis of tissue origin for plasma DNA carrying genetic variants is potentially useful for noninvasive prenatal testing, transplantation monitoring and cancer screening.

physiology↗

bioRxiv2018

Enteral Activation of WR-2721 Mediates Radioprotection and Improved Survival from Lethal Fractionated Radiation

Unresectable pancreatic cancer is almost universally lethal because chemotherapy and radiation cannot completely stop the growth of the cancer. The major problem with using radiation to approximate surgery in unresectable disease is that the radiation dose required to ablate pancreatic cancer exceeds the tolerance of the nearby duodenum. WR-2721, also known as amifostine, is a well-known radioprotector, but has significant clinical toxicities when given systemically. WR-2721 is a prodrug and is converted to its active metabolite, WR-1065, by alkaline phosphatases in normal tissues. The small intestine is highly enriched in these activating enzymes, and thus we reasoned that oral administration of WR-2721 just before radiation would result in localized production of the radioprotective WR-1065 in the small intestine, providing protective benefits without the significant systemic side effects. Here, we show that oral WR-2721 is as effective as intraperitoneal WR-2721 in promoting survival of intestinal crypt clonogens after morbid irradiation. Furthermore, oral WR-2721 confers full radioprotection and survival after lethal upper abdominal irradiation of 12.5Gy x 5 fractions (total of 62.5Gy, D2EQ=140.6Gy), which would likely ablate pancreatic cancer. We find that the efficacy of oral WR-2721 stems from its selective accumulation in the intestine, but not in tumors or other normal tissues, as determined by in vivo mass spectrometry analysis. Thus, we demonstrate that oral WR-2721 is a well-tolerated, and quantitatively selective, radioprotector of the intestinal tract that is capable of enabling clinically relevant ablative doses of radiation to the upper abdomen without unacceptable gastrointestinal toxicity.

cancer biology↗