ORCID Profile
0000-0001-5852-3726
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Applied Statistics | Biological Mathematics | Animal Physiology - Cell | Animal Neurobiology | Simulation and Modelling | Immunology | Biomechanical Engineering | Physiology | Artificial Intelligence and Image Processing | Cellular Immunology | Receptors and Membrane Biology | Cell Development, Proliferation and Death |
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Information and Computing Sciences | Expanding Knowledge in Engineering | Expanding Knowledge in the Mathematical Sciences | Immune System and Allergy | Expanding Knowledge in Technology
Related Links
Publications
Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.CRC-22-0305
Abstract: Solid cancer cells escape the primary tumor mass by transitioning from an epithelial-like state to an invasive migratory state. As they escape, metastatic cancer cells employ interchangeable modes of invasion, transitioning between fibroblast-like mesenchymal movement to amoeboid migration, where cells display a rounded morphology and navigate the extracellular matrix in a protease-independent manner. However, the gene transcripts that orchestrate the switch between epithelial, mesenchymal, and amoeboid states remain incompletely mapped, mainly due to a lack of methodologies that allow the direct comparison of the transcriptomes of spontaneously invasive cancer cells in distinct migratory states. Here, we report a novel single-cell isolation technique that provides detailed three-dimensional data on melanoma growth and invasion, and enables the isolation of live, spontaneously invasive cancer cells with distinct morphologies and invasion parameters. Via the expression of a photoconvertible fluorescent protein, compact epithelial-like cells at the periphery of a melanoma mass, elongated cells in the process of leaving the mass, and rounded amoeboid cells invading away from the mass were tagged, isolated, and subjected to single-cell RNA sequencing. A total of 462 differentially expressed genes were identified, from which two candidate proteins were selected for further pharmacologic perturbation, yielding striking effects on tumor escape and invasion, in line with the predictions from the transcriptomics data. This work describes a novel, adaptable, and readily implementable method for the analysis of the earliest phases of tumor escape and metastasis, and its application to the identification of genes underpinning the invasiveness of malignant melanoma. This work describes a readily implementable method that allows for the isolation of in idual live tumor cells of interest for downstream analyses, and provides the single-cell transcriptomes of melanoma cells at distinct invasive states, both of which open avenues for in-depth investigations into the transcriptional regulation of the earliest phases of metastasis.
Supplementary Figure 1 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932466.V1
Abstract: Supplementary Figure 1 shows data generation for the photoconvertible spheroid assay
CTCF genetic alterations in endometrial carcinoma are pro-tumorigenic
Publisher: Springer Science and Business Media LLC
Date: 20-03-2017
DOI: 10.1038/ONC.2017.25
FIGURE 5 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932472.V1
Abstract: Inhibition of OPRD1 reduces melanoma spheroid growth and invasion. b A, /b Representative maximum intensity projections of imaged hemispheroids for vehicle- and naltrindole-treated spheroids, scale bar 200 μm. b B, /b Relative spheroid volume over time for vehicle- and naltrindole-treated cells. Shaded areas represent SEM. b C, /b Cumulative number of escaped cells over time normalized to spheroid surface area. B and C, Datapoints represent the mean of eight replicates.
Keratins are asymmetrically inherited fate determinants in the mammalian embryo
Publisher: Springer Science and Business Media LLC
Date: 26-08-2020
DOI: 10.1038/S41586-020-2647-4
Abstract: To implant in the uterus, the mammalian embryo first specifies two cell lineages: the pluripotent inner cell mass that forms the fetus, and the outer trophectoderm layer that forms the placenta
SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling
Publisher: eLife Sciences Publications, Ltd
Date: 20-01-2022
DOI: 10.7554/ELIFE.67550
Abstract: T cell activation requires engagement of a cognate antigen by the T cell receptor (TCR) and the co-stimulatory signal of CD28. Both TCR and CD28 aggregate into clusters at the plasma membrane of activated T cells. While the role of TCR clustering in T cell activation has been extensively investigated, little is known about how CD28 clustering contributes to CD28 signalling. Here, we report that upon CD28 triggering, the BAR-domain protein sorting nexin 9 (SNX9) is recruited to CD28 clusters at the immunological synapse. Using three-dimensional correlative light and electron microscopy, we show that SNX9 generates membrane tubulation out of CD28 clusters. Our data further reveal that CD28 clusters are in fact dynamic structures and that SNX9 regulates their stability as well as CD28 phosphorylation and the resulting production of the cytokine IL-2. In summary, our work suggests a model in which SNX9-mediated tubulation generates a membrane environment that promotes CD28 triggering and downstream signalling events.
Supplementary Video 2B from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932412
Abstract: Supplementary Video 2B shows representative images of a Sulfasalazine-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Supplementary Figure 4 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932457.V1
Abstract: Supplementary Figure 4 shows an Epithelial cohort gallery
Supplementary Video 2A from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932415
Abstract: Supplementary Video 2A shows representative images of a vehicle-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Supplementary Video 1B from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932418
Abstract: Supplementary Video 1B shows representative images of a naltrindole-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Material-mediated histogenesis using mechano-chemically microstructured cell niches
Publisher: Cold Spring Harbor Laboratory
Date: 11-02-2021
DOI: 10.1101/2021.02.10.430691
Abstract: Stem-cell derived tissue models are commonly cultured under globally-delivered stimuli that trigger histogenesis via self-organizing activity. However, the culture of such tissue models is prone to stochastic behavior, limiting the reproducibility of cellular composition and resulting in non-physiological architectures. To overcome these shortcomings, we developed a method for printing cell niche microenvironments with microstructured cues that mediate local histogenic processes, including mechanosensing and differentiation of selected cell types. Microstructured cues include independently tunable mechano-chemical properties, with conjugated peptides, proteins, and morphogens across a range of Young’s moduli. By rationally designing niches, we mediate the structure of tissues derived from stem-cell-progenitor sources, including a bone-fat assembly from stromal mesenchyme, and embryonic tissues derived from hiPSC. We show that microstructured cues can recapitulate mechano-chemical signals resembling early embryonic histogenesis. This outcome includes a role for niche mechanics in human embryonic organization, where soft niche mechanics bias markers of mesendodermal differentiation and epithelial-to-mesenchymal-transition (EMT), as well as a demonstration of a material-mediated morphogen signaling centers able to induce foci of mesenchymal and EMT differentiation. Thus, microstructured materials can mediate local histogenic processes to enhance the structure and composition of tissue models.
Porous and strong three-dimensional carbon nanotube coated ceramic scaffolds for tissue engineering
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5TB01052G
Abstract: A method to coat high-quality uniform coatings of carbon nanotubes throughout 3D porous structures is developed. Testing of their physical and biological properties demonstrate their potential for application in tissue engineering.
FIGURE 6 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932469.V1
Abstract: SSZ treatment slows spheroid growth but induces tumor escape by elongated cells. b A, /b Representative maximum intensity projections of imaged hemispheroids for vehicle- and SSZ-treated spheroids. Scale bar, 200 μm. b B, /b Relative spheroid volume for vehicle- and SSZ-treated spheroids. b C, /b Cumulative escaped cells per surface area of imaged hemispheroid. b D, /b Mean escaped cell sphericity per day. B–D, Shaded areas represent SEM.
Targeting R ho‐GTP ases in immune cell migration and inflammation
Publisher: Wiley
Date: 02-07-0006
DOI: 10.1111/BPH.12658
Supplementary Figure 7 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932448.V1
Abstract: Supplementary Figure 7 shows metadata for isolated cells
Supplementary Video 2B from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696119
Abstract: Supplementary Video 2B shows representative images of a Sulfasalazine-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
FIGURE 4 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932475.V1
Abstract: Gene expression analysis for epithelial, escaping, and amoeboid cells. b A, /b Top: 462 genes were DE, 144 for escaping versus epithelial (Esc-v-Epi), 22 for amoeboid versus escaping (Amo-v-Esc), and 422 for amoeboid versus epithelial (Amo-v-Epi). Bottom: Venn diagram of the number of DE genes for the three pairwise comparisons. b B, /b Volcano plot for Esc-v-Epi DE analysis, with genes enriched in “escaping” cells shown as positive on the i x /i -axis. b C, /b Volcano plot for Amo-v-Esc DE analysis, with genes enriched in “amoeboid” cells shown as positive on the i x /i -axis. b D, /b Volcano plot for Amo-v-Epi DE analysis, with genes enriched in “amoeboid” cells shown as positive on the i x /i -axis. For B–D, significantly DE genes with an adjusted i P /i value 0.05 are shown in light blue, with no minimum value for log sub /sub FC. b E, /b Expression of EMT markers for epithelial, leaving, and amoeboid cells shown via dot plot. b F, /b Expression of cancer stem cell markers for epithelial, leaving, and amoeboid cells shown via dot plot.
FIGURE 2 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932481.V1
Abstract: Single-cell tagging and isolation based on morphologic and positional data. b A, /b Sphericity and distance to spheroid values are generated for each invading cell surrounding the spheroid. Left panel depicts maximum intensity projections for three invading cells (Fluo) of different sphericities, following cell surface segmentation (Fluo + Surf), and following surface pseudocoloring according to sphericity value (Surf + Pseudo). Right panels illustrate the smoothed spheroid surface (red) computed to calculate the nearest distance (dashed arrow) to the spheroid edge from in idual cells. b B, /b Left: The three photoconverted cell types “Epithelial,” “Escaping,” and “Amoeboid.” Middle: Representative images of the photoconversion of the three cell types and corresponding flow cytometry scatter plots of Dendra2 red versus green fluorescence obtained during single-cell sorting. Right: The number of cells photoconverted and recovered via single-cell sorting for escaping and amoeboid cells. b C, /b Sorting yields expressed as a percentage of photoconverted cells for escaping and amoeboid cells.
Supplementary Video 1A from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932421
Abstract: Supplementary Video 1A shows representative daily images of a vehicle-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Supplementary Figure 3 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932460.V1
Abstract: Supplementary Figure 3 shows validation of OPRD1 and SLC19A1 protein expression
Tropomyosin concentration but not formin nucleators mDia1 and mDia3 determines the level of tropomyosin incorporation into actin filaments
Publisher: Springer Science and Business Media LLC
Date: 24-04-2019
DOI: 10.1038/S41598-019-42977-2
Abstract: The majority of actin filaments in human cells exist as a co-polymer with tropomyosin, which determines the functionality of actin filaments in an isoform dependent manner. Tropomyosin isoforms are sorted to different actin filament populations and in yeast this process is determined by formins, however it remains unclear what process determines tropomyosin isoform sorting in mammalian cells. We have tested the roles of two major formin nucleators, mDia1 and mDia3, in the recruitment of specific tropomyosin isoforms in mammals. Despite observing poorer cell-cell attachments in mDia1 and mDia3 KD cells and an actin bundle organisation defect with mDia1 knock down depletion of mDia1 and mDia3 in idually and concurrently did not result in any significant impact on tropomyosin recruitment to actin filaments, as observed via immunofluorescence and measured via biochemical assays. Conversely, in the presence of excess Tpm3.1, the absolute amount of Tpm3.1-containing actin filaments is not fixed by actin filament nucleators but rather depends on the cell concentration of Tpm3.1. We conclude that mDia1 and mDia3 are not essential for tropomyosin recruitment and that tropomyosin incorporation into actin filaments is concentration dependent.
Supplementary Figure 5 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932454.V1
Abstract: Supplementary Figure 5 shows an Escaping cell cohort gallery
The Skin Immune Atlas: Three-Dimensional Analysis of Cutaneous Leukocyte Subsets by Multiphoton Microscopy
Publisher: Elsevier BV
Date: 2015
DOI: 10.1038/JID.2014.289
Supplementary Figure 3 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696167.V1
Abstract: Supplementary Figure 3 shows validation of OPRD1 and SLC19A1 protein expression
Supplementary Table 6 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932424
Abstract: Supplementary Table 6 shows Sorting events for each cell included in the final data set
Relationship between nanotopographical alignment and stem cell fate with live imaging and shape analysis
Publisher: Springer Science and Business Media LLC
Date: 02-12-2016
DOI: 10.1038/SREP37909
Abstract: The topography of a biomaterial regulates cellular interactions and determine stem cell fate. A complete understanding of how topographical properties affect cell behavior will allow the rational design of material surfaces that elicit specified biological functions once placed in the body. To this end, we fabricate substrates with aligned or randomly organized fibrous nanostructured topographies. Culturing adipose-derived stem cells (ASCs), we explore the dynamic relationship between the alignment of topography, cell shape and cell differentiation to osteogenic and myogenic lineages. We show aligned topographies differentiate cells towards a satellite cell muscle progenitor state - a distinct cell myogenic lineage responsible for postnatal growth and repair of muscle. We analyze cell shape between the different topographies, using fluorescent time-lapse imaging over 21 days. In contrast to previous work, this allows the direct measurement of cell shape at a given time rather than defining the morphology of the underlying topography and neglecting cell shape. We report quantitative metrics of the time-based morphological behaviors of cell shape in response to differing topographies. This analysis offers insights into the relationship between topography, cell shape and cell differentiation. Cells differentiating towards a myogenic fate on aligned topographies adopt a characteristic elongated shape as well as the alignment of cells.
Supplementary Table 5 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932427
Abstract: Supplementary Table 5 shows Differentially expressed genes for epithelial vs. amoeboid analysis
Supplementary Figure 3 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696167
Abstract: Supplementary Figure 3 shows validation of OPRD1 and SLC19A1 protein expression
Supplementary Video 1B from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696125.V1
Abstract: Supplementary Video 1B shows representative images of a naltrindole-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Supplementary Figure 8 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932445.V1
Abstract: Supplementary Figure 8 shows a summary of GSEA pathway analyses using Hallmarks gene set showing top 25 enriched pathways for each comparison
Supplementary Table 1 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932439.V1
Abstract: Supplementary Table 1 shows Cell type isolated, age (days) and number of cells sorted for each spheroid included in the study
Supplementary Figure 5 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696161
Abstract: Supplementary Figure 5 shows an Escaping cell cohort gallery
Supplementary Video 2B from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696119.V1
Abstract: Supplementary Video 2B shows representative images of a Sulfasalazine-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Supplementary Figure 4 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696164
Abstract: Supplementary Figure 4 shows an Epithelial cohort gallery
Supplementary Figure 4 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696164.V1
Abstract: Supplementary Figure 4 shows an Epithelial cohort gallery
Supplementary Table 4 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932430
Abstract: Supplementary Table 4 shows Differentially expressed genes for amoeboid vs. escaping analysis
FIGURE 3 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932478.V1
Abstract: Clustering of single cells and their gene expression heat map based on 462 DE genes. b A, /b UMAP clustering of 35 cells based on expression of 462 DE genes. b B, /b Unsupervised hierarchical clustering of 35 cells based on the expression of 462 DE genes. Cells initially erge into two groups, epithelial (L1) and invasive (R1), before further erging into escaping (R2) and amoeboid (R3). b C, /b Heat map of 462 DE genes for single cells arranged according to the unsupervised clustering in B.
Supplementary Table 3 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932433
Abstract: Supplementary Table 3 shows Differentially expressed genes for escaping vs. epithelial analysis
FIGURE 1 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932484.V1
Abstract: Methodologic pipeline for single-cell isolation of live escaping tumor cells. b , /b A clonal population of tumor cells are expanded from a single cell. b , /b Photoconvertible cells are seeded in ULA plates to generate spheroids. b , /b Spheroids are embedded within a 3D collagen matrix and imaged over time by light microscopy. b , /b Image analysis and extraction of quantitative metrics can be used to select cells of interest. b , /b Single cells of interest are photoconverted using a 405 nm laser. b , /b Collagen matrix is enzymatically digested to provide a solution of suspended single cells. b , /b Cells are sorted via FACS. b , /b Cells can be further cultured or immediately analyzed.
Supplementary Table 2 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932436.V1
Abstract: Supplementary Table 2 shows Transcript counts for common housekeeping genes, with cells excluded from the study due to low housekeeping expression highlighted in yellow
Supplementary Table 2 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932436
Abstract: Supplementary Table 2 shows Transcript counts for common housekeeping genes, with cells excluded from the study due to low housekeeping expression highlighted in yellow
Supplementary Figure 6 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932451.V1
Abstract: Supplementary Figure 6 shows an Amoeboid cell cohort gallery
Supplementary Table 1 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932439
Abstract: Supplementary Table 1 shows Cell type isolated, age (days) and number of cells sorted for each spheroid included in the study
Supplementary Video 2A from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696122.V1
Abstract: Supplementary Video 2A shows representative images of a vehicle-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Supplementary Figure 9 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932442.V1
Abstract: Supplementary Figure 9 shows Escaped cell analyses for naltrindole and sulfasalazine spheroid assays
Supplementary Figure 7 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696155.V1
Abstract: Supplementary Figure 7 shows metadata for isolated cells
Supplementary Figure 2 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696170
Abstract: Supplementary Figure 2 shows clustering of 44 single cells and their gene expression heat map based on 230 differentially expressed genes
Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption
Publisher: Elsevier BV
Date: 03-2021
Supplementary Figure 1 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696173
Abstract: Supplementary Figure 1 shows data generation for the photoconvertible spheroid assay
Flotillins promote T cell receptor sorting through a fast Rab5–Rab11 endocytic recycling axis
Publisher: Springer Science and Business Media LLC
Date: 26-09-2019
DOI: 10.1038/S41467-019-12352-W
Abstract: The targeted endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation. Despite this, the mechanisms that underlie the sorting of internalised receptors into recycling endosomes remain poorly understood. To build a comprehensive picture of TCR recycling during T cell activation, we developed a suite of new imaging and quantification tools centred on photoactivation of fluorescent proteins. We show that the membrane-organising proteins, flotillin-1 and -2, are required for TCR to reach Rab5-positive endosomes immediately after endocytosis and for transfer from Rab5- to Rab11a-positive compartments. We further observe that after sorting into in Rab11a-positive vesicles, TCR recycles to the plasma membrane independent of flotillin expression. Our data suggest a mechanism whereby flotillins delineate a fast Rab5-Rab11a endocytic recycling axis and functionally contribute to regulate the spatial organisation of these endosomes.
Partial loss of actin nucleator actin‐related protein 2/3 activity triggers blebbing in primary T lymphocytes
Publisher: Wiley
Date: 23-12-2020
DOI: 10.1111/IMCB.12304
Supplementary Figure 9 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932442
Abstract: Supplementary Figure 9 shows Escaped cell analyses for naltrindole and sulfasalazine spheroid assays
Supplementary Figure 5 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696161.V1
Abstract: Supplementary Figure 5 shows an Escaping cell cohort gallery
Supplementary Figure 8 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932445
Abstract: Supplementary Figure 8 shows a summary of GSEA pathway analyses using Hallmarks gene set showing top 25 enriched pathways for each comparison
Supplementary Figure 6 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696158.V1
Abstract: Supplementary Figure 6 shows an Amoeboid cell cohort gallery
TriKEs and BiKEs join CARs on the cancer immunotherapy highway
Publisher: Informa UK Limited
Date: 20-06-2016
Supplementary Figure 7 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932448
Abstract: Supplementary Figure 7 shows metadata for isolated cells
Detection and characterization of chemotaxis without cell tracking
Publisher: The Royal Society
Date: 03-2021
Abstract: Swarming has been observed in various biological systems from collective animal movements to immune cells. In the cellular context, swarming is driven by the secretion of chemotactic factors. Despite the critical role of chemotactic swarming, few methods to robustly identify and quantify this phenomenon exist. Here, we present a novel method for the analysis of time series of positional data generated from realizations of agent-based processes. We convert the positional data for each in idual time point to a function measuring agent aggregation around a given area of interest, hence generating a functional time series. The functional time series, and a more easily visualized swarming metric of agent aggregation derived from these functions, provide useful information regarding the evolution of the underlying process over time. We extend our method to build upon the modelling of collective motility using drift–diffusion partial differential equations (PDEs). Using a functional linear model, we are able to use the functional time series to estimate the drift and diffusivity terms associated with the underlying PDE. By producing an accurate estimate for the drift coefficient, we can infer the strength and range of attraction or repulsion exerted on agents, as in chemotaxis. Our approach relies solely on using agent positional data. The spatial distribution of diffusing chemokines is not required, nor do in idual agents need to be tracked over time. We demonstrate our approach using random walk simulations of chemotaxis and experiments investigating cytotoxic T cells interacting with tumouroids.
Data from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.C.6745837
Abstract: Solid cancer cells escape the primary tumor mass by transitioning from an epithelial-like state to an invasive migratory state. As they escape, metastatic cancer cells employ interchangeable modes of invasion, transitioning between fibroblast-like mesenchymal movement to amoeboid migration, where cells display a rounded morphology and navigate the extracellular matrix in a protease-independent manner. However, the gene transcripts that orchestrate the switch between epithelial, mesenchymal, and amoeboid states remain incompletely mapped, mainly due to a lack of methodologies that allow the direct comparison of the transcriptomes of spontaneously invasive cancer cells in distinct migratory states. Here, we report a novel single-cell isolation technique that provides detailed three-dimensional data on melanoma growth and invasion, and enables the isolation of live, spontaneously invasive cancer cells with distinct morphologies and invasion parameters. Via the expression of a photoconvertible fluorescent protein, compact epithelial-like cells at the periphery of a melanoma mass, elongated cells in the process of leaving the mass, and rounded amoeboid cells invading away from the mass were tagged, isolated, and subjected to single-cell RNA sequencing. A total of 462 differentially expressed genes were identified, from which two candidate proteins were selected for further pharmacologic perturbation, yielding striking effects on tumor escape and invasion, in line with the predictions from the transcriptomics data. This work describes a novel, adaptable, and readily implementable method for the analysis of the earliest phases of tumor escape and metastasis, and its application to the identification of genes underpinning the invasiveness of malignant melanoma. Significance: This work describes a readily implementable method that allows for the isolation of in idual live tumor cells of interest for downstream analyses, and provides the single-cell transcriptomes of melanoma cells at distinct invasive states, both of which open avenues for in-depth investigations into the transcriptional regulation of the earliest phases of metastasis. /
Supplementary Video 1B from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932418.V1
Abstract: Supplementary Video 1B shows representative images of a naltrindole-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Supplementary Table 1 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696146
Abstract: Supplementary Table 1 shows Cell type isolated, age (days) and number of cells sorted for each spheroid included in the study
Supplementary Figure 8 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696152.V1
Abstract: Supplementary Figure 8 shows a summary of GSEA pathway analyses using Hallmarks gene set showing top 25 enriched pathways for each comparison
Supplementary Figure 9 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696149
Abstract: Supplementary Figure 9 shows Escaped cell analyses for naltrindole and sulfasalazine spheroid assays
Nuclear F-actin counteracts nuclear deformation and promotes fork repair during replication stress
Publisher: Springer Science and Business Media LLC
Date: 30-11-2020
DOI: 10.1038/S41556-020-00605-6
Abstract: Filamentous actin (F-actin) provides cells with mechanical support and promotes the mobility of intracellular structures. Although F-actin is traditionally considered to be cytoplasmic, here we reveal that nuclear F-actin participates in the replication stress response. Using live and super-resolution imaging, we find that nuclear F-actin is polymerized in response to replication stress through a pathway regulated by ATR-dependent activation of mTORC1, and nucleation through IQGAP1, WASP and ARP2/3. During replication stress, nuclear F-actin increases the nuclear volume and sphericity to counteract nuclear deformation. Furthermore, F-actin and myosin II promote the mobility of stressed-replication foci to the nuclear periphery through increasingly diffusive motion and directed movements along the nuclear actin filaments. These actin functions promote replication stress repair and suppress chromosome and mitotic abnormalities. Moreover, we find that nuclear F-actin is polymerized in vivo in xenograft tumours after treatment with replication-stress-inducing chemotherapeutic agents, indicating that this pathway has a role in human disease.
Supplementary Table 6 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932424.V1
Abstract: Supplementary Table 6 shows Sorting events for each cell included in the final data set
4D analysis of malaria parasite invasion offers insights into erythrocyte membrane remodeling and parasitophorous vacuole formation
Publisher: Springer Science and Business Media LLC
Date: 15-06-2021
DOI: 10.1038/S41467-021-23626-7
Abstract: Host membrane remodeling is indispensable for viruses, bacteria, and parasites, to subvert the membrane barrier and obtain entry into cells. The malaria parasite Plasmodium spp . induces biophysical and molecular changes to the erythrocyte membrane through the ordered secretion of its apical organelles. To understand this process and address the debate regarding how the parasitophorous vacuole membrane (PVM) is formed, we developed an approach using lattice light-sheet microscopy, which enables the parasite interaction with the host cell membrane to be tracked and characterized during invasion. Our results show that the PVM is predominantly formed from the erythrocyte membrane, which undergoes biophysical changes as it is remodeled across all stages of invasion, from pre-invasion through to PVM sealing. This approach enables a functional interrogation of parasite-derived lipids and proteins in PVM biogenesis and echinocytosis during Plasmodium falciparum invasion and promises to yield mechanistic insights regarding how this is more generally orchestrated by other intracellular pathogens.
Supplementary Table 3 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696140
Abstract: Supplementary Table 3 shows Differentially expressed genes for escaping vs. epithelial analysis
Supplementary Table 2 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696143
Abstract: Supplementary Table 2 shows Transcript counts for common housekeeping genes, with cells excluded from the study due to low housekeeping expression highlighted in yellow
Supplementary Figure 3 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932460
Abstract: Supplementary Figure 3 shows validation of OPRD1 and SLC19A1 protein expression
Supplementary Figure 6 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932451
Abstract: Supplementary Figure 6 shows an Amoeboid cell cohort gallery
Supplementary Table 4 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932430.V1
Abstract: Supplementary Table 4 shows Differentially expressed genes for amoeboid vs. escaping analysis
Author response: TMEM87a/Elkin1, a component of a novel mechanoelectrical transduction pathway, modulates melanoma adhesion and migration
Publisher: eLife Sciences Publications, Ltd
Date: 10-03-2020
Supplementary Figure 5 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932454
Abstract: Supplementary Figure 5 shows an Escaping cell cohort gallery
Supplementary Table 5 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932427.V1
Abstract: Supplementary Table 5 shows Differentially expressed genes for epithelial vs. amoeboid analysis
Supplementary Figure 4 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932457
Abstract: Supplementary Figure 4 shows an Epithelial cohort gallery
Supplementary Video 2A from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932415.V1
Abstract: Supplementary Video 2A shows representative images of a vehicle-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Supplementary Table 2 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696143.V1
Abstract: Supplementary Table 2 shows Transcript counts for common housekeeping genes, with cells excluded from the study due to low housekeeping expression highlighted in yellow
Supplementary Video 1A from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696128.V1
Abstract: Supplementary Video 1A shows representative daily images of a vehicle-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Supplementary Figure 6 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696158
Abstract: Supplementary Figure 6 shows an Amoeboid cell cohort gallery
Supplementary Figure 9 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696149.V1
Abstract: Supplementary Figure 9 shows Escaped cell analyses for naltrindole and sulfasalazine spheroid assays
Supplementary Figure 2 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696170.V1
Abstract: Supplementary Figure 2 shows clustering of 44 single cells and their gene expression heat map based on 230 differentially expressed genes
The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation.
Publisher: The Company of Biologists
Date: 03-2020
DOI: 10.1242/JCS.238014
Abstract: It has become increasingly evident that T cell functions are subject to translational control in addition to transcriptional regulation. Here, by using live imaging of CD8+ T cells isolated from the Lifeact-EGFP mouse, we show that T cells exhibit a gain in fluorescence intensity following engagement of cognate tumour target cells. The GFP signal increase is governed by Erk1/2-dependent distal T cell receptor (TCR) signalling and its magnitude correlates with IFN-γ and TNF-α production, which are hallmarks of T cell activation. Enhanced fluorescence was due to increased translation of Lifeact-EGFP protein, without an associated increase in its mRNA. Activation-induced gains in fluorescence were also observed in naïve and CD4+ T cells from the Lifeact-EGFP reporter, and were readily detected by both flow cytometry and live cell microscopy. This unique, translationally controlled reporter of effector T cell activation simultaneously enables tracking of cell morphology, F-actin dynamics and activation state in in idual migrating T cells. It is a valuable addition to the limited number of reporters of T cell dynamics and activation, and opens the door to studies of translational activity and heterogeneities in functional T cell responses in situ.
Optimizing the Polymer Cloak for Upconverting Nanoparticles: An Evaluation of Bioactivity and Optical Performance
Publisher: American Chemical Society (ACS)
Date: 31-03-2021
Supplementary Figure 8 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696152
Abstract: Supplementary Figure 8 shows a summary of GSEA pathway analyses using Hallmarks gene set showing top 25 enriched pathways for each comparison
Supplementary Figure 7 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696155
Abstract: Supplementary Figure 7 shows metadata for isolated cells
Supplementary Table 3 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932433.V1
Abstract: Supplementary Table 3 shows Differentially expressed genes for escaping vs. epithelial analysis
Supplementary Figure 2 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932463
Abstract: Supplementary Figure 2 shows clustering of 44 single cells and their gene expression heat map based on 230 differentially expressed genes
Supplementary Figure 1 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932466
Abstract: Supplementary Figure 1 shows data generation for the photoconvertible spheroid assay
Tropomyosin Tpm 2.1 loss induces glioblastoma spreading in soft brain-like environments
Publisher: Springer Science and Business Media LLC
Date: 09-12-2018
DOI: 10.1007/S11060-018-03049-Z
Abstract: The brain is a very soft tissue. Glioblastoma (GBM) brain tumours are highly infiltrative into the surrounding healthy brain tissue and invasion mechanisms that have been defined using rigid substrates therefore may not apply to GBM dissemination. GBMs characteristically lose expression of the high molecular weight tropomyosins, a class of actin-associating proteins and essential regulators of the actin stress fibres and focal adhesions that underpin cell migration on rigid substrates. Here, we investigated how loss of the high molecular weight tropomyosins affects GBM on soft matrices that recapitulate the biomechanical architecture of the brain. We find that Tpm 2.1 is down-regulated in GBM grown on soft substrates. We demonstrate that Tpm 2.1 depletion by siRNA induces cell spreading and elongation in soft 3D hydrogels, irrespective of matrix composition. Tpm 1.7, a second high molecular weight tropomyosin is also down-regulated when cells are cultured on soft brain-like surfaces and we show that effects of this isoform are matrix dependent, with Tpm 1.7 inducing cell rounding in 3D collagen gels. Finally, we show that the absence of Tpm 2.1 from primary patient-derived GBMs correlates with elongated, mesenchymal invasion. We propose that Tpm 2.1 down-regulation facilitates GBM colonisation of the soft brain environment. This specialisation of the GBM actin cytoskeleton organisation that is highly suited to the soft brain-like environment may provide novel therapeutic targets for arresting GBM invasion.
FIGURE 6 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932469
Abstract: SSZ treatment slows spheroid growth but induces tumor escape by elongated cells. b A, /b Representative maximum intensity projections of imaged hemispheroids for vehicle- and SSZ-treated spheroids. Scale bar, 200 μm. b B, /b Relative spheroid volume for vehicle- and SSZ-treated spheroids. b C, /b Cumulative escaped cells per surface area of imaged hemispheroid. b D, /b Mean escaped cell sphericity per day. B–D, Shaded areas represent SEM.
ATR and mTOR regulate F-actin to alter nuclear architecture and repair replication stress
Publisher: Cold Spring Harbor Laboratory
Date: 24-10-2018
DOI: 10.1101/451708
Abstract: “Replication stress” describes phenomena that alter DNA replication rates 1–3 . Multiple architectural challenges within the confined nuclear volume must be navigated during replication to prevent or repair replication stress. Cellular mechanisms potentiating changes in nuclear architecture that facilitate DNA replication remain unclear. Here we show that the ATR, IPMK and mTOR kinases regulate actin polymerisation in human cells to alter nuclear architecture and promote replication fork repair. We demonstrate that replication stress activates mTOR, in an ATR and IPMK-dependent manner, to induce polymerisation of nuclear filamentous actin (F-actin). mTOR and ATR then counteract replication stress-induced nuclear envelope deformation and increase nuclear volume through their regulation of actin dynamics. Additionally, we reveal that FANCD2 labelled replication forks colocalise with actin filaments in late S-phase. mTOR and ATR then regulate the mobility, speed and directionality of stalled replication foci within the three-dimensional nuclear architecture. Importantly, we find nuclear F-actin also acts as a substrate for the directed migration of stalled replication foci to the nuclear periphery. Suppressing mTOR and ATR-dependent actin forces prevents replication fork restart and promotes chromosome segregation errors in primary and cancer cell lines. Together, these data reveal that ATR and mTOR regulate actin dynamics in the replication stress response to alter nuclear architecture and maintain genome stability.
Supplementary Video 2B from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932412.V1
Abstract: Supplementary Video 2B shows representative images of a Sulfasalazine-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Supplementary Table 1 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696146.V1
Abstract: Supplementary Table 1 shows Cell type isolated, age (days) and number of cells sorted for each spheroid included in the study
Cytotoxic T cells swarm by homotypic chemokine signalling
Publisher: eLife Sciences Publications, Ltd
Date: 13-10-2020
DOI: 10.7554/ELIFE.56554
Abstract: Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by lifying the direct recruitment of additional T cells independently of other leukocytes.
Memory B cells are reactivated in subcapsular proliferative foci of lymph nodes
Publisher: Springer Science and Business Media LLC
Date: 22-08-2018
DOI: 10.1038/S41467-018-05772-7
Abstract: Vaccine-induced immunity depends on the generation of memory B cells (MBC). However, where and how MBCs are reactivated to make neutralising antibodies remain unknown. Here we show that MBCs are prepositioned in a subcapsular niche in lymph nodes where, upon reactivation by antigen, they rapidly proliferate and differentiate into antibody-secreting plasma cells in the subcapsular proliferative foci (SPF). This novel structure is enriched for signals provided by T follicular helper cells and antigen-presenting subcapsular sinus macrophages. Compared with contemporaneous secondary germinal centres, SPF have distinct single-cell molecular signature, cell migration pattern and plasma cell output. Moreover, SPF are found both in human and mouse lymph nodes, suggesting that they are conserved throughout mammalian evolution. Our data thus reveal that SPF is a seat of immunological memory that may be exploited to rapidly mobilise secondary antibody responses and improve vaccine efficacy.
Supplementary Video 1B from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696125
Abstract: Supplementary Video 1B shows representative images of a naltrindole-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Supplementary Figure 1 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696173.V1
Abstract: Supplementary Figure 1 shows data generation for the photoconvertible spheroid assay
Supplementary Video 1A from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696128
Abstract: Supplementary Video 1A shows representative daily images of a vehicle-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation
Publisher: Elsevier BV
Date: 04-2018
DOI: 10.1016/J.CELL.2018.02.035
Abstract: Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation.
T cell morphodynamics reveal periodic shape oscillations in three-dimensional migration
Publisher: The Royal Society
Date: 05-2022
Abstract: T cells use sophisticated shape dynamics (morphodynamics) to migrate towards and neutralize infected and cancerous cells. However, there is limited quantitative understanding of the migration process in three-dimensional extracellular matrices (ECMs) and across timescales. Here, we leveraged recent advances in lattice light-sheet microscopy to quantitatively explore the three-dimensional morphodynamics of migrating T cells at high spatio-temporal resolution. We first developed a new shape descriptor based on spherical harmonics, incorporating key polarization information of the uropod. We found that the shape space of T cells is low-dimensional. At the behavioural level, run-and-stop migration modes emerge at approximately 150 s, and we mapped the morphodynamic composition of each mode using multiscale wavelet analysis, finding ‘stereotyped’ motifs. Focusing on the run mode, we found morphodynamics oscillating periodically (every approx. 100 s) that can be broken down into a biphasic process: front-widening with retraction of the uropod, followed by a rearward surface motion and forward extension, where intercalation with the ECM in both of these steps likely facilitates forward motion. Further application of these methods may enable the comparison of T cell migration across different conditions (e.g. differentiation, activation, tissues and drug treatments) and improve the precision of immunotherapeutic development.
Supplementary Table 6 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696131.V1
Abstract: Supplementary Table 6 shows Sorting events for each cell included in the final data set
FIGURE 2 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932481
Abstract: Single-cell tagging and isolation based on morphologic and positional data. b A, /b Sphericity and distance to spheroid values are generated for each invading cell surrounding the spheroid. Left panel depicts maximum intensity projections for three invading cells (Fluo) of different sphericities, following cell surface segmentation (Fluo + Surf), and following surface pseudocoloring according to sphericity value (Surf + Pseudo). Right panels illustrate the smoothed spheroid surface (red) computed to calculate the nearest distance (dashed arrow) to the spheroid edge from in idual cells. b B, /b Left: The three photoconverted cell types “Epithelial,” “Escaping,” and “Amoeboid.” Middle: Representative images of the photoconversion of the three cell types and corresponding flow cytometry scatter plots of Dendra2 red versus green fluorescence obtained during single-cell sorting. Right: The number of cells photoconverted and recovered via single-cell sorting for escaping and amoeboid cells. b C, /b Sorting yields expressed as a percentage of photoconverted cells for escaping and amoeboid cells.
Supplementary Video 2A from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696122
Abstract: Supplementary Video 2A shows representative images of a vehicle-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
FIGURE 5 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932472
Abstract: Inhibition of OPRD1 reduces melanoma spheroid growth and invasion. b A, /b Representative maximum intensity projections of imaged hemispheroids for vehicle- and naltrindole-treated spheroids, scale bar 200 μm. b B, /b Relative spheroid volume over time for vehicle- and naltrindole-treated cells. Shaded areas represent SEM. b C, /b Cumulative number of escaped cells over time normalized to spheroid surface area. B and C, Datapoints represent the mean of eight replicates.
FIGURE 4 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932475
Abstract: Gene expression analysis for epithelial, escaping, and amoeboid cells. b A, /b Top: 462 genes were DE, 144 for escaping versus epithelial (Esc-v-Epi), 22 for amoeboid versus escaping (Amo-v-Esc), and 422 for amoeboid versus epithelial (Amo-v-Epi). Bottom: Venn diagram of the number of DE genes for the three pairwise comparisons. b B, /b Volcano plot for Esc-v-Epi DE analysis, with genes enriched in “escaping” cells shown as positive on the i x /i -axis. b C, /b Volcano plot for Amo-v-Esc DE analysis, with genes enriched in “amoeboid” cells shown as positive on the i x /i -axis. b D, /b Volcano plot for Amo-v-Epi DE analysis, with genes enriched in “amoeboid” cells shown as positive on the i x /i -axis. For B–D, significantly DE genes with an adjusted i P /i value 0.05 are shown in light blue, with no minimum value for log sub /sub FC. b E, /b Expression of EMT markers for epithelial, leaving, and amoeboid cells shown via dot plot. b F, /b Expression of cancer stem cell markers for epithelial, leaving, and amoeboid cells shown via dot plot.
FIGURE 3 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932478
Abstract: Clustering of single cells and their gene expression heat map based on 462 DE genes. b A, /b UMAP clustering of 35 cells based on expression of 462 DE genes. b B, /b Unsupervised hierarchical clustering of 35 cells based on the expression of 462 DE genes. Cells initially erge into two groups, epithelial (L1) and invasive (R1), before further erging into escaping (R2) and amoeboid (R3). b C, /b Heat map of 462 DE genes for single cells arranged according to the unsupervised clustering in B.
Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption
Publisher: Elsevier BV
Date: 04-2021
T cell migration in intact lymph nodes in vivo
Publisher: Elsevier BV
Date: 10-2014
DOI: 10.1016/J.CEB.2014.05.002
Abstract: In the lymph node, T cells migrate rapidly and with striking versatility in a continuous scan for antigen presenting dendritic cells. The scanning process is greatly facilitated by the lymph node structure and composition. In vivo imaging has been instrumental in deciphering the spatiotemporal dynamics of intranodal T cell migration in both health and disease. Here we review recent developments in uncovering the migration modes employed by T cells in the lymph node, the underlying molecular mechanisms, and the scanning strategies utilised by T cells to ensure a timely response to antigenic stimuli.
Supplementary Table 5 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696134
Abstract: Supplementary Table 5 shows Differentially expressed genes for epithelial vs. amoeboid analysis
Supplementary Video 1A from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932421.V1
Abstract: Supplementary Video 1A shows representative daily images of a vehicle-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.
Supplementary Table 4 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696137
Abstract: Supplementary Table 4 shows Differentially expressed genes for amoeboid vs. escaping analysis
Supplementary Figure 2 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932463.V1
Abstract: Supplementary Figure 2 shows clustering of 44 single cells and their gene expression heat map based on 230 differentially expressed genes
Supplementary Table 5 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696134.V1
Abstract: Supplementary Table 5 shows Differentially expressed genes for epithelial vs. amoeboid analysis
Leukocyte migration in the interstitial space of non-lymphoid organs
Publisher: Springer Science and Business Media LLC
Date: 07-03-2014
DOI: 10.1038/NRI3641
Abstract: Leukocyte migration through interstitial tissues is essential for mounting a successful immune response. Interstitial motility is governed by a vast array of cell-intrinsic and cell-extrinsic factors that together ensure the proper positioning of immune cells in the context of specific microenvironments. Recent advances in imaging modalities, in particular intravital confocal and multi-photon microscopy, have helped to expand our understanding of the cellular and molecular mechanisms that underlie leukocyte navigation in the extravascular space. In this Review, we discuss the key factors that regulate leukocyte motility within three-dimensional environments, with a focus on neutrophils and T cells in non-lymphoid organs.
Supplementary Table 6 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696131
Abstract: Supplementary Table 6 shows Sorting events for each cell included in the final data set
Loss of calpains-1 and -2 prevents repair of plasma membrane scrape injuries, but not small pores, and induces a severe muscular dystrophy.
Publisher: American Physiological Society
Date: 06-2020
DOI: 10.1152/AJPCELL.00408.2019
Abstract: The ubiquitous calpains, calpain-1 and -2, play important roles in Ca 2+ -dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca 2+ -dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 ( CAPNS1 −/− ) virtually ablates Ca 2+ -dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptolysin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 ( CAPN1 −/− ) or -2 ( CAPN2 −/− ) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 ( CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca 2+ -signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.
TMEM87a/Elkin1, a component of a novel mechanoelectrical transduction pathway, modulates melanoma adhesion and migration
Publisher: eLife Sciences Publications, Ltd
Date: 04-2020
DOI: 10.7554/ELIFE.53308
Abstract: Mechanoelectrical transduction is a cellular signalling pathway where physical stimuli are converted into electro-chemical signals by mechanically activated ion channels. We describe here the presence of mechanically activated currents in melanoma cells that are dependent on TMEM87a, which we have renamed Elkin1. Heterologous expression of this protein in PIEZO1-deficient cells, that exhibit no baseline mechanosensitivity, is sufficient to reconstitute mechanically activated currents. Melanoma cells lacking functional Elkin1 exhibit defective mechanoelectrical transduction, decreased motility and increased dissociation from organotypic spheroids. By analysing cell adhesion properties, we demonstrate that Elkin1 deletion is associated with increased cell-substrate adhesion and decreased homotypic cell-cell adhesion strength. We therefore conclude that Elkin1 supports a PIEZO1-independent mechanoelectrical transduction pathway and modulates cellular adhesions and regulates melanoma cell migration and cell-cell interactions.
FIGURE 1 from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.23932484
Abstract: Methodologic pipeline for single-cell isolation of live escaping tumor cells. b , /b A clonal population of tumor cells are expanded from a single cell. b , /b Photoconvertible cells are seeded in ULA plates to generate spheroids. b , /b Spheroids are embedded within a 3D collagen matrix and imaged over time by light microscopy. b , /b Image analysis and extraction of quantitative metrics can be used to select cells of interest. b , /b Single cells of interest are photoconverted using a 405 nm laser. b , /b Collagen matrix is enzymatically digested to provide a solution of suspended single cells. b , /b Cells are sorted via FACS. b , /b Cells can be further cultured or immediately analyzed.
Mechanoimmunology: molecular-scale forces govern immune cell functions
Publisher: American Society for Cell Biology (ASCB)
Date: 08-08-2018
Abstract: Immune cell recognition of antigens is a pivotal process in initiating immune responses against injury, pathogens, and cancers. Breakthroughs over the past decade support a major role for mechanical forces in immune responses, laying the foundation for the emerging field of mechanoimmunology. In this Perspective, we discuss the mechanical forces acting at the level of ligand–receptor interactions and how they underpin receptor triggering, signal initiation, and immune cell activation. We also highlight the novel biophysical tools and advanced imaging techniques that have afforded us the recent progress in our understanding of the role of forces in immune cell functions.
Supplementary Table 3 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696140.V1
Abstract: Supplementary Table 3 shows Differentially expressed genes for escaping vs. epithelial analysis
Supplementary Table 4 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.23696137.V1
Abstract: Supplementary Table 4 shows Differentially expressed genes for amoeboid vs. escaping analysis
Data from Targeted Single-cell Isolation of Spontaneously Escaping Live Melanoma Cells for Comparative Transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 11-08-2023
DOI: 10.1158/2767-9764.C.6745837.V2
Abstract: Solid cancer cells escape the primary tumor mass by transitioning from an epithelial-like state to an invasive migratory state. As they escape, metastatic cancer cells employ interchangeable modes of invasion, transitioning between fibroblast-like mesenchymal movement to amoeboid migration, where cells display a rounded morphology and navigate the extracellular matrix in a protease-independent manner. However, the gene transcripts that orchestrate the switch between epithelial, mesenchymal, and amoeboid states remain incompletely mapped, mainly due to a lack of methodologies that allow the direct comparison of the transcriptomes of spontaneously invasive cancer cells in distinct migratory states. Here, we report a novel single-cell isolation technique that provides detailed three-dimensional data on melanoma growth and invasion, and enables the isolation of live, spontaneously invasive cancer cells with distinct morphologies and invasion parameters. Via the expression of a photoconvertible fluorescent protein, compact epithelial-like cells at the periphery of a melanoma mass, elongated cells in the process of leaving the mass, and rounded amoeboid cells invading away from the mass were tagged, isolated, and subjected to single-cell RNA sequencing. A total of 462 differentially expressed genes were identified, from which two candidate proteins were selected for further pharmacologic perturbation, yielding striking effects on tumor escape and invasion, in line with the predictions from the transcriptomics data. This work describes a novel, adaptable, and readily implementable method for the analysis of the earliest phases of tumor escape and metastasis, and its application to the identification of genes underpinning the invasiveness of malignant melanoma. Significance: This work describes a readily implementable method that allows for the isolation of in idual live tumor cells of interest for downstream analyses, and provides the single-cell transcriptomes of melanoma cells at distinct invasive states, both of which open avenues for in-depth investigations into the transcriptional regulation of the earliest phases of metastasis. /
Author response: Cytotoxic T cells swarm by homotypic chemokine signalling
Publisher: eLife Sciences Publications, Ltd
Date: 29-07-2020
Data from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics
Publisher: American Association for Cancer Research (AACR)
Date: 17-07-2023
DOI: 10.1158/2767-9764.C.6745837.V1
Abstract: Abstract Solid cancer cells escape the primary tumour mass by transitioning from an epithelial-like state to an invasive migratory state. As they escape, metastatic cancer cells employ interchangeable modes of invasion, transitioning between fibroblast-like mesenchymal movement to amoeboid migration, where cells display a rounded morphology and navigate the extracellular matrix in a protease-independent manner. However, the gene transcripts that orchestrate the switch between epithelial, mesenchymal and amoeboid states remain incompletely mapped, mainly due to a lack of methodologies that allow the direct comparison of the transcriptomes of spontaneously invasive cancer cells in distinct migratory states. Here, we report a novel single cell isolation technique that provides detailed 3D data on melanoma growth and invasion, and enables the isolation of live, spontaneously invasive cancer cells with distinct morphologies and invasion parameters. Via the expression of a photoconvertible fluorescent protein, compact epithelial-like cells at the periphery of a melanoma mass, elongated cells in the process of leaving the mass, and rounded amoeboid cells invading away from the mass were tagged, isolated and subjected to single cell RNA-sequencing. 462 differentially expressed genes were identified, from which two candidate proteins were selected for further pharmacological perturbation, yielding striking effects on tumour escape and invasion, in line with the predictions from the transcriptomics data. This work describes a novel, adaptable and readily implementable method for the analysis of the earliest phases of tumour escape and metastasis, and its application to the identification of genes underpinning the invasiveness of malignant melanoma. /
Related Organisations
National Research And Innovation Agency, Indonesia
Location: Indonesia
Related Funding Activities
Discovery Projects - Grant ID: DP180102458
Start Date: 06-2018
End Date: 06-2021
Amount: $387,834.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE170100206
Start Date: 05-2017
End Date: 12-2017
Amount: $550,000.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE210100001
Start Date: 07-2021
End Date: 07-2022
Amount: $875,000.00
Funder: Australian Research Council
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