Biology subjects

PADDISON, P.

Publications and source records attributed to PADDISON, P..

9 recordsLinked to original sources

bioRxiv2025

Characterization of quiescent subpopulations and proliferative compartments in glioblastoma

Glioblastoma (GBM) quiescent (Q) cell populations are hypothesized to contain cancer stem-like cells (CSC) that drive tumor growth, cellular heterogeneity, and recurrence. However, GBM tumors do not neatly resolve into developmental hierarchies and Q stem-like activities are difficult to assess. Here, we evaluated tumor Q subpopulations in patient-derived GBM xenograft tumors using live cell reporters, DNA label retention assays, and single cell genomics. Compared to adult neural stems cells (NSCs), GBM Q populations contain hybrid transcriptional states composed of networks found in both dormant and activated adult NSCs, resulting in constitutive expression of key Q egress transcription factors and their targets (e.g., AP-1 and CCND1/2). As a result, even the longest Q-residing cells ([~]12 days) in xenograft tumors continuously cycle and fail to enter dormant Q states. We provide evidence and hypothesize that transient Q states in primary tumors arise as part of distinct proliferative compartments rather than deterministic developmental hierarchies driven by CSC activity. We further speculate that increases in basal translation rates drive Q instability in GBM tumors.

cancer biology↗

bioRxiv2025

Cellular heterogeneity and therapeutic response profiling of human IDH+ glioma stem cell cultures

Summary/AbstractGlioblastoma stem cell (GSC) cultures are initiated from glioblastoma (GBM) surgical resection tissue. They can capture and propagate key GBM primary tumor molecular and cellular features. We have deeply characterized four isocitrate dehydrogenase (IDH)-expressing (or IDH+) GSC cultures from unrelated adults to serve as cellular models for the majority of adult primary GBM. We demonstrate that GSC cultures can be continuously propagated in defined, serum-free media and 5% oxygen without requiring specialized growth substrates; have well-defined genomic and mtDNA variants and gene/protein expression profiles; and highly reproducible dose-survival curves when treated with the GBM standard-of-care therapies of ionizing radiation (IR) and temozolomide (TMZ). We also illustrate how expressed lentiviral barcodes, mtDNA variants and single cell gene expression profiling can be used to define and track cellular heterogeneity over 40 days after IR treatment. These well-characterized IDH+ GSC cultures can support many high throughput in vitro assay formats, including xenograft, organoid and other GBM disease modeling protocols. They should prove a useful resource to better understand GBM biology, and to identify new and more effective GBM therapies and treatment regimens. HighlightsO_LIGlioblastoma (GBM)-derived IDH-expressing Glioma Stem Cell (GSC) cultures can capture and propagate GBM genomic variants, gene and protein expression programs. C_LIO_LIMitochondrial DNA (mtDNA) variants identified by high accuracy Duplex DNA sequencing are abundant and sub-clonally organized in GSC cultures. C_LIO_LIGSC cultures have highly reproducible dose-survival curves for ionizing radiation and temozolomide, the GBM standard-of-care therapies. C_LIO_LIGSC culture cellular heterogeneity can be captured, characterized and tracked by using expressed lentiviral barcodes, mtDNA variants and scRNA sequencing. C_LI eTOC blurbIDH-expressing glioma stem cell cultures (GSCs) are experimentally tractable and versatile glioblastoma (GBM) cellular disease models. Deeply characterized GSC cultures can enable new work to understand GBM biology, and help identify new and more effective GBM therapies and treatment regimens. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/667532v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@82aceeorg.highwire.dtl.DTLVardef@1277c18org.highwire.dtl.DTLVardef@144188eorg.highwire.dtl.DTLVardef@1a84bb3_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Breaking out of the cell cycle: Including quiescence in cell cycle classification

Single-cell transcriptomics has unveiled a vast landscape of cellular heterogeneity in which the cell cycle is a significant component. We trained a high-resolution cell cycle classifier (ccAFv2) using single cell RNA-seq (scRNA-seq) characterized human neural stem cells. The ccAFv2 classifies six cell cycle states (G1, Late G1, S, S/G2, G2/M, and M/Early G1) and a quiescent-like G0 state (Neural G0), and it incorporates a tunable parameter to filter out less certain classifications. The ccAFv2 classifier performed better than or equivalent to other state-of-the-art methods even while classifying more cell cycle states, including G0. We demonstrate that the ccAFv2 classifier effectively generalizes the S, S/G2, G2/M, and M/Early G1 states across cell types derived from all three germ layers. While the G0, G1, and Late G1 states perform well in neuroepithelial cell types, their accuracy is lower in other cell types. However, misclassifications are confined to the G0, G1, and Late G1 states. We showcased the versatility of ccAFv2 by successfully applying it to classify cells, nuclei, and spatial transcriptomics data in humans and mice, using various normalization methods and gene identifiers. We provide methods to regress the cell cycle expression patterns out of single cell or nuclei data to uncover underlying biological signals. The classifier can be used either as an R package integrated with Seurat or a PyPI package integrated with SCANPY. We proved that ccAFv2 has enhanced accuracy, flexibility, and adaptability across various experimental conditions, establishing ccAFv2 as a powerful tool for dissecting complex biological systems, unraveling cellular heterogeneity, and deciphering the molecular mechanisms by which proliferation and quiescence affect cellular processes.

cell biology↗

bioRxiv2023

Functional genomic analysis of adult and pediatric brain tumor isolates

BackgroundAdult and pediatric tumors display stark differences in their mutation spectra and chromosome alterations. Here, we attempted to identify common and unique gene dependencies and their associated biomarkers among adult and pediatric tumor isolates using functional genetic lethal screens and computational modeling. MethodsWe performed CRISRP-Cas9 lethality screens in two adult glioblastoma (GBM) tumor isolates and five pediatric brain tumor isolates representing atypical teratoid rhabdoid tumors (ATRT), diffuse intrinsic pontine glioma, GBM, and medulloblastoma. We then integrated the screen results with machine learning-based gene-dependency models generated from data from >900 cancer cell lines. ResultsWe found that >50% of candidate dependencies of 280 identified were shared between adult GBM tumors and individual pediatric tumor isolates. 68% of screen hits were found as nodes in our network models, along with shared and tumor-specific predictors of gene dependencies. We investigated network predictors associated with ADAR, EFR3A, FGFR1 (pediatric-specific), and SMARCC2 (ATRT-specific) gene dependency among our tumor isolates. ConclusionsThe results suggest that, despite harboring disparate genomic signatures, adult and pediatric tumor isolates share a preponderance of genetic dependences. Further, combining data from primary brain tumor lethality screens with large cancer cell line datasets produced valuable insights into biomarkers of gene dependency, even for rare cancers. Importance of the StudyOur results demonstrate that large cancer cell lines data sets can be computationally mined to identify known and novel gene dependency relationships in adult and pediatric human brain tumor isolates. Gene dependency networks and lethality screen results represent a key resource for neuro-oncology and cancer research communities. We also highlight some of the challenges and limitations of this approach.

cancer biology↗

bioRxiv2022

FBXO42 activity is required to prevent mitotic arrest, spindle assembly checkpoint activation, and lethality in glioblastoma and other cancers

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. To identify genes differentially required for the viability of GBM stem-like cells (GSCs), we performed functional genomic lethality screens comparing GSCs and control human neural stem cells. Among top scoring hits in a subset of GBM cells was the F-box-containing gene FBXO42, which was also essential in [~]15% of cell lines derived from a broad range of cancers. Mechanistic studies revealed that, in sensitive cells, FBXO42 activity prevents chromosome alignment defects, mitotic cell cycle arrest, and cell death. The cell cycle arrest, but not the cell death, triggered by FBXO42 inactivation could be suppressed by brief exposure to a chemical inhibitor of Mps1, a key spindle assembly checkpoint (SAC) kinase. FBXO42s cancer-essential function requires its F-box and Kelch domains, which are necessary for FBXO42s substrate recognition and targeting by SCF ubiquitin ligase complex. However, none of FBXO42s previously proposed targets, including ING4, p53, and RBPJ, were responsible for the observed phenotypes. Instead, our results suggest that FBOX42 activity suppresses the accumulation of one or more proteins that perturb chromosome-microtubule dynamics in cancer cells, which, in turn, leads to induction of the SAC and cell death.

cancer biology↗

bioRxiv2022

An optimized gene knock-out strategy for human CD34+ hematopoietic stem and progenitor cells.

Human CD34+ hematopoietic stem and progenitor cells (HSPCs) are a standard source of cells for clinical HSC transplantations as well as experimental xenotransplantation to generate "humanized mice". To further extend the range of applications of these humanized mice, we developed a protocol to efficiently edit the genomes of human CD34+ HSPCs before transplantation. In the past, manipulating HSPCs has been complicated by the fact that they are inherently difficult to transduce with lentivectors, and rapidly lose their stemness and engraftment potential during in vitro culture. However, with optimized nucleofection of sgRNA:Cas9 ribonucleoprotein complexes, we are now able to edit a candidate gene in CD34+ HSPCs with almost 100% efficiency, and without affecting their potential for engraftment and multilineage differentiation in mice. The result is a humanized mouse from which we knocked out a gene of interest from their human immune system.

bioengineering↗

bioRxiv2022

KAT5 activity regulates G0-like states in human gliomas

In solid tumors, G0-like states are likely critical for maintaining developmental hierarchies and cellular heterogeneity and promoting tumor growth/recurrence, yet little is known about tumor G0 states or regulation of their ingress/egress. To discover G0-like states and their regulators for glioblastoma (GBM), we analyzed G0 populations in an orthotopic model of GBM using single cell RNA-seq and performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) for genes that trap cells in G0 when inhibited. We identify the protein acetyltransferase KAT5 as a key regulator of transcriptional, epigenetic, and proliferative heterogeneity impacting transitions into G0-like states. KAT5 activity suppresses the emergence of non-dividing subpopulations with oligodendrocyte progenitor and radial glial cell characteristics both in vitro and in a human GSC brain tumor model. In primary gliomas, KAT5 activity is dynamic with KAT5low tumor cells displaying quiescent properties, while KAT5 activity overall increases from low to high grade tumors and is associated with worse patient outcomes.

cancer biology↗

bioRxiv2020

A simple and highly efficient method for multi-allelic CRISPR-Cas9 editing in primary cell cultures

ABSTRACTBackground CRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. None-the-less, limitations of the delivery and efficacy of these technologies restrict their application in primary cells.Aims To create an optimized protocol for penetrant, reproducible, and fast targeted insertiondeletion mutation (indel) formation in cell cultures derived from primary cells, using patient-derived glioblastoma (GBM) stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs) for proof-of-concept experiments.Methods We employed transient nucleofection of Cas9:sgRNA ribonucleoprotein complexes using chemically synthesized 2’-O-methyl 3’phosphorothioate-modified sgRNAs and purified Cas9 protein. Indel frequency and size distribution were measured via computational deconvolution of Sanger sequencing trace data. Western blotting was used to evaluate protein loss. RNA-seq in edited NSCs was used to assess gene expression changes resulting from knockout of tumor suppressors commonly altered in GBM.Results We found that with this optimized technique, we can routinely achieve >90% indel formation in only 3 days, without the need to create clonal lines for simple loss-of-function experiments. We observed near-total protein loss of target genes in cell pools. Additionally, we found that this approach allows for the creation of targeted genomic deletions. We also demonstrated the utility of this method for quickly creating a series of gene knockouts that allow for the study of oncogenic activities.Conclusion Our data suggest that this relatively simple method can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells (such as GSCs). This represents an extremely useful tool for the cancer research community when wishing to inactivate not only coding genes, but also non-coding RNAs, UTRs, enhancers, and promoters. This method can be readily applied to diverse cell types by varying the nucleofection conditions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2018

CRISPR-Cas9 Screens Reveal Genes Regulating a G0-like State in Human Neural Progenitors

Single cell RNA-seq has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNA-seq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From this data, we created a cell cycle classifier, which, in addition to traditional cell cycle phases, also identifies a putative quiescent-like state in neuroepithelial-derived cell types during mammalian neurogenesis and in gliomas. This state, Neural G0, is enriched for expression of quiescent NSC genes and other neurodevelopmental markers found in non-dividing neural progenitors. For gliomas, Neural G0 cell populations and gene expression is significantly associated with less aggressive tumors and extended patient survival. Genetic screens to identify modulators of Neural G0 revealed that knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, down regulation of quiescence-associated markers, and loss of Neural G0 gene expression. Thus, Neural G0 represents a dynamic quiescent-like state found in neuro-epithelial derived cells and gliomas.

cell biology↗