scholarly journals EPCO-27. GLIOMA SINGLE CELL MULTI-OMIC ANALYSES REVEALS REGULATORS OF PLASTICITY AND ADAPTIVE STRESS RESPONSE

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii75-ii75
Author(s):  
Kevin Johnson ◽  
Kevin Anderson ◽  
Elise Courtois ◽  
Floris Barthel ◽  
Frederick Varn ◽  
...  
Keyword(s):  
Stress Response ◽  
Single Cell ◽  
Tumor Cells ◽  
Binding Sites ◽  
Copy Number ◽  
Epigenetic Mechanisms ◽  
Cellular Plasticity ◽  
Epigenetic Instability ◽  
Extracellular Stimuli ◽  
Cell Stress Response

Abstract Extensive intra- and intertumoral heterogeneity in glioma contributes to therapeutic resistance and poor patient outcomes. Alterations to DNA methylation (DNAme) modulate epigenetic homeostasis, allowing tumor cells to sample alternative cell states to promote tumorigenesis. However, the epigenetic mechanisms that promote cellular plasticity and regulate cell states are still poorly understood. To characterize the epigenetic mechanisms underlying glioma heterogeneity we profiled 914 single-cell methylomes, 55,284 single-cell transcriptomes, and bulk whole genomes across 11 patient samples spanning initial and recurrent time points and 3 molecular subtypes delineated by IDH mutation status. Local DNAme disorder, defined as epimutation burden, was increased in tumor cells relative to nontumor cells, higher in IDH wild-type than in IDH mutant glioma and was positively associated with copy number alteration (CNA). Epimutation was positively associated with transcriptional variability and enriched at genes involved in cellular differentiation. Epimutation was also increased in the binding sites of transcription factors (TFs) associated with response to extracellular stimuli, suggesting that stochastic DNAme alterations enable cellular plasticity and diverse responses to microenvironmental stressors. Integrative clustering of DNAme and scRNAseq profiles defined stem-like and differentiated-like cell states which exhibited differences in TF activity. Stem-like cells were enriched for differentially methylated binding sites of TFs associated with hypoxia response. scDNAme and scRNAseq-derived copy number profiles were compared with bulk copy number profiles and inferred tumor phylogenies to assess how the timing of CNAs impact epigenetic instability, with results suggesting that early CNA events propagate both genetic and epigenetic heterogeneity. Bulk longitudinal data was used to validate the relationship of epigenetic instability with CNA burden as well as differentially methylated binding sites of cell stress response TFs. Our work suggests that local DNAme disorder promotes cellular plasticity and enables adaptive response to cellular stress such as hypoxia.

scholarly journals Single-cell multimodal glioma analyses reveal epigenetic regulators of cellular plasticity and environmental stress response

2020 ◽  
Author(s):  
Kevin C. Johnson ◽  
Kevin J. Anderson ◽  
Elise T. Courtois ◽  
Floris P. Barthel ◽  
Frederick S. Varn ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stress Response ◽  
Environmental Stress ◽  
Single Cell ◽  
Intratumoral Heterogeneity ◽  
Cellular Plasticity ◽  
Environmental Stress Response ◽  
Integrative Framework ◽  
Epigenetic Instability ◽  
Somatic Copy Number Alterations

ABSTRACTGlioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to universal therapeutic resistance. Here, we integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes, and bulk multi-omic profiles across 11 adult IDH-mutant or IDH-wild-type gliomas to delineate sources of intratumoral heterogeneity. We found that local DNA methylation instability, or epimutation burden, was elevated in more aggressive tumors, reflected intratumoral variability, linked with transcriptional disruption, and associated with environmental stress response. We show that the activation of cell-state specific transcription factors is impacted by epimutations and that loosened epigenetic control may facilitate cellular plasticity. Our analyses support that somatic copy number alterations (SCNAs) promote epigenetic instability and that SCNAs largely precede epigenetic and transcriptomic diversification during glioma evolution. We confirmed the link between genetic and epigenetic instability by analyzing larger cohorts of bulk longitudinally collected and spatially separated DNA methylation data. Increased DNA methylation instability was associated with accelerated disease progression, and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work provides an integrative framework to better understand glioma evolution and highlights the importance of epigenetic heterogeneity in shaping therapeutic response.

scholarly journals Whole genome sequencing of single circulating tumor cells from neuroendocrine neoplasms

2021 ◽  
Author(s):  
Alexa Childs ◽  
Christopher D. Steele ◽  
Clare Vesely ◽  
Francesca M. Maria Rizzo ◽  
Leah Ensell ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Copy Number ◽  
White Blood Cells ◽  
Neuroendocrine Neoplasms ◽  
Whole Genome ◽  
Bulk Analysis ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen

Single-cell profiling of circulating tumor cells (CTCs) as part of a minimally invasive liquid biopsy presents an opportunity to characterize and monitor tumor heterogeneity and evolution in individual patients. In this study, we aimed to compare single-cell copy number variation (CNV) data with tissue, and define the degree of intra- and inter-patient genomic heterogeneity. We performed next generation sequencing (NGS) whole genome CNV analysis of 125 single CTCs derived from seven patients with neuroendocrine neoplasms (NEN) alongside matched white blood cells (WBC), formalin fixed paraffin embedded (FFPE) and fresh frozen (FF) samples. CTC CNV profiling demonstrated recurrent chromosomal alterations in previously reported NEN copy number hotspots, including the prognostically relevant loss of chromosome 18. Unsupervised hierarchical clustering revealed CTCs with distinct clonal lineages as well as significant intra- and inter-patient genomic heterogeneity, including subclonal alterations not detectable by bulk analysis and previously unreported in NEN. Notably, we also demonstrate the presence of genomically distinct CTCs according to the enrichment strategy utilized (EpCAM-dependent versus size-based). This work has significant implications for the identification of therapeutic targets, tracking of evolutionary change and the implementation of CTC-biomarkers in cancer.

Prevalence and tissue concordance of BRCA2 copy number loss evaluated by single-cell, shallow whole genome sequencing of circulating tumor cells (CTCs) in castration-resistant prostate cancer (CRPC).

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 5531-5531
Author(s):  
Ethan Barnett ◽  
Joseph Schonhoft ◽  
Nikolaus D. Schultz ◽  
Jerry Lee ◽  
Samir Zaidi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Whole Genome Sequencing ◽  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Genome Sequencing ◽  
Copy Number ◽  
Cellular Heterogeneity ◽  
Cancer Tissue ◽  
Whole Genome

5531 Background: Genomic studies have shown that up to 25% of prostate cancer tissue specimens harbor alterations in DNA Damage Repair (DDR) genes, which may sensitize the tumor to poly ADP-ribose polymerase inhibitors (PARPi). Trials evaluating PARPi in patients with DDR deficiencies have shown varied response rates and differences regarding which genomic alterations predict for sensitivity to these agents, with the majority of objective responses seen in BRCA2-altered tumors. These results highlight the need to develop biomarker assays which can predict benefit from PARPi therapy. Tissue and cell-free DNA (cfDNA) have been the most utilized sources of tumor material for analysis in this setting, but success rates of obtaining sufficient tumor for analysis from bone are low and detecting tumor-derived copy number variants (CNVs) in cfDNA is challenging. Circulating tumor cells (CTCs) represent an alternate source of genetic information, for which assays are available to isolate and sequence individual cells in a manner that eliminates background noise from stroma and healthy cells, while capturing inter-cellular heterogeneity. Methods: Blood samples, collected from 138 progressing metastatic CRPC patients within 30 days of a pre-treatment biopsy intended for sequencing using MSK-IMPACT, were sent to EPIC Sciences for CTC analysis. Detected CTCs underwent single cell, low pass whole genome sequencing. Prevalence and concordance of BRCA2 copy-loss, regardless of whether single copy or homozygous, was compared in matched tissue and CTC samples. Results: BRCA2 copy-loss was identified in 21% (23/108) and 50% (58/115) of successfully sequenced tissue and CTC samples, respectively. In the 58 patients with CTC-detected BRCA2 loss, BRCA2 loss was detected in 36% (220/565) of the sequenced CTCs, representing a median of 46% (range 4-100%) of CTCs found in each individual sample. When both sequencing assays were successful, BRCA2 loss was detected in CTCs in 84% (16/19) of the tissue-positive cases, whereas tissue sequencing detected BRCA2 loss in 35% (16/46) of CTC-positive cases. Conclusions: Data from this study supports the notion that single-cell CTC sequencing can detect BRCA2 copy-loss at a high frequency, including cases that were negative in tissue, while also characterizing inter-cellular heterogeneity. Further studies will investigate whether CTC BRCA2 copy-loss can predict the likelihood of response to PARPi.

scholarly journals Desensitization of P2U receptor in neuronal cell line. Different control by the agonists ATP and UTP, as demonstrated by single-cell Ca2+ responses

1996 ◽  
Vol 320 (1) ◽  
pp. 215-219 ◽  
Author(s):  
Uwe CZUBAYKO ◽  
Georg REISER
Keyword(s):  
Single Cell ◽  
Cell Line ◽  
Binding Sites ◽  
Neuronal Cell ◽  
Nucleotide Receptor ◽  
Maximal Amplitude ◽  
Neuronal Cell Line ◽  
Natural Ligands ◽  
Extracellular Stimuli ◽  
Comparable Size

The neuronal cell line NG108-15 (180CC15) responds to extracellular stimuli of ATP or UTP with a transient increase in the level of cytosolic Ca2+. Desensitization was investigated by recording single-cell Ca2+ responses induced by consecutive, regularly spaced (100 s intervals), brief pulses of the nucleotides. The two natural ligands of the P2U receptor, ATP and UTP, were applied at a concentration that evoked responses of a comparable size. With two pulses of UTP (10 µM), a substantial decrease (of 43%) was observed in the size of the second response. The magnitude of response was determined by measuring either the maximal amplitude or the total response, represented by the area of the Ca2+ transient. The analogous studies with ATP pulses showed a much smaller decrease (of 12%). Comparable experiments performed to investigate the mutual interaction between ATP and UTP revealed that after stimulation with ATP the response to UTP was slightly (12%) diminished, whereas the response to ATP after UTP was greatly (52%) decreased. The different degree of desensitization by either UTP or ATP of P2U receptors could be due to (1) a difference in the mode of activation of the receptor by the two ligands or (2) recruitment of another effector mechanism besides elevated Ca2+. Our results indicate the existence of a novel mechanism of receptor control, involving different modes of the receptor, that are induced by two different, activating ligands. We also investigated the cross-talk between the bradykinin B2 receptor and the nucleotide receptor. ATP and UTP, even when eliciting responses of comparable size in the neuronal cell line, affect the desensitization of the bradykinin receptor differently. This suggests regulatory binding sites for the nucleotides on either the nucleotide receptor or the peptide receptor.

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