Computer vision reveals hidden variables underlying NF-κB activation in single cells
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ABSTRACT:
SUBMITTER: Patel P
PROVIDER: S-EPMC8535821 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1-2 nanoliters from a thin (~100 μm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 μm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved.
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Project description:IntroductionImproving treatments for Diffuse Large B-Cell Lymphoma (DLBCL) is challenged by the vast heterogeneity of the disease. Nuclear factor-κB (NF-κB) is frequently aberrantly activated in DLBCL. Transcriptionally active NF-κB is a dimer containing either RelA, RelB or cRel, but the variability in the composition of NF-κB between and within DLBCL cell populations is not known.ResultsHere we describe a new flow cytometry-based analysis technique termed "NF-κB fingerprinting" and demonstrate its applicability to DLBCL cell lines, DLBCL core-needle biopsy samples, and healthy donor blood samples. We find each of these cell populations has a unique NF-κB fingerprint and that widely used cell-of-origin classifications are inadequate to capture NF-κB heterogeneity in DLBCL. Computational modeling predicts that RelA is a key determinant of response to microenvironmental stimuli, and we experimentally identify substantial variability in RelA between and within ABC-DLBCL cell lines. We find that when we incorporate NF-κB fingerprints and mutational information into computational models we can predict how heterogeneous DLBCL cell populations respond to microenvironmental stimuli, and we validate these predictions experimentally.DiscussionOur results show that the composition of NF-κB is highly heterogeneous in DLBCL and predictive of how DLBCL cells will respond to microenvironmental stimuli. We find that commonly occurring mutations in the NF-κB signaling pathway reduce DLBCL's response to microenvironmental stimuli. NF-κB fingerprinting is a widely applicable analysis technique to quantify NF-κB heterogeneity in B cell malignancies that reveals functionally significant differences in NF-κB composition within and between cell populations.
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Project description:Transcriptional profiling of human control and Néstor-Guillermo Progeria Syndrome (NGPS) fibroblasts and induced pluripotent stem cells (iPSCs). Somatic cell reprogramming involves rejuvenation of adult cells and relies on the ability to erase age-associated molecular marks. Accordingly, reprogramming efficiency declines with ageing, and age-associated features such as genetic instability, cell senescence or telomere shortening negatively affect this process. However, the regulatory mechanisms that constitute age-associated barriers for cell reprogramming remain largely unknown. Here, by using cells from patients with premature ageing, we demonstrate that NF-κB activation is a critical barrier for the generation of induced pluripotent stem cells (iPSCs) in ageing. We show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs generation of iPSCs by eliciting reprogramming repressors DOT1L and YY1, reinforcing cell senescence signals and down-regulating pluripotency genes. We also show that genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo Progeria Syndrome (NGPS) and Hutchinson-Gilford Progeria Syndrome (HGPS) patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo ameliorates the accelerated ageing phenotype and extends lifespan in a progeroid animal model. Collectively, our results provide evidence for a novel role of NF-κB in the control of cell fate transitions and reinforce the interest of studying age-associated molecular impairments to implement cell reprogramming methodologies, and to identify new targets of rejuvenation strategies. Control and NGPS fibroblasts were reprogrammed. RNA was extracted and transcriptional profiling was obtained with GeneChip Human Exon 1.0 ST Arrays.
2015-07-01 | E-GEOD-65170 | biostudies-arrayexpress
Project description:Transcriptional profiling of human control and Néstor-Guillermo Progeria Syndrome (NGPS) mesenchymal stem cells (MSCs). Somatic cell reprogramming involves rejuvenation of adult cells and relies on the ability to erase age-associated molecular marks. Accordingly, reprogramming efficiency declines with ageing, and age-associated features such as genetic instability, cell senescence or telomere shortening negatively affect this process. However, the regulatory mechanisms that constitute age-associated barriers for cell reprogramming remain largely unknown. Here, by using cells from patients with premature ageing, we demonstrate that NF-κB activation is a critical barrier for the generation of induced pluripotent stem cells (iPSCs) in ageing. We show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs generation of iPSCs by eliciting reprogramming repressors DOT1L and YY1, reinforcing cell senescence signals and down-regulating pluripotency genes. We also show that genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo Progeria Syndrome (NGPS) and Hutchinson-Gilford Progeria Syndrome (HGPS) patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo ameliorates the accelerated ageing phenotype and extends lifespan in a progeroid animal model. Collectively, our results provide evidence for a novel role of NF-κB in the control of cell fate transitions and reinforce the interest of studying age-associated molecular impairments to implement cell reprogramming methodologies, and to identify new targets of rejuvenation strategies. Control and NGPS MSCs were differentiated into bone in the presence or absence of sodium salicylate. Total RNA was extracted and global gene expression was analyzed.
2015-07-01 | E-GEOD-65172 | biostudies-arrayexpress
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Project description:Rad50 is a component of MRN (Mre11-Rad50-Nbs1), which participates in DNA double-strand break repair and DNA-damage checkpoint activation. Here, we sought to investigate the clinical and functional significance of Rad50 in high-grade serous ovarian cancer (HGSOC). We found that Rad50 was frequently upregulated in HGSOCs and enhanced Rad50 expression inversely correlated with patient survival. In addition, ectopic expression of Rad50 promoted proliferation/invasion and induced EMT of ovarian cancer cells, whereas knockdown of Rad50 led to decreased aggressive behaviors. Mechanistic investigations revealed that Rad50 induced aggressiveness in HGSOC via activation of NF-κB signaling pathway. Moreover, we identified CARD9 as an interacting protein of Rad50 in ovarian cancer cells and the activation of NF-κB pathway by Rad50 is CARD9 dependent. Our findings provide evidence that Rad50 exhibits oncogenic property via NF-κB activation in HGSOC.
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